Metabolic recovery in goldfish: A comparison of recovery from severe hypoxia exposure and exhaustive exercise

Abstract

Severe hypoxia exposure and exhaustive exercise in goldfish both elicit a strong activation of substrate-level phosphorylation with the majority of the metabolic perturbations occurring in the white muscle. Approximately half of the muscle glycogen breakdown observed during severe hypoxia exposure was accounted for by ethanol production and loss to the environment, which limited the extent of muscle glycogen recovery when animals were returned to normoxic conditions. Ethanol production in goldfish is not solely a response to anoxia/hypoxia exposure however, as a transient increase in ethanol production was observed during the early stages of recovery from exhaustive exercise. These data suggest that ethanol production is a ubiquitous “anaerobic” end product, which accumulates whenever metabolic demands exceed mitochondrial oxidative potential. Exhaustive exercise and hypoxia exposure both caused a 7 to 8 μmol g^− 1 wet mass increase in muscle [lactate] and the rates of recovery following these perturbations were similar. The rates of muscle PCr and pHi recovery after hypoxia exposure and exhaustive exercise were similar with levels returning to controls values within 0.5 h. Surprisingly, liver [glycogen] was not depleted during exposure to severe hypoxia, however, during recovery from both hypoxia and exercise dramatically different responses in liver [glycogen] were noted. During the early stages of recovery, liver [glycogen] transiently increased to high levels after exhaustive exercise, while during recovery from hypoxia there was a transient decrease in liver glycogen over the same time frame. Overall, this points to the liver playing a dramatically different role in facilitating recovery from exercise compared with hypoxia exposure.

Introduction

Energy production during exposure to severe hypoxia and exhaustive exercise occurs primarily via substrate-level phosphorylation (phosphocreatine (PCr) hydrolysis and glycolysis), which compensates for ATP demands that exceed oxidative capacity. During severe hypoxia exposure, goldfish suppress metabolic rate by up to 60% (Van Waversveld et al., 1989) and rely on large liver and muscle glycogen reserves to support substrate-level phosphorylation to maintain cellular energy balance (Lutz and Nilsson, 1997). In contrast, ATP demands in muscle during exhaustive exercise increase beyond the oxidative capacity and thus substrate-level phosphorylation must be activated to meet the energetic demands of muscle contraction (Richards et al., 2002a). Although the metabolic reasons for the activation of substrate-level phosphorylation differ during exposure to severe hypoxia and exhaustive exercise, the end metabolic profiles observed in muscle are similar with low [glycogen] and [PCr] and high [lactate] and metabolic [H⁺] (van den Thillart et al., 1989, Richards et al., 2002a, Richards et al., 2003, Richards et al., 2007).

During recovery from hypoxia and exhaustive exercise, pathways must be activated to resynthesize ATP, PCr, and glycogen. To this end, the early portions of recovery are characterized by a period of excess O₂ consumption (Scarabello et al., 1991, van den Thillart and Verbeek, 1991, van Ginneken et al., 1995), which in part represents a stimulation of oxidative phosphorylation to support the energetic costs of recovery. Generally, muscle [PCr] returns to resting values quickly during recovery from both hypoxia exposure and exhaustive exercise (within 1 to 2 h if not sooner), but recovery of muscle [lactate] and [glycogen] requires a longer period (> 2 h; Richards et al., 2002b). Lactate is preferentially retained in muscle during recovery from exercise or hypoxia exposure (Richards et al., 2007) and there is accumulating evidence to suggest that muscle glycogenesis occurs in situ and relies on lactate as the substrate (Schulte et al., 1992).

Members of the family Cyprinidae, especially the crucian carp (Carassius carassius L.) and the common goldfish (Carassius auratus L.), have long been recognized as unique among vertebrates for their incredible anoxia tolerance. The common goldfish, for example, can survive complete anoxia at room temperature (20 °C) for many hours (van den Thillart et al., 1983) and as environmental temperature decreases, survival can be extended to weeks or possibly even months (Blazka, 1958, Walker and Johanson, 1977). Prolonged anoxia survival at cold temperatures is likely related to three main factors: 1. the storage of high concentrations of fermentable substrates (e.g. liver [glycogen] of ∼ 800 μmol g^− 1 wet mass; van den Thillart et al., 1980a), 2. the suppression of whole-animal metabolic rate (Van Waversveld et al., 1989), and 3. the ability to convert anaerobically derived lactate to ethanol and CO₂ (Shoubridge and Hochachka, 1980).

Ethanol production by the crucian carp and goldfish begins with the movement of lactate from other tissues to red and white skeletal muscle, where lactate is converted to pyruvate via lactate dehydrogenase. Pyruvate is then decarboxylated to acetaldehyde and CO₂ via an inefficient or modified pyruvate dehydrogenase complex (van Waarde et al., 1993, Lutz and Nilsson, 1997). Typically, PDH converts pyruvate to acetyl-CoA with acetaldehyde as a bound intermediate, but during anerobiosis acetaldehyde “leaks” out of the PDH complex, diffuses into the cytoplasm and is converted to ethanol by alcohol dehydrogenase. Ethanol then freely diffuses across the gills, limiting lactate accumulation. Loss of ethanol to the environment, however, can represent a substantial loss of carbon, especially over months of anoxia exposure, and this may seriously impact the ability of the animal to recover in a normoxic environment.

Few studies have examined the effects of exhaustive exercise on Cyprinids, and those that have mostly focused on the non-ethanol producing common carp (Cyprinus carpio; Driedzic and Hochachka, 1976, Sugita et al., 2001, van Ginneken et al., 2004) and no studies, to our knowledge, have examined whether ethanol production occurs during “anaerobic” exhaustive exercise. For the most part, the metabolic profiles observed in carp white muscle during exhaustive exercise are similar to those observed in other fish (e.g. trout), albeit the magnitude of the effects are smaller. Huber and Guderley (1993) noted significant accumulation of lactate during ∼ 30 min of exhaustive exercise in goldfish, but did not measure whether lactate was converted to ethanol. It is likely, however, that even if lactate conversion to ethanol occurs, due to the rapid rate of glycolysis necessary to support muscle contraction, lactate would be preferentially accumulated over ethanol.

The objectives of this study were to compare the recovery profiles of white muscle [ATP], [PCr], [pHi], and glycogen in goldfish after exposure to 10 h of severe hypoxia at 10 °C or a bout of exhaustive exercise. We predicted that severe hypoxia exposure would lead to ethanol production and the loss of carbon to the environment and as a result, muscle glycogen recovery after hypoxia exposure would be impaired relative to exhaustive exercise where no or limited ethanol production was predicted to occur. Specifically, we examined the rate of ethanol production in goldfish during and after severe hypoxia exposure and exhaustive exercise to determine the relative amounts of ethanol produced and its relation to glycogen recovery. Changes in liver glycogen were also examined to gain insight into the potential role of the liver in fuelling metabolic recovery from hypoxia exposure and exhaustive exercise.