Elizabeth L. Crockett

Some pathways of energy metabolism are cold adapted in Antarctic fishes

Biochemical indices of metabolic cold adaptation were studied in muscular tissues of ecotypically similar Antarctic and Temperate Zone marine fishes. Pairwise comparisons were made between sluggish bottom-dwelling (polar: Notothenia gibberifrons; temperate: Myoxocephalus octodecimspinosus) and more active pelagic (polar: Trematomus newnesi; temperate: Tautoga onitis) species to ensure that results reflect differences in thermal habitats rather than life histories. Maximal activities of enzymes from central pathways of aerobic energy metabolism (citrate synthase, cytochrome oxidase) were 1.5-5-fold higher in oxidative muscles from polar species than from Temperate Zone counterparts when assayed at 1° C. Under similar conditions, enzyme markers for fatty acid oxidation (carnitine palmitoyltransferase, 3-hydroxyacyl-CoA dehydrogenase) were 1.3-27-fold higher in tissues from polar species, while those for both aerobic (hexokinase) and anaerobic (6-phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) metabolism of carbohydrate were generally lower in tissues of polar than of Temperate Zone fishes. These data suggest significant metabolic cold adaptation of aerobic energy metabolism reliant upon fatty fuels inpolar species, but a lack of such adaptation in pathways of carbohydrate metabolism. In the Temperate Zone species, acute thermal sensitivities between 1° and 10° C of most catalysts from central pathways of aerobic energy metabolism were lower (Q10s generally < 2) than those from pathways of anaerobic metabolism (Q10 generally > 2). If these patterns of thermal sensitivities are widespread, they may help explain selective expansion of aerobic metabolism to support activity at low body temperatures.

Temperature acclimation alters oxidative capacities and composition of membrane lipids without influencing activities of enzymatic antioxidants or susceptibility to lipid peroxidation in fish muscle

SUMMARY Cold acclimation of ectotherms results typically in enhanced oxidative capacities and lipid remodeling, changes that should increase the risk of lipid peroxidation (LPO). It is unclear whether activities of antioxidant enzymes may respond in a manner to mitigate the increased potential for LPO. The current study addresses these questions using killifish (Fundulus heteroclitus macrolepidotus) and bluegill (Lepomis macrochirus) acclimated to 5 and 25°C for 9 days and 2 months, respectively. Because the effects of temperature acclimation on pro- and antioxidant metabolism may be confounded by variable activity levels among temperature groups, one species (killifish) was also subjected to a 9-day exercise acclimation. Oxidative capacity of glycolytic (skeletal) muscle (indicated by the activity of cytochrome c oxidase) was elevated by 1.5-fold in killifish, following cold acclimation, but was unchanged in cardiac muscle and also unaffected by exercise acclimation in either tissue. No changes in citrate synthase activity were detected in either tissue following temperature acclimation. Enzymatic antioxidants (catalase and superoxide dismutase) of either muscle type were unaltered by temperature or exercise acclimation. Mitochondria from glycolytic muscle of cold-acclimated killifish were enriched in highly oxidizable polyunsatured fatty acids (PUFA), including diacyl phospholipids (total carbons:total double bonds) 40:8 and 44:12. Increased oxidative capacity, coupled with elevated PUFA content in mitochondria from cold-acclimated animals did not, however, impact LPO susceptibility when measured with C11-BODIPY. The apparent mismatch between oxidative capacity and enzymatic antioxidants following temperature acclimation will be addressed in future studies.

The cold but not hard fats in ectotherms: consequences of lipid restructuring on susceptibility of biological membranes to peroxidation, a review

The production of reactive oxygen species is a regular feature of life in the presence of oxygen. Some reactive oxygen species possess sufficient energy to initiate lipid peroxidation in biological membranes, self-propagating reactions with the potential to damage membranes by altering their physical properties and ultimately their function. Two of the most prominent patterns of lipid restructuring in membranes of ectotherms involve contents of polyunsaturated fatty acids and ratios of the abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine. Since polyunsaturated fatty acids and phosphatidylethanolamine are particularly vulnerable to oxidation, it is likely that higher contents of these lipids at low body temperature elevate the inherent susceptibility of membranes to lipid peroxidation. Although membranes from animals living at low body temperatures may be more prone to oxidation, the generation of reactive oxygen species and lipid peroxidation are sensitive to temperature. These scenarios raise the possibility that membrane susceptibility to lipid peroxidation is conserved at physiological temperatures. Reduced levels of polyunsaturated fatty acids and phosphatidylethanolamine may protect membranes at warm temperatures from deleterious oxidations when rates of reactive oxygen species production and lipid peroxidation are relatively high. At low temperatures, enhanced susceptibility may ensure sufficient lipid peroxidation for cellular processes that require lipid oxidation products.

Exposure to critical thermal maxima increases oxidative stress in hearts of white- but not red-blooded Antarctic notothenioid fishes.

SUMMARY Antarctic icefishes have a significantly lower critical thermal maximum (CTmax) compared with most red-blooded notothenioid fishes. We hypothesized that the lower thermal tolerance of icefishes compared with red-blooded notothenioids may stem from a greater vulnerability to oxidative stress as temperature increases. Oxidative muscles of icefishes have high volume densities of mitochondria, rich in polyunsaturated fatty acids, which can promote the production of reactive oxygen species (ROS). Moreover, icefishes have lower levels of antioxidants compared with red-blooded species. To test our hypothesis, we measured levels of oxidized proteins and lipids, and transcript levels and maximal activities of antioxidants in heart ventricle and oxidative pectoral adductor muscle of icefishes and red-blooded notothenioids held at 0°C and exposed to their CTmax. Levels of oxidized proteins and lipids increased in heart ventricle of some icefishes but not in red-blooded species in response to warming, and not in pectoral adductor muscle of any species. Thus, increases in oxidative damage in heart ventricles may contribute to the reduced thermal tolerance of icefishes. Despite an increase in oxidative damage in hearts of icefishes, neither transcript levels nor activities of antioxidants increased, nor did they increase in any tissue of any species in response to exposure to CTmax. Rather, transcript levels of the enzyme superoxide dismutase (SOD) decreased in hearts of icefishes and the activity of SOD decreased in hearts of the red-blooded species Gobionotothen gibberifrons. These data suggest that notothenioids may have lost the ability to elevate levels of antioxidants in response to heat stress.

Inter-relationship between mitochondrial function and susceptibility to oxidative stress in red- and white-blooded Antarctic notothenioid fishes

SUMMARY It is unknown whether Antarctic fishes can defend themselves against oxidative stress induced by elevations in temperature. We hypothesized that Antarctic icefishes, lacking the oxygen-binding protein hemoglobin, might be more vulnerable to temperature-induced oxidative stress compared with red-blooded notothenioids because of differences in their mitochondrial properties. Mitochondria from icefishes have higher densities of phospholipids per mg of mitochondrial protein compared with red-blooded species, and these phospholipids are rich in polyunsaturated fatty acids (PUFA), which can promote the formation of reactive oxygen species (ROS). Additionally, previous studies have shown that multiple tissues in icefishes have lower levels of antioxidants compared with red-blooded species. We quantified several properties of mitochondria, including proton leak, rates of ROS production, membrane composition and susceptibility to lipid peroxidation (LPO), the activity of superoxide dismutase (SOD) and total antioxidant power (TAOP) in mitochondria isolated from hearts of icefishes and red-blooded notothenioids. Mitochondria from icefishes were more tightly coupled than those of red-blooded fishes at both 2°C and 10°C, which increased the production of ROS when the electron transport chain was disrupted. The activity of SOD and TAOP per mg of mitochondrial protein was equivalent between icefishes and red-blooded species, but TAOP normalized to mitochondrial phospholipid content was significantly lower in icefishes compared with red-blooded fishes. Additionally, membrane susceptibility to peroxidation was only detectable in icefishes at 1°C and not in red-blooded species. Together, our results suggest that the high density of mitochondrial phospholipids in hearts of icefishes may make them particularly vulnerable to oxidative stress as temperatures rise.

Habitat temperature is an important determinant of cholesterol contents in copepods.

SUMMARY Effects of habitat and acclimation temperature on cholesterol contents were examined in oceanic and inshore species of copepods. The cholesterol content of five species of thermally acclimated copepods was determined, and nine species (representing six families) were sampled to assess the role of habitat temperature. The species selected have maximum habitat temperatures (and temperature tolerances) that vary at least twofold. Levels of dietary cholesterol required to achieve maximum growth were also studied at different acclimation temperatures in a eurythermal copepod. Both eggs and copepodites of Calanus finmarchicus had higher cholesterol levels at the warm acclimation temperature (16°C) than at the cooler temperature (6°C). Neither Acartia tonsa, Acartia hudsonica, Temora longicornis nor Eurytemora affinis altered cholesterol contents with acclimation temperature. Maximum growth rates were achieved at fourfold higher concentrations of dietary cholesterol in warm-acclimated Eurytemora affinis than in cold-acclimated animals. The most consistent trend is the positive relationship between cholesterol content and habitat temperature. Species residing in warmer habitats (e.g. Centropages typicus, Eurytemora affinis) had approximately twice the cholesterol of species living in colder waters (e.g. Calanus glacialis, Euchaeta norvegica). A similar pattern was observed for comparisons of species within genera (Calanus, Acartia and Centropages), with the species abundant at lower latitudes having more cholesterol than the northern congener. These data indicate that habitat temperature is an important determinant of cholesterol content, and cholesterol endows membranes with the stability required for a range of body temperatures.

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Abstract Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na+,K+)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na+,K+)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na+,K+)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na+,K+)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change.

A cholesterol-enriched diet enhances egg production and egg viability without altering cholesterol Content of biological membranes in the copepod Acartia hudsonica.

Copepods may lack the capacity for de novo synthesis of cholesterol, while at the same time their dietary levels of sterol vary. We tested the hypothesis that copepods maintain the cholesterol contents of their biological membranes despite varying dietary levels of cholesterol. Acartia hudsonica were acclimated for 5 d to phytoplankton alone or phytoplankton supplemented with cholesterol, at a level sufficient to induce a maximal response on egg production rates. Biological membranes were prepared from the copepods and cholesterol contents assayed. Egg production and hatch rates were measured (the former to confirm that supplemented cholesterol was being assimilated). Analyses of marker enzymes indicate that the majority of membrane‐associated cholesterol in the copepod resides in the plasma membrane. In membranes fractions, cholesterol normalized to protein or activity of Na+/K+‐ATPase is not significantly different for supplemented and unsupplemented groups (29 and 33 μg cholesterol mg−1 protein, respectively; 0.24 and 0.25 mg cholesterol U−1 Na+/K+‐ATPase, respectively). At the same time, acclimating animals to a diet enriched with cholesterol enhances egg production by up to 1.8‐fold and egg viability by 1.5‐fold. We conclude that a cholesterol‐enriched diet stimulates both egg production and hatching rates without altering cholesterol contents of plasma membranes in the copepod A. hudsonica.

The glutathione-dependent system of antioxidant defense is not modulated by temperature acclimation in muscle tissues from striped bass, Morone saxatilis.

Cold temperature generally induces an enhancement of oxidative capacities, a greater content of intracellular lipids, and a remodeling of lipids in biological membranes. These physiological responses may pose a heightened risk of lipid peroxidation (LPO), while warm temperature could result in greater risk of LPO since rates involving reactive oxygen species and LPO will be elevated. The current study examines responses of the glutathione system of antioxidant defense after temperature acclimation. We measured total glutathione (tGSH), and protein levels of GPx1, GPx4, and GST (cardiac and skeletal muscles), and enzymatic activity (skeletal muscle) of glutathione-dependent antioxidants (GPx, GPx4, and GST) in tissues from striped bass (Morone saxatilis) acclimated for six weeks to 7 °C or 25 °C. tGSH of cardiac muscle from cold-acclimated animals was 1.2-times higher than in warm-bodied counterparts, but unchanged with temperature acclimation in skeletal muscle. A second low molecular weight antioxidant, ascorbate was 1.4- and 1.5-times higher in cardiac and skeletal muscle, respectively in warm- than cold-acclimated animals. Despite 1.2-times higher oxidative capacities (as indicated by citrate synthase activity), in skeletal muscle from cold- versus warm-acclimated fish, levels and activities of antioxidant enzymes were similar between acclimation groups. Lipid peroxidation products (as indicated by TBARS), normalized to tissue wet weight, were more than 2-times higher in skeletal muscle from cold- than warm-acclimated animals, however, when normalized to phospholipid content there was no statistical difference between acclimation groups. Our results demonstrate that the physiological changes, associated with acclimation to low temperature in the eurythermal striped bass, are not accompanied by an enhanced antioxidant defense in the glutathione-dependent system.

Relationship between oxidizable fatty acid content and level of antioxidant glutathione peroxidases in marine fish

SUMMARY Biological membranes can be protected from lipid peroxidation by antioxidant enzymes including catalase (CAT) and selenium-dependent glutathione peroxidases 1 and 4 (GPx1 and GPx4). Unlike GPx1, GPx4 can directly detoxify lipid hydroperoxides in membranes without prior action of phospholipase A2. We hypothesized that (1) GPx4 is enhanced in species that contain elevated levels of highly oxidizable polyunsaturated fatty acids (PUFA) and (2) activities of antioxidant enzymes are prioritized to meet species-specific oxidative stresses. In this study we examined (i) activities of the oxidative enzyme citrate synthase (CS) and antioxidant (CAT, GPx1 and GPx4) enzymes, (ii) GPx4 protein expression, and (iii) phospholipid composition in livers of five species of marine fish (Myxine glutinosa, Petromyzon marinus, Squalus acanthias, Fundulus heteroclitus and Myoxocephalus octodecemspinosus) that contain a range of PUFA. GPx4 activity was, on average, 5.8 times higher in F. heteroclitus and S. acanthias than in the other three marine fish species sampled. Similarly, activities of CAT and GPx1 were highest in S. acanthias and F. heteroclitus, respectively. GPx4 activity for all species correlates with membrane unsaturation, as well as oxidative activity as indicated by CS. These data support our hypothesis that GPx4 level in marine fish is a function, at least in part, of high PUFA content in these animals. GPx1 activity was also correlated with membrane unsaturation, indicating that marine species partition resources among glutathione-dependent defenses for protection from the initial oxidative insult (e.g. H2O2) and to repair damaged lipids within biological membranes.