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

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.

Effects of food intake and hydration state on behavioral thermoregulation and locomotor activity in the tropidurid lizard Tropidurus catalanensis.

Theoretical models predict that lizards adjust their body temperature through behavioral thermoregulation as a function of food availability. However, behavioral thermoregulation is also governed by interactions among physiological and ecological factors other than food availability, such as hydration state, and sometimes it can even conflict with the locomotor activity of animals. Here, we aimed to investigate the role of food intake and hydration state on behavioral thermoregulation and voluntary locomotor activity in the lizard Tropidurus catalanensis We hypothesized that food intake can influence behavioral thermoregulation via an interaction with hydration state. We also hypothesized that lizards should endeavor to spend as little time as possible to reach their preferred body temperature to defend other physiological and/or ecological functions. We collected lizards in the field and brought them to the laboratory to measure the preferred temperature selected in a thermal gradient and the total distance traveled by them in fed and unfed conditions and with variable hydration state. Our results showed that food consumption was the most important predictor of preferred temperature. In contrast, either the hydration state alone or its interaction with food consumption did not have important effects on the lizards' thermal preference. Also, we found that the total distance traveled by lizards was not affected by food intake and was barely affected by the hydration state. We provide an experimental approach and a robust analysis of the factors that influence behavioral thermoregulation and locomotor activity in a tropical lizard.

Do prolonged fasting periods influence the postprandial metabolic responses in turtles? What can Trachemys scripta elegans teach us about this?

The postprandial period is characterized by a modification of the gastrointestinal activity after food intake, accompanied by an increase in metabolic rate, secretion of acids, and absorption of nutrients. For ectothermic vertebrates, those changes are particularly prominent given the relatively low metabolic cost and the low frequency of food uptake. However, prolonged fasting periods decrease energy reserves and may compromise the upregulation of costly processes, such as the increase in metabolic rate after resuming the meal intake. Assuming that the main source of energy needed to support such events is provided from the animal's own body reserves, our aim with this study is to test the hypothesis that the longer the period of fasting, the smaller the metabolic rate increase during the postprandial period, since lesser energy reserves trigger these increases. For this, we measured the oxygen consumption rates (V̇O2 ) of red-eared slider turtles, Trachemys scripta elegans, submitted to different periods of fasting (47 and 102 days), before and after the ingestion of meals equivalent to 5% of their body masses. Despite the longer fasting period, which led to a reduction of 10.77% in the body mass of the turtles, there were no differences between the two experimental groups regarding maximum V̇O2 values after food intake (V̇O2 peak), postprandial metabolic scope, mean time to V̇O2 peak, and postprandial duration. Results indicate that 102 fasting days does not compromise aerobic metabolic increase during postprandial period and does not impair digestive process of the turtles, even with a loss of body mass.

Preparation for oxidative stress in Proceratophrys cristiceps (Anura, Odontophrynidae) naturally estivating in the Brazilian Caatinga.

Preparation for oxidative stress (POS), i.e., the buildup of endogenous antioxidants during metabolic depression or low oxygen stress conditions, has been observed in at least 8 animal phyla under controlled conditions in laboratory. Despite the expected implications on the endurance to extreme environments and ecosystem occupation, the extent to which POS occurs in animals under natural conditions remains unexplored. Therefore, we took advantage of the natural history of the Brazilian Caatinga's frog Proceratophrys cristiceps to investigate the modulation of endogenous antioxidants and redox balance in their skeletal muscle and to verify if POS occurs under natural conditions. Expectedly, estivating frogs had low levels of the oxidative metabolism enzymes. Citrate synthase and isocitrate dehydrogenase activities were 36% and 25% lower than those in active frogs respectively. We found an overall upregulation of antioxidants in estivating P. cristiceps. Reduced glutathione concentration was 61% higher in estivating frogs than that in active animals. During estivation the activities of the hydroperoxide detoxification enzymes catalase, glutathione peroxidase, and glutathione H2O2-peroxidase were 48%, 57%, and 78% greater than those during the rainy season. Moreover, estivating frogs had a 47% lower ratio of disulfide to total glutathione levels than active frogs. Our findings confirm the occurrence of 'preparation for oxidative stress' in naturally estivating frogs and paves the way for further research on the redox biology of animals under natural settings. Such approach might reveal biochemical strategies under ecologically relevant scenarios.

Blood oxygen affinity increases during digestion in the South American rattlesnake, Crotalus durissus terrificus.

Digesting snakes experience massive increases in metabolism that can last for many days and are accompanied by adjustments in the oxygen transport cascade. Accordingly, we examined the oxygen-binding properties of the blood in the South American rattlesnake (Crotalus durissus terrificus) during fasting and 24 and 48h after the snakes have ingested a rodent meal corresponding to 15% (±2%) of its own body mass. In general, oxygen-hemoglobin (Hb-O2) affinity was significantly increased 24h post-feeding, and then returned toward fasting values within 48h post-feeding. Content of organic phosphates ([NTP] and [NTP]/[Hb]), hemoglobin cooperativity (Hill's n), and Bohr Effect (ΔlogP50/ΔpH) were not affected by feeding. The postprandial increase in Hb-O2 affinity in the South American rattlesnake can be almost entirely ascribed by the moderate alkaline tide that follows meal ingestion. In general, digesting snakes were able to regulate blood metabolites at quite constant levels (e.g., plasma osmolality, lactate, glucose, and total protein levels). The level of circulating lipids, however, was considerably increased, which may be related to their mobilization, since lipids are known to be incorporated by the enterocytes after snakes have fed. In conclusion, our results indicate that the exceptional metabolic increment exhibited by C. d. terrificus during meal digestion is entirely supported by the aerobic pathways and that among the attending cardiorespiratory adjustments, pulmonary Hb-O2 loading is likely improved due to the increment in blood O2 affinity.

The antiapoptotic effect of granulocyte colony-stimulating factor reduces infarct size and prevents heart failure development in rats.

BACKGROUND/AIM: Granulocyte colony-stimulating factor (G-CSF) reduces myocardial injury and improves cardiac function after myocardial infarction (MI). We investigated the early alterations provided by G-CSF and the chronic repercussions in infarcted rats. METHODS: Male Wistar rats (200-250g) received vehicle (MI) or G-CSF (MI-GCSF) (50 µg/kg, sc) at 7, 3 and 1 days before MI surgery. Afterwards MI was produced and infarct size was measured 1 and 15 days after surgery. Expression of anti- and proapoptotic proteins was evaluated immediately before surgery. 24 hours after surgery, apoptotic nuclei were evaluated. Two weeks after MI, left ventricular (LV) function was evaluated, followed by in situ LV diastolic pressure-volume evaluation. RESULTS: Infarct size was decreased by 1 day pre-treatment before occlusion (36±2.8 vs. 44±2.1% in MI; P<0.05) and remained reduced at 15 days after infarction (28±2.2 vs. 36±1.4% in MI; P<0.05). G-CSF pretreatment increased Bcl-2 and Bcl-xL protein expression, but did not alter Bax in LV. Apoptotic nuclei were reduced by treatment (Sham: 0.46±0.42, MI: 15.5±2.43, MI-GCSF: 5.34±3.34%; P<0.05). Fifteen days after MI, cardiac function remained preserved in G-CSF pretreated rats. The LV dilation was reduced in MI-G-CSF group as compared to MI rats, being closely associated with infarct size. CONCLUSION: The early beneficial effects of G-CSF were essentials to preserve cardiac function at a chronic stage of myocardial infarction.

MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic ß-cell proliferation in islets from early lactating mothers.

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phospho-ERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.

Energy and water in aestivating amphibians.

The physiological mechanisms, behavioral adjustments, and ecological associations that allow animal species to live in extreme environments have evoked the attention of many zoologists. Often, extreme environments are defined as those believed to be limiting to life in terms of water, energetic availability, and temperature. These three elements seem extreme in a number of arid and semi-arid settings that even so have been colonized by amphibians. Because this taxon is usually seen as the quintessential water-dependent ectotherm tetrapods, their presence in a number of semi-arid environments poses a number of intriguing questions regarding microhabitat choice and physiological plasticity, particularly regarding the ecological and physiological correlates of behaviors granting avoidance of the harshest conditions of semi-arid environments. Such avoidance states, generally associated to the concept of aestivation, are currently seen as a diverse and complex phenomena varying from species to species and involving numerous behavioral and metabolic adjustments that enhance survival during the drought. This chapter reviews the physiological ecology of anuran aestivation, mainly from the perspective of water and energy balance.

Granulocyte colony-stimulating factor reduces mortality by suppressing ventricular arrhythmias in acute phase of myocardial infarction in rats.

Our aim was to evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on early cardiac arrhythmias after myocardial infarction (MI) and the impact on survival. Male Wistar rats received repeated doses of 50 mug/kg G-CSF (MI-GCSF group) or vehicle (MI group) at 7, 3, and 1 days before surgery. MI was induced by permanent occlusion of left coronary artery. The electrocardiogram was obtained before occlusion and then for 30 minutes after surgery. Events and duration of ventricular arrhythmias were analyzed. The levels of connexin43 (Cx43) were measured by Western blot immediately before MI production. Survival was significantly increased in MI-GCSF pretreated group (74% versus 52.9% MI, P < 0.05). G-CSF pretreatment also significantly reduced the ventricular premature beats when compared with the untreated-MI group (201 +/- 47 versus 679 +/- 117, P < 0.05). The number and the duration of ventricular tachycardia were smaller in the MI-G-CSF group, as well as the number of ventricular fibrillation episodes (10% versus 69% in MI, P < 0.05). Cx43 levels were significantly increased by G-CSF treatment (1.27 +/- 0.13 versus 0.86 +/- 0.11; P < 0.05). The MI size 24 hours after occlusion was reduced by G-CSF pretreatment (36 +/- 3% versus 44 +/- 2% of left ventricle in MI group; P < 0.05). The increase of Cx43 expression in the heart may explain the reduced incidence in ventricular arrhythmias in the early phases after coronary artery occlusion in rats, thus increasing survival after MI.

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulum Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period.

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PRL) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to non-pregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (L3). The expression of the sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in beta-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of P19 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

Physiological basis for diurnal activity in dispersing juvenile Bufo granulosus in the Caatinga, a Brazilian semi-arid environment.

Diurnal activity is characteristic of many toad species, including Bufo granulosus from the Brazilian semi-arid biome called the Caatinga. Because of their patterns of activity, juvenile toads are exposed to hot and dehydrating conditions. Our investigation focuses on temperature and water relationships, and is based on the prediction that anuran diurnal activity in a semi-arid environment must be associated with morphological, physiological and behavioral traits enhancing thermal tolerances, capacity for performance at high temperatures and water balance. To test specific hypothesis related with this prediction, we investigated postmetamorphic B. granulosus and collected data on thermal tolerances and preferences, thermal safety margins, thermal dependence of locomotor behavior, thermal and kinetic properties of citrate synthase (CS), and skin morphophysiology. This information was compared with additional data from adult conspecifics and adult toads from sympatric species or from species from more moderate environments. We found that juvenile B. granulosus exhibit the highest critical maximum temperature reported for toads (44.2 degrees C) and are well suited to move at high temperatures. However, and in contrast with juveniles of other Bufo species, they do not show thermal preferences in a gradient and appear to hydroregulate more than thermoregulate. The CS of adult and juvenile toads shows typical patterns of thermal sensibility, but the thermal stability of this enzyme is much higher in juveniles than in adult Bufo of any other species studied. The inguinal skin exhibits a complex folding pattern and seems highly specialized for capillary water uptake. Diurnal activity in juvenile B. granulosus is possible given high thermal tolerances, keen ability to detect and uptake water, and avoidance behaviors.

Physiological basis for diurnal activity in dispersing juvenile Bufo granulosus in the Caatinga, a Brazilian semi-arid environment

Diurnal activity is characteristic of many toad species, including Bufo granulosus from the Brazilian semi-arid biome called the Caatinga. Because of their patterns of activity, juvenile toads are exposed to hot and dehydrating conditions. Our investigation focuses on temperature and water relationships, and is based on the prediction that anuran diurnal activity in a semi-arid environment must be associated with morphological, physiological and behavioral traits enhancing thermal tolerances, capacity for performance at high temperatures and water balance. To test specific hypothesis related with this prediction, we investigated postmetamorphic B. granulosus and collected data on thermal tolerances and preferences, thermal safety margins, thermal dependence of locomotor behavior, thermal and kinetic properties of citrate synthase (CS), and skin morphophysiology. This information was compared with additional data from adult conspecifics and adult toads from sympatric species or from species from more moderate environments. We found that juvenile B. granulosus exhibit the highest critical maximum temperature reported for toads (44.2 °C) and are well suited to move at high temperatures. However, and in contrast with juveniles of other Bufo species, they do not show thermal preferences in a gradient and appear to hydroregulate more than thermoregulate. The CS of adult and juvenile toads shows typical patterns of thermal sensibility, but the thermal stability of this enzyme is much higher in juveniles than in adult Bufo of any other species studied. The inguinal skin exhibits a complex folding pattern and seems highly specialized for capillary water uptake. Diurnal activity in juvenile B. granulosus is possible given high thermal tolerances, keen ability to detect and uptake water, and avoidance behaviors.

Interindividual differences in leg muscle mass and pyruvate kinase activity correlate with interindividual differences in jumping performance of Hyla multilineata.

Frog jumping is an excellent model system for examining the structural basis of interindividual variation in burst locomotor performance. Some possible factors that affect jump performance, such as total body size, hindlimb length, muscle mass, and muscle mechanical and biochemical properties, were analysed at the interindividual (intraspecies) level in the tree frog Hyla multilineata. The aim of this study was to determine which of these physiological and anatomical variables both vary between individuals and are correlated with interindividual variation in jump performance. The model produced via stepwise linear regression analysis of absolute data suggested that 62% of the interindividual variation in maximum jump distance could be explained by a combination of interindividual variation in absolute plantaris muscle mass, total hindlimb muscle mass (excluding plantaris muscle), and pyruvate kinase activity. When body length effects were removed, multiple regression indicated that the same independent variables explained 43% of the residual interindividual variation in jump distance. This suggests that individuals with relatively large jumping muscles and high pyruvate kinase activity for their body size achieved comparatively large maximal jump distances for their body size.

Prolactin-modulated gene expression profiles in pancreatic islets from adult female rats.

The effects of prolactin (PRL) on transcript profile expression in 24h cultured pancreatic adult rat islets were investigated by cDNA expression array analysis to identify possible candidate mRNA species that encode proteins involved in the maturation and growth of the endocrine pancreas. The expression of 54 out of 588 genes was altered by treatment with PRL. The differentially expressed transcripts identified were distributed in six main categories involved in cell proliferation and differentiation, namely, cell cycle regulation, signal transduction, transcription factors and coactivators, translational machinery, Ca(2+)-mediated exocytosis, and immuno-response. Treatment with PRL also reduced the expression of genes related to apoptosis. Several genes, whose expression was previously not known to be modulated by PRL were also identified including macrophage migration inhibitory factor and Ca(2+)/calmodulin-dependent protein kinase IV. These genes have recently been shown to play a crucial role in insulin secretion and insulin gene expression, respectively. Treatment with PRL also modified the expression of AKT2 and bone morphogenetic protein receptor 1A that control glucose homeostasis and directly affect the behavior of endocrine pancreas and/or the sensitivity of target tissues to insulin. In conclusion, PRL induces several patterns of gene expression in pancreatic islet cells. The analysis of these different patterns will be useful for understanding the complex mechanism of action of PRL in the maturation and differentiation of pancreatic islets.

Locomotor performance of closely related Tropidurus species: relationships with physiological parameters and ecological divergence.

Tropidurid lizards have colonized a variety of Brazilian open environments without remarkable morphological variation, despite ecological and structural differences among habitats used. This study focuses on two Tropidurus sister-species that, despite systematic proximity and similar morphology, exhibit great ecological divergence and a third ecologically generalist congeneric species providing an outgroup comparison. We quantified jumping capacity and sprint speed of each species on sand and rock to test whether ecological divergence was also accompanied by differences in locomotor performance. Relevant physiological traits possibly associated with locomotor performance - metabolic scopes and fiber type composition, power output and activity of the enzymes citrate synthase, pyruvate kinase and lactate dehydrogenase of the iliofibularis muscle - were also compared among the three Tropidurus species. We found that the two sister-species exhibited remarkable differences in jumping performance, while Tropidurus oreadicus, the more distantly related species, exhibited intermediate values. Tropidurus psamonastes, a species endemic to sand dunes, exhibited high absolute sprint speeds on sand, jumped rarely and possessed a high proportion of glycolytic fibers and low activity of citrate synthase. The sister-species Tropidurus itambere, endemic to rocky outcrops, performed a large number of jumps and achieved lower absolute sprint speed than T. psamonastes. This study provides evidence of rapid divergence of locomotor parameters between sister-species that use different substrates, which is only partially explained by variation in physiological parameters of the iliofibularis muscle.