Antioxidant potential is positively correlated with mitochondrial enzyme activity in Antarctic and non-Antarctic notothenioid fishes

Antarctic notothenioid fishes possess high oxidative capacities, large amounts of intracellular lipid combined with biological membranes enriched in polyunsaturated fatty acids, all of which could make these animals susceptible to oxidative injury, particularly in the form of lipid peroxidation. The central objective in this study was to examine capacities for oxidative metabolism and total antioxidant defense in Antarctic and non-Antarctic notothenioids in order to test the hypothesis that the cold-bodied Antarctic fishes possess elevated activities of citrate synthase (CS), matched by a more robust antioxidant (AOX) defense, than non-Antarctic species. CS activities and total AOX capacities were measured in brain and heart of 4 Antarctic species and 2 non-Antarctic species collected on the 2004 ICEFISH cruise. While no statistical differences are found among Antarctic and non-Antarctic fishes in either CS or AOX capacities, AOX capacity in both tissues expands with CS activity among individuals measured when all species are combined. There is also a 4.5-fold greater AOX capacity, when normalized to CS activity, in brain than in heart indicating the requirement for extra AOX defense in a tissue well known for its particularly high levels of phospholipids more prone to lipid peroxidation.     

Enzymatic capacities for beta-oxidation of fatty fuels are low in the gill of teleost fishes despite presence of fatty acid-binding protein.

A variety of circulating fuels can support the work of the teleost gill. Previous work indicates, however, that unlike other aerobic tissues from teleosts, the gill may have a limited capacity to oxidize fatty fuels. We determined capacities for catabolism of carbohydrate, fatty acids, and amino acids in four species of temperate marine or euryhaline teleosts representing distinct lineages. In addition, we assessed the capacity for fatty acid oxidation in the gill from an Antarctic species. Activities of rate-limiting or regulatory enzymes from pathways of energy metabolism were measured at physiological temperatures (15 degrees or 1 degrees C). In the temperate species, ATP yields from glucose are 3- to 30-fold greater (varying with species) than ATP yields from a monounsaturated fatty acid, while ATP generation from glutamate is 2-50 times greater than similar capacities for the lipid fuel. Like the temperate species, capacity for beta-oxidation of fatty acids is limited in the Antarctic species. A positive linear correlation between activities of citrate synthase (central pathway of oxidative metabolism) and hexokinase (glycolysis) adds further support to the hypothesis that glucose is a preferred metabolic fuel in gill. Our results also demonstrate that fatty acid-binding protein is present in the gill of teleost fishes. It is likely that this protein plays a more important role facilitating anabolic pathways in lipid metabolism rather than fatty acid oxidation in the gill of teleost fishes.     

Effects of temperature acclimation on cardiorespiratory performance of the Antarctic notothenioid Trematomus bernacchii

Notothenioid fishes of the Southern Ocean have evolved under cold and stable temperatures for millions of years. Due to rising temperatures in the Southern Ocean, investigating thermal limits and the capacities for inducing a temperature acclimation response in notothenioids has become of increasing interest. Here, we investigated effects of temperature acclimation on cardiorespiratory responses and cardiac and skeletal muscle energy metabolism in a benthic Antarctic notothenioid, Trematomus bernacchii. We acclimated specimens to ?1, 2 and 4.5 °C for 14 days and quantified heart rates and ventilation rates during an acute increase in temperature. Ventilation rates showed an effect of acclimation both at initial steady-state acclimation conditions and during an acute temperature increase, suggesting a partial thermal compensatory response. However, acclimation did not affect heart rates at steady-state acclimation conditions and the temperatures at which onset of cardiac arrhythmia occurred, suggesting lack of inducible thermal tolerance in cardiac performance. Citrate synthase (CS), lactate dehydrogenase (LDH) and 3-hydroxyacyl dehydrogenase activities in skeletal muscle tissues suggested acclimation-induced shifts in metabolic fuel preferences, and a marked increase in LDH activity with acclimation to 4.5 °C showed an increase in anaerobic metabolism. In heart tissue, CS and LDH activities decreased with acclimation to 4.5 °C, suggesting reduced cardiac ATP production. Overall, the data suggest a partial acclimatory response to temperature by T. bernacchii and support the hypothesis that reduced cardiac acclimatory capacity may play a role in limiting the thermal plasticity of T. bernacchii.     

Aerobic mitochondrial capacities in Antarctic and temperate eelpout (Zoarcidae) subjected to warm versus cold acclimation

Capacities and effects of cold or warm acclimation were investigated in two zoarcid species from the North Sea (Zoarces viviparus) and the Antarctic (Pachycara brachycephalum) by investigating temperature dependent mitochondrial respiration and activities of citrate synthase (CS) and NADP⁺ -dependent isocitrate dehydrogenase (IDH) in the liver. Antarctic eelpout were acclimated to 5°C and 0°C (controls) for at least 10 months, whereas boreal eelpout, Z. viviparus (North Sea) were acclimated to 5°C and to 10°C (controls). Liver sizes were found to be increased in both species in the cold, with a concomitant rise in liver mitochondrial protein content. As a result, total liver state III rates were elevated in both cold-versus and warm-exposed P. brachycephalum and Z. viviparus, with the highest rates in boreal eelpout acclimated to 5°C. CS and IDH activities in the total liver were similar in Z. viviparus acclimated to 5°C and 10°C, but decreased in those warm acclimated versus control P. brachycephalum. Enzyme capacities in the total liver were higher in eelpout from Antarctica than those from the North Sea. In conclusion, cold compensation of aerobic capacities in the liver seems to be linked to an increase in organ size with unchanged specific mitochondrial protein content. Despite its life in permanently cold climate, P. brachycephalum was able to reduce liver aerobic capacities in warm climate and thus, displayed a capacity for temperature acclimation.     

Metabolic shifts in the Antarctic fish <Emphasis Type="Italic">Notothenia rossii</Emphasis> in response to rising temperature and <Emphasis Type="Italic">P</Emphasis> CO<Subscript>2</Subscript>

Introduction

Ongoing ocean warming and acidification increasingly affect marine ecosystems, in particular around the Antarctic Peninsula. Yet little is known about the capability of Antarctic notothenioid fish to cope with rising temperature in acidifying seawater. While the whole animal level is expected to be more sensitive towards hypercapnia and temperature, the basis of thermal tolerance is set at the cellular level, with a putative key role for mitochondria. This study therefore investigates the physiological responses of the Antarctic Notothenia rossii after long-term acclimation to increased temperatures (7°C) and elevated P CO₂ (0.2 kPa CO₂) at different levels of physiological organisation.

Results

For an integrated picture, we analysed the acclimation capacities of N. rossii by measuring routine metabolic rate (RMR), mitochondrial capacities (state III respiration) as well as intra- and extracellular acid–base status during acute thermal challenges and after long-term acclimation to changing temperature and hypercapnia. RMR was partially compensated during warm- acclimation (decreased below the rate observed after acute warming), while elevated P CO₂ had no effect on cold or warm acclimated RMR. Mitochondrial state III respiration was unaffected by temperature acclimation but depressed in cold and warm hypercapnia-acclimated fish. In both cold- and warm-exposed N. rossii, hypercapnia acclimation resulted in a shift of extracellular pH (pH_e) towards more alkaline values. A similar overcompensation was visible in muscle intracellular pH (pH_i). pH_i in liver displayed a slight acidosis after warm normo- or hypercapnia acclimation, nevertheless, long-term exposure to higher P CO₂ was compensated for by intracellular bicarbonate accumulation.

Conclusion

The partial warm compensation in whole animal metabolic rate indicates beginning limitations in tissue oxygen supply after warm-acclimation of N. rossii. Compensatory mechanisms of the reduced mitochondrial capacities under chronic hypercapnia may include a new metabolic equilibrium to meet the elevated energy demand for acid–base regulation. New set points of acid–base regulation under hypercapnia, visible at the systemic and intracellular level, indicate that N. rossii can at least in part acclimate to ocean warming and acidification. It remains open whether the reduced capacities of mitochondrial energy metabolism are adaptive or would impair population fitness over longer timescales under chronically elevated temperature and P CO₂.

     

Identification of a metabolic bottleneck for cold acclimation in Arabidopsis thaliana

Central carbohydrate metabolism of Arabidopsis thaliana is known to play a crucial role during cold acclimation and the acquisition of freezing tolerance. During cold exposure, many carbohydrates accumulate and a new metabolic homeostasis evolves. In the present study, we analyse the diurnal dynamics of carbohydrate homeostasis before and after cold exposure in three natural accessions showing distinct cold acclimation capacity. Diurnal dynamics of soluble carbohydrates were found to be significantly different in cold-sensitive and cold-tolerant accessions. Although experimentally determined maximum turnover rates for sucrose phosphate synthase in cold-acclimated leaves were higher for cold-tolerant accessions, model simulations of diurnal carbohydrate dynamics revealed similar fluxes. This implied a significantly higher capacity for sucrose synthesis in cold-tolerant than cold-sensitive accessions. Based on this implication resulting from mathematical model simulation, a critical temperature for sucrose synthesis was calculated using the Arrhenius equation and experimentally validated in the cold-sensitive accession C24. At the critical temperature suggested by model simulation, an imbalance in photosynthetic carbon fixation ultimately resulting in oxidative stress was observed. It is therefore concluded that metabolic capacities at least in part determine the ability of accessions of Arabidopsis thaliana to cope with changes in environmental conditions.     

Thermal acclimation,mitochondrial capacities and organ metabolic profiles in a reptile (<Emphasis Type="Italic">Alligator mississippiensis</Emphasis>)

Reptiles thermoregulate behaviourally, but change their preferred temperature and the optimal temperature for performance seasonally. We evaluated whether the digestive and locomotor systems of the alligator show parallel metabolic adjustments during thermal acclimation. To this end, we allowed juvenile alligators to grow under thermal conditions typical of winter and summer, providing them with seasonally appropriate basking opportunities. Although mean body temperatures of alligators in these groups differed by approximately 10°C, their growth and final anatomic status was equivalent. While hepatic mitochondria isolated from cold-acclimated alligators had higher oxidative capacities at 30°C than those from warm-acclimated alligators, the capacities did not differ at 20°C. Cold acclimation decreased maximal oxidative capacities of muscle mitochondria. For mitochondria from both organs and acclimation groups, palmitate increased oligomycin-inhibited respiration. GDP addition reduced palmitate-uncoupled rates more in liver mitochondria from warm- than cold-acclimated alligators. In muscle mitochondria, carboxyatractyloside significantly reduced palmitate-uncoupled rates. This effect was not changed by thermal acclimation. The aerobic capacity of liver, skeletal muscle and duodenum, as estimated by activities of cytochrome c oxidase (COX), increased with cold acclimation. At acclimation temperatures, the activities of COX and citrate synthase (CS) in these organs were equivalent. By measuring COX and CS in isolated mitochondria and tissue extracts, we estimated that cold acclimation did not change the mitochondrial content in liver, but increased that of muscle. The thermal compensation of growth rates and of the aerobic capacity of the locomotor and digestive systems suggests that alligators optimised metabolic processes for the seasonally altered, preferred body temperature. The precision of this compensatory response exceeds that typically shown by aquatic ectotherms whose body temperatures are at the mercy of their habitat.     

Locomotor performance and muscle metabolic capacities: impact of temperature and energetic status

In aquatic ectotherms, muscle metabolic capacities are strongly influenced by exogenous factors, principally temperature and food availability. Seasonal changes in temperature lead many organisms to modify their metabolic machinery so as to maintain capacity even in "slower" cold habitats. Modifications of mitochondrial capacities are central in this response. The increases in protein-specific oxidative capacities of mitochondria during cold acclimation of temperate fishes do not occur during the evolutionary adaptation to cold in Antarctic species. Instead, Antarctic fishes tend to increase the proportion of fibre volume devoted to mitochondria, perhaps to facilitate intracellular distribution of oxygen and metabolites. Variation in energetic status can drastically modify muscle metabolic status, with glycolytic muscle changing more than oxidative muscle. This in turn impacts swimming performance. A decrease in the condition of cod leads endurance at speeds above Ucrit to drop by 70%. Sprint swimming is less affected, perhaps as it does not exhaust glycolytic muscle. We used interindividual variation in muscle metabolic capacities to identify correlates of swimming performance in stickleback and cod. Activities of cytochrome c oxidase in glycolytic muscle are a correlate of sprint swimming in stickleback (Gasterosteus aculeatus) and cod (Gadus morhua), whereas lactate dehydrogenase activities in glycolytic muscle are a correlate of cod endurance swimming. In scallops, gonadal maturation leads to virtually complete mobilisation of glycogen from muscle. This does not reduce the capacity of the scallops, Chlamys islandica and Euvola ziczac, to mount escape responses, but significantly slows their recuperation from exhaustive exercise. Muscle metabolic capacities fall in parallel with glycogen mobilisation. In the compromise between muscles' dual roles as a motor and a macromolecular reserve, a significant loss in locomotory ability occurs during gametogenesis and spawning. Reproductive fitness takes the upper hand over maintenance of performance.     

Use of helium-oxygen to examine the effect of cold acclimation on the summit metabolism of a marsupial, Dasyuroides byrnei

To determine whether marsupial mammals increase their metabolic capabilities during cold acclimation, the metabolism of both warm and cold acclimated Dasyuroides byrnei was examined by exposure to cold in a helium-oxygen atmosphere. Mean values of heat production and conductance were significantly higher in a helium-oxygen atmosphere than in air. Body temperature did not change until metabolic capacity was exhausted. Both cold and warm acclimated groups could maintain a metabolic scope of 10-11 times the basal or standard level for this species. Such a metabolic scope is much higher than levels recorded for placental mammals. At very low ambient temperatures cold acclimated D. byrnei could sustain a high level of heat production longer than could warm acclimated animals. While there are some similarities between marsupial mammals and placental mammals in their responses to cold acclimation, an increase in maximum metabolism, as reported for placentals, does not seem to occur in marsupials.     

Mitochondrial Acclimation Capacities to Ocean Warming and Acidification Are Limited in the Antarctic Nototheniid Fish,Notothenia rossii and Lepidonotothen squamifrons

Antarctic notothenioid fish are characterized by their evolutionary adaptation to the cold, thermostable Southern Ocean, which is associated with unique physiological adaptations to withstand the cold and reduce energetic requirements but also entails limited compensation capacities to environmental change. This study compares the capacities of mitochondrial acclimation to ocean warming and acidification between the Antarctic nototheniid Notothenia rossii and the sub-Antarctic Lepidonotothen squamifrons, which share a similar ecology, but different habitat temperatures. After acclimation of L. squamifrons to 9°C and N. rossii to 7°C (normocapnic/hypercapnic, 0.2 kPa CO₂/2000 ppm CO₂) for 4–6 weeks, we compared the capacities of their mitochondrial respiratory complexes I (CI) and II (CII), their P/O ratios (phosphorylation efficiency), proton leak capacities and mitochondrial membrane fatty acid compositions. Our results reveal reduced CII respiration rates in warm-acclimated L. squamifrons and cold hypercapnia-acclimated N. rossii. Generally, L. squamifrons displayed a greater ability to increase CI contribution during acute warming and after warm-acclimation than N. rossii. Membrane unsaturation was not altered by warm or hypercapnia-acclimation in both species, but membrane fatty acids of warm-acclimated L. squamifrons were less saturated than in warm normocapnia−/hypercapnia-acclimated N. rossii. Proton leak capacities were not affected by warm or hypercapnia-acclimation of N. rossii. We conclude that an acclimatory response of mitochondrial capacities may include higher thermal plasticity of CI supported by enhanced utilization of anaplerotic substrates (via oxidative decarboxylation reactions) feeding into the citrate cycle. L. squamifrons possesses higher relative CI plasticities than N. rossii, which may facilitate the usage of energy efficient NADH-related substrates under conditions of elevated energy demand, possibly induced by ocean warming and acidification. The observed adjustments of electron transport system complexes with a higher flux through CI under warming and acidification suggest a metabolic acclimation potential of the sub-Antarctic L. squamifrons, but only limited acclimation capacities for N. rossii.     

Plasticity of muscle function in a thermoregulating ectotherm (Crocodylus porosus): biomechanics and metabolism

Thermoregulation and thermal sensitivity of performance are thought to have coevolved so that performance is optimized within the selected body temperature range. However, locomotor performance in thermoregulating crocodiles (Crocodylus porosus) is plastic and maxima shift to different selected body temperatures in different thermal environments. Here we test the hypothesis that muscle metabolic and biomechanical parameters are optimized at the body temperatures selected in different thermal environments. Hence, we related indices of anaerobic (lactate dehydrogenase) and aerobic (cytochrome c oxidase) metabolic capacities and myofibrillar ATPase activity to the biomechanics of isometric and work loop caudofemoralis muscle function. Maximal isometric stress (force per muscle cross-sectional area) did not change with thermal acclimation, but muscle work loop power output increased with cold acclimation as a result of shorter activation and relaxation times. The thermal sensitivity of myofibrillar ATPase activity decreased with cold acclimation in caudofemoralis muscle. Neither aerobic nor anaerobic metabolic capacities were directly linked to changes in muscle performance during thermal acclimation, although there was a negative relationship between anaerobic capacity and isometric twitch stress in cold-acclimated animals. We conclude that by combining thermoregulation with plasticity in biomechanical function, crocodiles maximize performance in environments with highly variable thermal properties.     

Activity metabolism of lizards after thermal acclimation

1. 1. I tested the hypothesis that thermal acclimation necessarily exerts the same effect on resting metabolism and on aerobic and anaerobic activity metabolism in Anolis carolinensis and Sceloporus jarrovi, diurnally active iguanid lizards that exhibit reduced winter activity but not prolonged dormancy and that are often active on warm winter days.

2. 2. Cold acclimation reduced the total metabolic scope for activity in these lizards by 33–40%. The benefits of winter activity must outweigh the increased risk of predation that presumably accompanies this reduction in capacity for intense, short term muscular exercise.

3. 3. Acclimation did not influence resting metabolism, aerobic activity metabolism, and anaerobic activity metabolism in parallel ways.

Acclimation temperature affects the metabolic response of amphibian skeletal muscle to insulin

Frog skeletal muscle mainly utilizes the substrates glucose and lactate for energy metabolism. The goal of this study was to determine the effect of insulin on the uptake and metabolic fate of lactate and glucose at rest in skeletal muscle of the American bullfrog, Lithobates catesbeiana, under varying temperature regimens. We hypothesize that lactate and glucose metabolic pathways will respond differently to the presence of insulin in cold versus warm acclimated frog tissues, suggesting an interaction between temperature and metabolism under varying environmental conditions. We employed radiolabeled tracer techniques to measure in vitro uptake, oxidation, and incorporation of glucose and lactate into glycogen by isolated muscles from bullfrogs acclimated to 5 °C (cold) or 25 °C (warm). Isolated bundles from Sartorius muscles were incubated at 5 °C, 15 °C, or 25 °C, and in the presence and absence of 0.05 IU/mL bovine insulin. Insulin treatment in the warm acclimated and incubated frogs resulted in an increase in glucose incorporation into glycogen, and an increase in intracellular [glucose] of 0.5 μmol/g (P<0.05). Under the same conditions lactate incorporation into glycogen was reduced (P<0.05) in insulin-treated muscle. When compared to the warm treatment group, cold acclimation and incubation resulted in increased rates of glucose oxidation and glycogen synthesis, and a reduction in free intracellular glucose levels (P<0.05). When muscles from either acclimation group were incubated at an intermediate temperature of 15 °C, insulin's effect on substrate metabolism was attenuated or even reversed. Therefore, a significant interaction between insulin and acclimation condition in controlling skeletal muscle metabolism appears to exist. Our findings further suggest that one of insulin's actions in frog muscle is to increase glucose incorporation into glycogen, and to reduce reliance on lactate as the primary metabolic fuel.     

Dynamic metabolic transformation in tumor invasion and metastasis in mice with LM-8 osteosarcoma cell transplantation

While extensive evidence indicates that tumor cells shift their global metabolic programs, the molecular details of the metabolic transformation in tumor invasion, progression, and metastasis remain largely unknown. Characterization of the time-dependent metabolic shift during the tumor invasion, development, and metastasis will describe an important aspect of tumor phenotypes and potentially allow us to design therapies that inhibit tumor cell movement. In this study, a metabonomic study was performed to characterize the global metabolic changes during the process of tumor invasion and metastasis to lung in a mouse model with subcutaneous transplantation of murine osteosarcoma cell line (LM8). The serum metabolic profiling revealed that many key metabolites in glycolysis and tricarboxylic acid (TCA) cycle, as well as most of the amino acids were elevated at rapidly growing stage of tumor, presumably resulting from a high energy demand and turnover of anabolic metabolism during the tumor cell proliferation. Serum levels of succinic acid and proline significantly increased (with fold change FC = 10.75 and 4.43, relative to controls) among all the metabolites in the third week. The serum metabolic profile of lung metastasis at week 4 was different from that at week 3, in that most of previously increased serum metabolites were found decreased, except for cholesterol and several free fatty acids, suggesting lowered carbohydrate and amino acids metabolism, but an elevated lipid metabolism associated with tumor metastasis.     

柠檬酸合酶的分子生物学研究进展

柠檬酸合酶（citrate synthase,CS）是细胞内多种重要代谢途径的关键酶。CS可催化草酰乙酸和乙酰辅酶A之间的缩合反应生成柠檬酸和辅酶A。通常革兰氏阳性细菌、古菌以及真核细胞的CS为同源二聚体,而革兰氏阴性细菌的CS为同源六聚体。根据其在细胞内的定位不同,CS可分为线粒体CS、乙醛酸循环体CS、过氧化物酶体CS。这些同工酶在能量代谢、植物脂肪的代谢、脂肪酸的氧化及细胞解毒过程中起着重要作用。不同来源的CS空间结构、催化机制和动力学性质十分相似。针对其生化特性、空间结构特点、催化机制以及分子进化等研究进展进行综述。     

Multigenerational exposure to increased temperature reduces metabolic rate but increases boldness in Gambusia affinis

Acute exposure to warming temperatures increases minimum energetic requirements in ectotherms. However, over and within multiple generations, increased temperatures may cause plastic and evolved changes that modify the temperature sensitivity of energy demand and alter individual behaviors. Here, we aimed to test whether populations recently exposed to geothermally elevated temperatures express an altered temperature sensitivity of metabolism and behavior. We expected that long‐term exposure to warming would moderate metabolic rate, reducing the temperature sensitivity of metabolism, with concomitant reductions in boldness and activity. We compared the temperature sensitivity of metabolic rate (acclimation at 20 vs. 30°C) and allometric slopes of routine, standard, and maximum metabolic rates, in addition to boldness and activity behaviors, across eight recently divergent populations of a widespread fish species (Gambusia affinis). Our data reveal that warm‐source populations express a reduced temperature sensitivity of metabolism, with relatively high metabolic rates at cool acclimation temperatures and relatively low metabolic rates at warm acclimation temperatures compared to ambient‐source populations. Allometric scaling of metabolism did not differ with thermal history. Across individuals from all populations combined, higher metabolic rates were associated with higher activity rates at 20°C and bolder behavior at 30°C. However, warm‐source populations displayed relatively bolder behavior at both acclimation temperatures compared to ambient‐source populations, despite their relatively low metabolic rates at warm acclimation temperatures. Overall, our data suggest that in response to warming, multigenerational exposure (e.g., plasticity, adaptation) may not result in trait change directed along a simple “pace‐of‐life syndrome” axis, instead causing relative decreases in metabolism and increases in boldness. Ultimately, our data suggest that multigenerational warming may produce a novel combination of physiological and behavioral traits, with consequences for animal performance in a warming world.