In the present work, we performed quantitative metabolomic analysis for eleven biological tissues of S. lucioperca. The advantage of the quantitative approach over commonly used semi-quantitative measurements is that the obtained data on the metabolite concentrations expressed in nmoles per gram of a tissue can be directly used by any researcher as a reference to the baseline level of metabolites in that tissue. Quantitative data also allow for the comparing the tissues with very dissimilar metabolomic compositions.
The metabolomic analysis performed in the present work demonstrates that although the majority of metabolites are common for all tissues, their concentrations in tissues may vary at a large scale. Moreover, there are some tissue-specific compounds with very high abundance in only 1–2 types of tissue. The examples of such metabolites are glycine, histidine, creatine, and betaine in muscle, ovothiol A in lens, NAA in lens and brain, glucose in liver. Apparently, these compounds are important for biological functions specific for these particular tissues.
Two groups of metabolites, osmolytes and antioxidants, play the key role in the cell protection against osmotic and oxidative stresses. In this work, the following compounds were conventionally assigned to osmolytes: taurine, myo-inositol, NAH, NAA, betaine, threonine-phosphoethanolamine (Thr-PETA), and Ser-PETA. Obviously, this assignment is rather arbitrary: some of these compounds, besides osmotic protection, perform other cellular functions, including cell signaling, providing substrate for biosynthesis, and so on17,18,19. At the same time, the tissues under study contain metabolites with concentrations of the same level or even higher than the concentrations of compounds assigned to osmolytes: lactate, glucose, acetate, creatine. These metabolites are mostly related to the reactions of cellular energy generation, and their concentrations should strongly depend on the fish activity. For that reason, in this work we did not include them into the list of osmolytes. Figure 6 shows the concentrations of osmolytes in different fish tissues (excluding acellular tissues AH and VH), and demonstrates that the composition of osmolytes in tissues strongly depends on the cell type.
Concentrations of major osmolytes (in µmol/g) in S. lucioperca tissues.
We found in the fish tissues the following compounds with antioxidative properties: glutathione (GSH), ascorbate, OSH, and NADH. The detection of minor amounts of one more well-known thiol antioxidant, ergothioneine, in the gills of another freshwater fish, R. rutilus lacustris, has recently been reported15; however, in the present work ergothioneine was not found neither by NMR nor by LC–MS in any of the studied tissues of S. lucioperca. NADH was found only in NMR spectra of liver, muscle, heart, and lens, and its concentration in these tissues does not exceed 15 nmol/g. GSH, OSH, and ascorbate are present in much higher concentrations in the majority of the fish tissues, so these three compounds play the main role in the cellular defense against the oxidative stress. The presence and the relative abundance of GSH and OSH in the fish tissues were confirmed by LC–MS data.
The metabolomic features of particular fish tissues are discussed below.
AH and VH
AH and VH are acellular fluids with minimal metabolic activity. AH is produced in the ciliary epithelium through both the active secretion and the passive diffusion/ultrafiltration of blood plasma20,21,22,23. Consequently, the metabolomic composition of AH is similar to that of plasma10. VH is also connected with blood via the hematoophthalmic barrier24 and with AH, and one can see (Table 1, Fig. 2) that the metabolomic compositions of AH and VH are close to each other. Thus, it is safe to assume that the levels of metabolites in AH and VH reflect their levels in blood plasma, which circulates through the majority of fish tissues. Significant deviations of metabolite levels in tissue as compared to plasma should be attributed to the intracellular metabolic activity specific for this particular tissue.
Lens
The eye lens is one of the most anatomically isolated tissues. The lens mostly consists of metabolically inert fiber cells without nuclei and organelles with the exception of metabolically active epithelial monolayer. The data present in Table 1 indicate that the lens contains very high levels of proteinogenic amino acids: for some amino acids (for example, branched-chain amino acids, glutamine, aspartate) their levels in the lens are more than ten-fold higher than that in AH. Moreover, the concentrations of the majority of amino acids in the lens are higher than in any other fish tissue. The elevated levels of amino acids in the lens has been noticed many years ago25, and it was attributed to the active amino acid transport from AH to lens26,27,28,29. These amino acids are presumably needed to synthesize high protein content (up to 40% of the total lens weight), which is in turn needed to provide high refraction coefficient.
The fish lens contains a unique set of osmolytes and antioxidants. The lens osmolytes are myo-inositol, NAH, NAA, Thr-PETA, and Ser-PETA. We have previously shown15 that the concentrations of osmolytes in the fish lens undergo significant seasonal variations. At the late winter time, when the fish was caught for this study, the most abundant lens osmolyte is myo-inositol. High concentrations of this compound are also found in other fish tissues, including brain, gill, and spleen. Thr-PETA and Ser-PETA are also among the most abundant metabolites in the majority of the fish tissues. In opposite, NAH and NAA are present in high concentrations only in the fish lens and brain. At the same time, the concentration of taurine, which is the most abundant osmolyte in all other fish tissues, in the lens is rather low.
The major antioxidant of the fish lens is OSH12. It has been shown that the level of OSH in S. lucioperca lens vary from 3 µmol/g at autumn to 1.5 µmol/g at winter15, which is in a good agreement with our present data (Table 1). The concentration of the second most abundant lens antioxidant, GSH, is 3–4 times lower than that of OSH. Taking into account the properties of OSH30,31,32,33, it has been proposed12,34 that OSH is a primary protector against the oxidative stress, while the main function of GSH in the lens is the maintenance of OSH in the reduced state. It should be noticed that although OSH was also found in other fish tissues (Table 1, Fig. 2), its concentration in these tissues is significantly lower than in the lens. Therefore, in respect to fish, OSH can truly be called “lenticular antioxidant”.
High concentrations of amino acids, osmolytes, antioxidants and some other compounds in the lens indicate that these metabolites are either synthesized in metabolically active epithelial cells, or pumped into the lens from AH against the concentration gradient with the use of specific transporters also located in the epithelial layer. The fiber cells of the lens are metabolically passive, and fresh metabolites can appear in these cells only due to the diffusion from the epithelial layer toward the lens center. Therefore, one can expect that the concentrations of the most important metabolites decrease from the lens cortex toward the lens nucleus. To check this assumption, we measured the metabolomic profiles for cortex and nucleus separately. The measurements were performed for three lenses; then the ratios of the metabolite concentrations in the cortex to that in the nucleus were calculated and averaged. The results of the calculations are shown in Fig. 7 (only for metabolites with the highest and the lowest cortex/nucleus ratios) and Supplementary Table S2 (for all metabolites). Indeed, the levels of the majority of metabolites in the lens nucleus are significantly lower than in the cortex. For five metabolites, namely ATP, NAA, inosinate, ADP, and GSH the difference exceeds the factor of thirty; that means that these compounds are almost completely depleted during their diffusion toward the lens nucleus.
Barplot for statistically significant differences in the metabolomic content of lens cortex and nucleus. Bars show the averaged ratio of metabolite concentrations in the cortex to that in the nucleus of the S. lucioperca lens.
Brain
The brain tissue similarly to the lens is isolated from the vascular system by means of the hematoencephalic barrier. However, in opposite to the lens, brain is very metabolically active tissue, as in particular indicated by the high level of lactate (14 µmol/g). Similar lactate concentrations were found only in muscle and heart (Table 1). Besides lactate, the most abundant metabolites of the fish brain are osmolytes myo-inositol, Ser-PETA, taurine, Thr-PETA, NAH, and NAA; the concentrations of these compounds in brain are in the range from 2.5 to 13 µmol/g (Table 1). The brain tissue also contains high levels of glutamate and creatine, which are used by brain cells for the cellular energy generation. The level of antioxidant ascorbate in brain (400 nmol/g) is significantly higher than in other fish tissues, which indicates the importance of ascorbate for the brain correct operation. Besides ascorbate, the brain tissue also contains OSH and GSH, but at significantly lower concentrations (100–200 nmol/g).
Blood-rich organs: liver, spleen, milt, muscle, heart, gill, kidney
Figure 4 demonstrates that from the metabolomic viewpoint, gill, kidney, milt, and spleen are the most similar tissues. However, the quantitative analysis indicates significant differences. In particular, spleen does not contain measurable by NMR amounts of antioxidants OSH and GSH. The levels of osmolytes are also different: the concentration of taurine in spleen is threefold higher than in gill and milt, while the level of myo-inositol in milt is much lower than in spleen and gill (Fig. 6). Significant differences are also found for some amino acids (alanine, creatine), organic acids (lactate, GABA), and nucleosides (ATP, ADP, AMP, inosine).
One of the important liver functions is the maintaining the glucose level in blood regulated by producing glucose from stored glycogen. Correspondingly, the level of glucose in liver is extremely high (40 µmol/g), which is higher than in any other tissue by at least an order of magnitude. Liver also contains elevated (as compared to other tissues) concentrations of threonine, glutamate, succinate, fumarate, AMP, and nicotinamide.
The biological functions of muscle and heart are relatively similar; however, the metabolomic compositions of these tissues differ significantly. The main osmolyte in muscle cells is taurine (30 µmol/g), while in heart the osmotic protection is shared between taurine (15 µmol/g) and Ser-PETA (12 µmol/g). Muscle contains very high levels of glycine and histidine. Glycine is known to protect muscles from wasting under various wasting conditions35,36, while histidine and histidine-related compounds were reported to play the role of intracellular proton buffering constituents in vertebrate muscle37. Very likely that both glycine and histidine also participate in the osmotic protection of the muscle cells. The level of creatine—the energy source—in muscle (23 µmol/g) is five-fold higher than in heart.
Source: Ecology - nature.com
