Effects of Long-Term Hypoxia on Enzymes of Carbohydrate Metabolism in the Gulf Killifish, Fundulus grandis

The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l(-1) dissolved oxygen (approximately 3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and the changes were smaller in magnitude, perhaps due to preferential perfusion of heart and brain during hypoxia. The specific activities of some gluconeogenic enzymes increased in liver during long-term hypoxic exposure, which may be coupled to increased protein catabolism in skeletal muscle. These results demonstrate that when intact fish are subjected to prolonged hypoxia, enzyme activities respond in a tissue-specific fashion reflecting the balance of energetic demands, metabolic role and oxygen supply of particular tissues. Furthermore, within glycolysis, the effects of hypoxia varied among enzymes, rather than being uniformly distributed among pathway enzymes

Population Proteomics: Quantitative Variation Within and Among Populations in Cardiac Protein Expression

Population analysis of gene expression is typically achieved by quantifying levels of mRNA; however, gene expression is also a function of protein translation and turnover. Therefore, a complete understanding of population variation in gene expression requires quantitative knowledge of protein expression within and among natural populations. We used two dimensional fluorescence difference gel electrophoresis (2D-DIGE) to quantitatively compare expression of heart ventricle proteins among 18 individuals in three populations of the teleost fish Fundulus. Among populations, expressions between orthologous proteins and mRNAs were generally positively correlated. Additionally, similar to the pattern of cardiac mRNA expression for the same populations, we found considerable variation in protein expression both within and among populations: Of 408 protein features in 2D gels, 34% are significantly different (P \u3c 0.01) among individuals within a population, 9% differ between populations, and 12% have a pattern of expression that suggests they have evolved by natural selection. Although similar to mRNA expression, the frequency of significant differences among populations is larger for proteins. Similar to mRNA expressions, expressions of most proteins are correlated to the expressions of many other proteins. However, the correlations among proteins are more extensive than the correlation for similar RNAs. These correlations suggest a greater coordinate regulation of protein than mRNA expression. The larger frequency of significant differences among populations and the greater frequency of correlated expression among proteins versus among RNAs suggest that the molecular mechanisms affecting protein expression enhance the differences among populations, and these regulatory steps could be a source of variation for adaptation

Biochemical Correlates of Estivation Tolerance in the Mountainsnail Oreohelix (Pulmonata: Oreohelicidae)

Biochemical changes occurring over 7 months of estivation were studied in two species of land snail, Oreohelix strigosa (Gould) and 0. subrudis (Reeve), to determine whether differential mortality during estivation is related to different energetic strategies. Laboratory- maintained snails, which were fed ad libitum prior to es- tivation, were compared with snails collected from the field and induced to estivate without augmenting their energy reserves. In all groups, polysaccharide was catab- olized early in estivation, and protein was the primary metabolic substrate after polysaccharide reserves were de- pleted. Lipid was catabolized at a low rate throughout estivation. Rates of catabolism were largely statistically equivalent between species. Urea and purine bases ac- cumulated during estivation as a result of protein catab- olism, with the former being quantitatively more impor- tant. In both laboratory-maintained and field-collected snails, the rate of urea accumulation was greater in O. subrudis, resulting in higher tissue urea contents in this species at the end of the 7-month experiment. The tissue concentrations of urea at 7 months ranged from about 150 to 300 mM and were positively correlated (r = 0.99, P = 0.006) with mortality in these snails. Methylamine compounds, a class of compounds that can offset disrup- tive effects of elevated urea, were measured in one group of 0. strigosa at 7 months of estivation and found to be low relative to urea levels. We suggest, therefore, that in the absence of elevated levels of counteracting com- pounds, urea may reach toxic levels and may be one factor limiting the duration of estivation that is survived by these land snails

Fundulus as the Premier Teleost Model in Environmental Biology: Opportunities for New Insights Using Genomics

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms

Oxygen limitation and tissue metabolic potential of the African fish Barbus neumayeri: roles of native habitat and acclimatization

Oxygen availability in aquatic habitats is a major environmental factor influencing the ecology, behaviour, and physiology of fishes. This study evaluates the contribution of source population and hypoxic acclimatization of the African fish, Barbus neumayeri, in determining growth and tissue metabolic enzyme activities. Individuals were collected from two sites differing dramatically in concentration of dissolved oxygen (DO), Rwembaita Swamp (annual average DO 1.35 mgO2 L-1) and Inlet Stream West (annual average DO 5.58 mgO2 L-1) in Kibale National Park, Uganda, and reciprocally transplanted using a cage experiment in the field, allowing us to maintain individuals under natural conditions of oxygen, food availability, and flow. Fish were maintained under these conditions for four weeks and sampled for growth rate and the activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome c oxidase (CCO) in four tissues, liver, heart, brain, and skeletal muscle

Exercise-and Hypoxia-Induced Anaerobic Metabolism and Recovery: A Student Laboratory Exercise Using Teleost Fish

Anaerobic metabolism is recruitedin vertebrates under conditions of intense exercise or lowered environmental oxygen availability (hypoxia), typically resulting in the accumulation of lactate in blood and tissues. Lactate will be cleared over time after the reoxygenation of tissues, eventually returning to control levels. Here, we present a laboratory exercise developed as part of an upper-level vertebrate physiology class that demonstrates the effects of exercise and hypoxia exposure on blood lactate in fish and the subsequent decrease in lactate during recovery. Typically, the results obtained by students demonstrate that both treatments cause significant increases in blood lactate concentrations (two to three times higher than control values) that decrease back to normal values within 3 h of recovery under normoxia. The procedures described are generally applicable to other fish species and provide an alternative to using humans or other mammalian species to investigate anaerobic metabolism

Oxygen limitation and tissue metabolic potential of the African fish Barbus neumayeri: roles of native habitat and acclimatization

Oxygen availability in aquatic habitats is a major environmental factor influencing the ecology, behaviour, and physiology of fishes. This study evaluates the contribution of source population and hypoxic acclimatization of the African fish, Barbus neumayeri, in determining growth and tissue metabolic enzyme activities. Individuals were collected from two sites differing dramatically in concentration of dissolved oxygen (DO), Rwembaita Swamp (annual average DO 1.35 mgO2 L-1) and Inlet Stream West (annual average DO 5.58 mgO2 L-1) in Kibale National Park, Uganda, and reciprocally transplanted using a cage experiment in the field, allowing us to maintain individuals under natural conditions of oxygen, food availability, and flow. Fish were maintained under these conditions for four weeks and sampled for growth rate and the activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome c oxidase (CCO) in four tissues, liver, heart, brain, and skeletal muscle

Effects of Long-Term Hypoxia on Enzymes of Carbohydrate Metabolism in the Gulf Killifish, Fundulus grandis

The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l(-1) dissolved oxygen (approximately 3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and the changes were smaller in magnitude, perhaps due to preferential perfusion of heart and brain during hypoxia. The specific activities of some gluconeogenic enzymes increased in liver during long-term hypoxic exposure, which may be coupled to increased protein catabolism in skeletal muscle. These results demonstrate that when intact fish are subjected to prolonged hypoxia, enzyme activities respond in a tissue-specific fashion reflecting the balance of energetic demands, metabolic role and oxygen supply of particular tissues. Furthermore, within glycolysis, the effects of hypoxia varied among enzymes, rather than being uniformly distributed among pathway enzymes