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. 2021 Dec 9:12:769833.
doi: 10.3389/fphys.2021.769833. eCollection 2021.

Metabolic and Redox Biomarkers in Skeletal Muscle Underlie Physiological Adaptations of Two Estivating Anuran Species in a South American Semi-arid Environment

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Metabolic and Redox Biomarkers in Skeletal Muscle Underlie Physiological Adaptations of Two Estivating Anuran Species in a South American Semi-arid Environment

Daniel C Moreira et al. Front Physiol. .

Abstract

The upregulation of endogenous antioxidants (i.e., preparation for oxidative stress, POS) is part of the biochemical responses underlying the adaptation of animals to adverse environments. Despite the phylogenetic diversity of animals in which POS has been described, most studies focus on animals under controlled laboratory conditions. To address this limitation, we have recently assessed the redox metabolism in the skeletal muscle of Proceratophrys cristiceps estivating under natural settings in the Caatinga. Here, we analyzed biochemical biomarkers in the muscle of another Caatinga species, Pleurodema diplolister, during the rainy (active) and dry (estivating frogs) seasons. We aimed to determine whether P. diplolister enhances its antioxidants during estivation under field conditions and to identify any effect of species on the biochemical responses of P. diplolister and P. cristiceps associated with estivation. To do so, we measured the activities of representative enzymes of intermediary metabolism and antioxidant systems, as well as glutathione and protein carbonyl levels, in the skeletal muscle of P. diplolister. Our findings revealed the suppression of oxidative metabolism and activation of antioxidant enzymes in estivating P. diplolister compared with active specimens. No changes in oxidative damage to proteins were observed and estivating P. diplolister had lower levels of disulfide glutathione (GSSG) and disulfide-to-total glutathione ratio (GSSG/tGSH) than those observed in active individuals. When data for P. diplolister and P. cristiceps were assembled and analyzed, significant effects of species were detected on the activities of metabolic enzymes (citrate synthase, isocitric dehydrogenase, malic enzyme, and creatine kinase) and antioxidant enzymes (catalase, glutathione peroxidase and glutathione transferase), as well as on GSSG/tGSH ratio. Such effects might underlie the physiological and behavioral differences between these two species that share the same microhabitat and survival strategy (i.e., to estivate) during the dry season. Despite some peculiarities, which reflect the physiological diversity of the mechanisms associated with estivation in the Brazilian Caatinga, both P. diplolister and P. cristiceps seem to balance the suppression of oxidative pathways, the maintenance of the capacity of oxygen-independent pathways, and the activation of endogenous antioxidants to preserve muscle function and be ready to resume activity whenever the unpredictable rainy period arrives.

Keywords: Caatinga; Pleurodema diplolister; Proceratophrys cristiceps; amphibia; antioxidant; glutathione; preparation for oxidative stress; reactive oxygen species.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Total rainfall (mm) for each month from 2005 to 2012 as recorded in the Florânia meteorological station (6°6′36ʺS and 36°48′36ʺW) of the National Meteorological Institute (INMET), the closest station to the collection site. The Caatinga is characterized by a rainy season within the first 3–4months of the year and high annual variability of total precipitation levels. Data are shown as boxes (from the 25th to the 75th percentiles) and whiskers (from the lowest to the highest value) with the line inside the box marking the median.
Figure 2
Figure 2
Activities of enzymes that catalyze key steps in metabolic pathways in the skeletal muscle of Pleurodema diplolister collected during the dry season (estivating) or the rainy season (active). (A) Citrate synthase. (B) Isocitric dehydrogenase. (C) Pyruvate kinase. (D) Creatine kinase. (E) Malic enzyme. (F) Lactic dehydrogenase. The asterisk (*) denotes significant differences between active and estivating groups. N=6.
Figure 3
Figure 3
Levels of glutathione (total and reduced) and glutathione transferase activity in the skeletal muscle of Pleurodema diplolister collected during the dry season (estivating) or the rainy season (active). (A) Total glutathione (tGSH). (B) Reduced glutathione (GSH). (C) Glutathione transferase. There were no significant differences between active and estivating groups. N=6.
Figure 4
Figure 4
Redox imbalance and oxidative stress markers in the skeletal muscle of Pleurodema diplolister collected during the dry season (estivating) or the rainy season (active). (A) Disulfide glutathione (GSSG). (B) Ratio between GSSG and total glutathione (GSSG/tGSH). (C) Protein carbonyl. The asterisk (*) denotes significant differences between active and estivating groups. N=6.
Figure 5
Figure 5
Activities of core antioxidant enzymes in the skeletal muscle of Pleurodema diplolister collected during the dry season (estivating) or the rainy season (active). (A) Total superoxide dismutase (tSOD). (B) Catalase. (C) H2O2-Glutathione peroxidase (GPX). (D) Total glutathione peroxidase (tGPX). The asterisk (*) denotes significant differences between active and estivating groups. N=6.
Figure 6
Figure 6
Principal component analysis of biomarkers of key metabolic pathways, endogenous antioxidants, redox imbalance, and oxidative stress. Each circle corresponds to an individual. Note that the group of active Proceratophrys cristiceps has only five individuals, since one of the analyzed variables (catalase activity) was measured in only five animals in this group. The resulting coefficients for each principal component are shown in Table 3. Data for P. cristiceps, previously published in Moreira et al. (2020) and summarized in Table 1, were included in the analysis.
Figure 7
Figure 7
Graphical summary of the biochemical adaptations of two frog species that estivate during the seasonal droughts in the Caatinga. Free-ranging adult specimens of Pleurodema diplolister and Proceratophrys cristiceps were collected during the rainy (active) and the dry season (estivating) in the Caatinga. The whole skeletal mass surrounding the femur was dissected and homogenized for the measurement of biochemical variables. The activities of intermediary metabolism and antioxidant enzymes, the levels of total (tGSH), reduced (GSH) and disulfide (GSSG) glutathione, and the degree of oxidative damage to proteins were measured. Species had an effect on the activities of citrate synthase, isocitric dehydrogenase, glutathione peroxidase, glutathione transferase, catalase, malic enzyme and creatine kinase, as well as on GSSG/tGSH ratio. Estivation had an effect on the activities of citrate synthase, isocitric dehydrogenase, glutathione peroxidase and catalase, as well as on the levels of tGSH, GSH, GSSG, and GSSG/tGSH. The upward arrows indicate variables whose values are higher in P. diplolister (vs. P. cristiceps) or in estivating frogs (vs. active frogs). The downward arrows indicate variables whose values are lower in P. diplolister (vs. P. cristiceps) or in estivating frogs (vs. active frogs).

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