In the present study, we aimed to decipher the molecular mechanisms of the response to acute oxidative stress in a fish model and correlate them with the phenotype observed in aging animals. We discuss our results in the context of mounting evidence linking oxidative stress to aging. In particular, we study the role of the urea cycle enzymes in stress response to acute hypoxia in fish and distinguish between the patterns of Arg1 and Arg2 expression. Additionally, we propose an original model for the evolutionary role of arginases in the stress response mechanisms, which is especially relevant in light of recent observations showing a significant elevation in arginase activity and Arg1 expression levels in the muscles of aging mice and following oxidative damage in in vitro models [47]. Likewise, recent research on the role of arginase in the development of neurodegenerative diseases points to a translational potential of our study [8, 48].
We have mentioned above that simple organisms possess only a mitochondrial Arg2, which indicates its bacterial origin [49]. The arginase coding gene duplication followed the partition of vertebrates and invertebrates (Fig. 1) [41]. Vertebrates typically express a cytosolic isoform, Arg1, additionally to Arg2 [19]. Accordingly, it is believed that the mitochondrial arginase is the ancestral isoform [24], which we also prove in our evolutionary analysis (Fig. 1). Moreover, we demonstrate that Arg2 is the predominant isoform in zebrafish (Fig. 6c), which further indicates its origin.
Even though, in most ammoniotelic teleost, arginase activity is exclusively mitochondrial [50], several species express intra- and extra-mitochondrially enzymatically active arginase isoforms [51, 52]. Moreover, some air-breathing walking fish demonstrate unique mitochondrial localization for both Arg1 and Arg2 [53]. Here, we demonstrate a distinct Arg1 expression in the zebrafish liver mitochondria (Fig. 6). We prove our findings with a bioinformatics tool and evidence an apparent mitochondrial signature in both isoforms in zebrafish (Fig. 5). It is plausible to assume that two arginase isoforms fulfill several overlapping functions in fish, and many evolutionary transitional species show significant functional differences between Arg1 and Arg2. Therefore, we suggest that a comparative analysis of the remote evolutionary species may resolve this functional conundrum related to the concomitant presence of two seemingly similar isoforms.
Of note, both arginase isoforms were shown in the murine brain tissue by several groups [54, 55]. Moreover, the brain arginase enzymatic activity is accounted for both isoforms [24, 56]. Remarkably, the levels of two central urea cycle enzymes, namely ornithine transcarbamylase (OTC) and carbamoyl phosphate synthetase (CPS), in the mammal brain are shallow [57], which resembles the pattern in adult fish and points to the unique function of arginase in the mammal central nervous system (CNS) transcending beyond the urea cycle.
ROS have been shown to instigate Arg1 expression levels and activity [58]. Arg2 levels also escalate significantly as a reaction to hypoxia, as well as bacterial lipopolysaccharides, tumor necrosis factor-alpha (TNFα), and oxidized low-density lipoproteins [59]. Remarkably, Arg2 activation in mammals is associated with its translocation from the mitochondria to the cytosol [55, 60].
In the present study, we consistently demonstrate via several methods a significant upsurge in the levels of Arg2 as a reaction to acute oxidative stress and following aging in zebrafish brain and muscles. Accordingly, we suggest that being an ancestral isoform, Arg2 fulfills the primary protective antioxidative function in fish. In mammals, in contrast, this function shifts towards the Arg1, which is a vital enzyme in mice and humans. Though, mammalian Arg2 becomes seemingly futile - its deletion leads to no apparent phenotype and even prolongs rodents' lifespan [61]. In this context, it might be interesting to look at the arginase function in amphibian animals, representing an intermediate evolutionary stage.
It is well-established that the chief function of arginase in ureotelic animals is to deal with an excess of ammonia, being the last enzyme of the urea cycle (Fig. S1)[62]. Nevertheless, recent discoveries indicate the enzyme's role in diverse physiological functions and pathological processes that overstep the urea cycle. It has been shown that Arg2 is essential for polyamine synthesis [24]. Of note, the principal biosynthetic polyamines' pathway utilizes arginine as a precursor of putrescine and comprises arginase and ODC. ODC is the rate-limiting enzyme of the polyamines' biosynthesis [17], highly conserved in fish and mammals [18]. It is noteworthy that ODC is dependent upon the principal arginase product, ornithine, concentrations. Therefore, arginase represents an efficient upstream gate-keeping enzyme of the polyamine pathway.
Short-term PSR has been proposed to be universally beneficial for an individual's survival [8, 9]. Consequently, we suggest that arginase elevation is the first step in a typical PSR that has evolved as a ubiquitous adaptive mechanism. High-order polyamines, spermidine, and spermine serve as universal free radical scavenging agents and potent regulators of various cellular functions. Spermine has been shown to diminish NOS levels and activity [63], which explains the decline in NOS1 and NOS2b levels observed in our study (Fig. 2c). Inactive NOS improves arginine bioavailability for arginase and further facilitates polyamine synthesis. Of note, arginase and NOS compete for a mutual substrate, arginine. Accordingly, Arg2 overexpression causes substrate deprivation of NOS and eventuates into NOS1 underexpression [64].
Remarkably, arginase activity in teleosts is mainly found in high metabolic rate tissues, including muscles [65], pointing to a peculiar function of arginase in these tissues, which is presumably related to its antioxidant properties. Likewise, the expression profile pattern for Arg2 in aging fish resembles the hypoxia paradigm (Fig. 4c), which points to analogous mechanisms. This observation supports our view on aging as a consequence of emergent oxidative stress [66]. Aging has a progressive impact upon the antioxidant properties of various tissues and ROS homeostasis. Highly active metabolically tissues of the brain and muscles are susceptible to age-related changes. Consequently, these homeostatic imbalances lead to musculoskeletal pathologies (including sarcopenia) and neurodegenerative diseases (including Alzheimer's disease) [8]. Recent preclinical studies indicate promising potential for arginase inhibitors in the treatment of age-associated sarcopenia [47] and neurodegeneration [67]. This approach leads to fine-tuning of arginase activity and improvement in the general bioavailability of arginine that is a mighty natural antioxidant [68].
In the present study, we provide evidence that acute hypoxia induces polyamine synthesis and arrests the urea cycle in adult zebrafish, in contrast with embryos, which do not demonstrate a substantial reduction in ASS1 levels (Fig. 2a). We have mentioned above that the urea cycle is rudimental in adult zebrafish; however, it is active and vital in young animals. Therefore, its decline is much less prominent in young fish following acute hypoxia.
It is also noteworthy that acute hypoxia predictably caused elevation in the levels of CAT and HIF-1 (Fig. 2b, c). CAT is ubiquitous in all phyla that are exposed to oxygen. The enzyme decomposes hydrogen peroxide to generate water and oxygen, which protects cells from oxidative damage. Its expression levels are also sensitive to ROS concentration, though its activity is substantially reduced in aging and degenerating tissues making catalase a putative pathogenic factor [69]. HIF-1 is a transcription factor with a central role in the coordination of cellular response to oxygen levels. Thus fish, an ancient product of the Cambrian explosion, apparently apply various strategies to cope with oxidative stress, and PSR plays one of the central roles in this fabulously coordinated process. Remarkably, the stress-associated upsurge in the levels of arg2 and odc1 in fish exceeds substantially the increase in the classical antioxidants’ levels (Fig. 2). This phenomenon signifies the key role of PSR in the evolutionary-conserved machinery to cope with the environmental challenges.
As noted above, adult fish dispose ammonia directly into the environment, indicating unconventional functions for the urea cycle enzymes in these animals. Though, some species maintain the urea cycle gene expression throughout the life cycle to cope with stressful environmental conditions, which points to its evolutionary role. Still, the question of why some fish periodically synthesize the energetically expensive urea is debated in the literature. Also, the precise role of Arg2 in mammals remains a mystery. Incontestably, the presence and conservation throughout the evolution of apparently futile enzymes indicates their alternative but crucial functional significance, which is yet to be revealed.