Characterization of the hypoxia-inducible factor-1 pathway in hearts of Antarctic notothenioid fishes.

The ability of Antarctic notothenioid fishes to mount a robust molecular response to hypoxia is largely unknown. The transcription factor, hypoxia-inducible factor-1 (HIF-1), a heterodimer of HIF-1α and HIF-1ß subunits, is the master regulator of oxygen homeostasis in most metazoans. We sought to determine if, in the hearts of Antarctic notothenioids, HIF-1 is activated and functional in response to either an acute heat stress or hypoxia. The red-blooded Notothenia coriiceps and the hemoglobinless icefish, Chaenocephalus aceratus, were exposed to their critical thermal maximum (CTMAX) or hypoxia (5.0 ± 0.3 mg of O2 L-1) for 2 h. Additionally, N. coriiceps was exposed to 2.3 ± 0.3 mg of O2 L-1 for 12 h, and red-blooded Gobionotothen gibberifrons was exposed to both levels of hypoxia. Levels of HIF-1α were quantified in nuclei isolated from heart ventricles using western blotting. Transcript levels of genes involved in anaerobic metabolism, and known to be regulated by HIF-1, were quantified by real-time PCR, and lactate levels were measured in heart ventricles. Protein levels of HIF-1α increase in nuclei of hearts of N. coriiceps and C. aceratus in response to exposure to CTMAX and in hearts of N. coriiceps exposed to severe hypoxia, yet mRNA levels of anaerobic metabolic genes do not increase in any species, nor do lactate levels increase, suggesting that HIF-1 does not stimulate metabolic remodeling in hearts of notothenioids under these conditions. Together, these data suggest that Antarctic notothenioids may be vulnerable to hypoxic events, which are likely to increase with climate warming.

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

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 degrees 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 polyunsaturated 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.

Effects of acclimation temperature on enzymatic capacities and mitochondrial membranes from the body wall of the earthworm Lumbricus terrestris.

Many ectotherms respond to low temperature by adjusting capacities of enzymes from energy metabolism, restructuring membrane phospholipids and modulating membrane fluidity. Although much is known about the temperature biology of earthworms, it is not known to what extent earthworms employ compensatory changes in enzymatic capacities and membrane physical properties after exposure to low temperature. We examined activities of enzymes from glycolysis and central oxidative pathways as well as fluidity and phospholipid fatty acid composition of mitochondrial membranes prepared from the body wall of the temperate oligochaete Lumbricus terrestris after a one month acclimation to 5 degrees and 15 degrees C. No compensation occurs in central pathways of oxidative metabolism since activities of cytochrome-c oxidase and citrate synthase, when measured at a common temperature, are similar for 5 degrees C and 15 degrees C-acclimated animals. In contrast, activity of pyruvate kinase is elevated 1.3-fold after acclimation to 5 degrees C. Mitochondrial membranes display inverse compensation with respect to temperature (membranes from 5 degrees C animals are more ordered than membranes from 15 degrees C animals). Our results, in combination with earlier reports, indicate that routine metabolism in L. terrestris may be maintained at reduced temperatures with little or no change in enzymatic capacities and inverse compensation of mitochondrial membranes.

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.

Sensitive assay for cholesterol in biological membranes reveals membrane-specific differences in kinetics of cholesterol oxidase.

Quantification of cholesterol in biological membranes from a variety of sources is an important step toward understanding cholesterol's roles in membrane function. We extend to biological membranes the fluorometric/enzymatic approach (cholesterol oxidase) to measure cholesterol, originally described for whole cells (Heider and Boyett [1978] J. Lipid Res., 19:514-518; Gamble et al. [1978] J. Lipid Res., 19:1068-1070) and serum (Huang et al. [1975] Clin Chem., 21:1605-1608). This method has a detection limit of 0.3 microgram cholesterol. As revealed by comparison with high-performance liquid chromatography, the fluorometric/enzymatic method with biological membranes is accurate (within 4% and 8% for intestinal and hepatic plasma membranes, respectively). The assay may be completed within 3 to 4 hours and requires neither lipid extraction nor chromatographic techniques. Kinetics of the cholesterol oxidase reaction are membrane-specific, and first-order rate constants (k) are positively correlated with membrane order.

Peroxisomal beta-oxidation is a significant pathway for catabolism of fatty acids in a marine teleost.

Hepatic beta-oxidation is characterized in a marine teleost, Myoxocephalus octodecimspinosus, to determine mitochondrial and peroxisomal substrate selectivity as well as metabolic partitioning. Substrate selectivity is broad for peroxisomal beta-oxidation. Acyl CoA oxidase activities, with all unsaturated substrates measured, are at least 35% of activity with palmitoyl CoA (16:0), a saturated substrate. Mitochondrial selectivities are more pronounced. Carnitine palmitoyltransferase activity with a monounsaturate, palmitoleoyl CoA (16:1), is nearly 40% greater than activity with palmitoyl CoA, whereas activities with two polyunsaturates are < 10% of activity with the saturate. The presence of polyunsaturated acyl CoA esters inhibits up to 70% the oxidation of palmitoyl CoA by intact peroxisomes. Acyl CoA hydrolase activity is localized to peroxisomal fractions prepared by density-gradient centrifugation. Hydrolytic activity in these fractions is nearly twofold the activity of beta-oxidation. Estimates for metabolic partitioning suggest that at least 50% of hepatic beta-oxidation may be initiated by the peroxisomal compartment.

Substrate selectivities differ for hepatic mitochondrial and peroxisomal beta-oxidation in an Antarctic fish, Notothenia gibberifrons.

Hepatic mitochondrial and peroxisomal beta-oxidation were examined in an Antarctic marine teleost, Notothenia gibberifrons. Enzymic profiles and rates of beta-oxidation by intact organelles were determined by using a range of fatty acyl-CoA substrates to evaluate substrate preferences. Partitioning of beta-oxidation between organelles was estimated. Substrate selectivities are broader for peroxisomal beta-oxidation than for mitochondrial beta-oxidation. Mitochondria show marked preference for the oxidation of a monounsaturated substrate, palmitoleoyl-CoA (C16:1), and two polyunsaturates, eicosapentaenoyl-CoA (C20:5) and docosahexaenoyl-CoA (C22:6). Carnitine palmitoyltransferase activities with palmitoleoyl-CoA (C16:1) are 2.4-fold higher than activities with palmitoyl-CoA (C16:0). Most polyunsaturated acyl-CoA esters measured appear to inhibit by over 40% the oxidation of palmitoyl-CoA by peroxisomes. Our findings suggest that the polyunsaturates, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), found in high concentrations in Antarctic fishes [Lund and Sidell (1992) Mar. Biol. 112, 377-382], are utilized as fuels to support aerobic energy metabolism. Metabolic capacities of rate-limiting enzymes and beta-oxidation rates by intact organelles indicate that up to 30% of hepatic beta-oxidation in N. gibberifrons can be initiated by the peroxisomal pathway.