6.1.1. Organization
Intermediary metabolism seems complex to the uninitiated eye and to students with bad memories of memorizing the intermediates of the Krebs cycle. However, this apparent complexity is markedly reduced when meta- bolic pathways are viewed in a functional context in which pathways are clas- sified according to their catabolic or anabolic nature (Fig. 6.1). Metabolic pathways are multienzymatic sequences in which the product of one en- zyme is the substrate of the next, leading to the accumulation of many small chemical changes in the original molecule. In the case of catabolic sequences, these changes serve to transfer part of the chemical energy of the substrate to ATP (or its equivalents) or to NADPH for use in other cell functions. In anabolic sequences, precursor molecules are converted into compounds from which macromolecules, including proteins, complex carbohydrates, nucleic acids, and lipids, are built. Most of the pathways in in- termediary metabolism are linear, although certain important processes are circular.
FIG. 6.1
General outline of metabolic processes, showing the central role of the coupling agents, ATP/ADP, NADH/NAD, and NADPH/NADP. Based on Atkinson (1977).
The central pathways of metabolism are comparatively few in number and their organization is highly conserved. The metabolic machinery in fish is much the same as that in mammals. The important functional differences between intermediary metabolism in fish and the more completely studied mammals lie in the means by which control is exercised, in the sensitivity of metabolic demand to biotic and abiotic factors, and in the exact roles of tissues and organs. Among abiotic factors, temperature is particularly central in its impact on intermediary metabolism in fish, given that the majority of fish are in thermal equilibrium with their environment. Its pervasive impacts on protein structure and function, in both the cytosolic and the membrane fractions of the cell, are the subjects of an excellent, comprehensive review (Somero 1997). Fish, living in water, have the advantage of an excellent medium into which their nitrogenous wastes can be excreted. Thus fish eliminate most of their excess nitrogen as ammonia from the gills, thereby simplifying their intermediary metabolism. Finally, many aspects of muscle metabolism are better understood in fish than in mammals, given that fish muscles are separated according to fiber type.
6.1.2. Mechanisms of Metabolic Control
Flux through metabolic pathways can be controlled in many fashions, all of which modify the rate at which the enzymes in the pathway catalyze their reactions. Enzymes will increase their catalytic activity when substrate and cofactor levels rise, up to concentrations at which the enzyme is saturated with substrate. Allosteric modulators can modify the activity of some enzymes by dramatically shifting the substrate affinity curves. Shifts in the intracellu- lar binding of enzymes may modify their catalytic activity or the efficiency of transfer of substrates between enzymes. Phosphorylation–dephosphory- lation reactions catalyzed by intracellular protein kinases and phosphatases change the activity of certain enzymes. Rapid hormonal control of metabolic pathways is generally exercised through such posttranscriptional modifica- tions.
Classical studies of metabolic control emphasize the role of key regulatory sites, such as in glycolysis in which the “nonequilibrium” reactions catalyzed by glycogen phosphorylase, phosphofructokinase, and pyruvate kinase are thought to be major sites of control. Control of flux through these “key”
control sites is postulated to occur by changes in metabolic signals, such as outlined above. The comparative studies of Newsholme and co-workers were based on the concept that the maximal activity of these enzymes set the maximal capacity for flux through the pathways in which they partici- pate (Newsholme and Crabtree, 1986). Whereas the complex allosteric and posttranslational control mechanisms regulating these enzymes certainly
confer considerable potential for modulating metabolic flux, their activities are only closely related to maximal pathway flux in muscles of organisms which have specialized high metabolic rates such as hummingbirds, tuna, and honeybees (Suarezet al.,1997). Therefore, tissue activities of regulatory enzymes are best used to indicate the metabolic specialization of a tissue or organ (i.e., carbohydrate or lipid catabolism, aerobic or glycolytic), rather than to measure the capacity for flux in a given pathway.
Metabolic flux may also be controlled by loci which catalyze near-equi- librium reactions. Such reactions are typically catalyzed by enzymes with maximal capacities 2–3 orders of magnitude higher than the net flux through the pathway. This “excess” capacity was explained by Haldane (1930) when he demonstrated that net forward flux at such reactions is possible only when their maximal capacity greatly exceeds the pathway flux. Therefore, modification of the capacities of these loci can lead them to assume greater importance in metabolic control than “apparently” warranted by either their maximal capacities or their kinetic properties. A clear indication of the importance of kinetic changes in the properties of equilibrium reactions is provided by the functional impact of the lactate dehydrogenase allozymes in theFundulus heteroclitussystem on the eastern seaboard of the United States (Powers and Schulte, 1998). Application of the Haldane equation to phos- phoglucoisomerase from honeybee flight muscle indicates that although its maximal capacity is 20-fold higher than its maximum pathway flux, under intracellular conditions it supports a maximum flux only 5% above the max- imum pathway flux (Suarez and Staples, 1997). The use of metabolic control theory to establish the relative contributions of different components of a series of reactions to metabolic control has been particularly successful with mitochondrial physiology (Brandet al.,1993), although less has been done with fish systems than could be desired.
Few of the above control mechanisms modify the maximal capacity of the enzymes or pathways. Changes of enzyme concentrations through longer- acting control mechanisms or by modifications of the microenvironment in which the enzymes operate (i.e., membrane lipid composition) are means by which the overall capacity of a metabolic pathway can be changed. Such changes occur during development and growth or in response to shifts in environmental conditions. For example, changes in food availability lead to marked changes in the metabolic capacities of fish muscle (see below).
Oxidative (red) fibers conserve their metabolic capacities during starvation, whereas glycolytic (white) fibers undergo marked decreases in metabolic ca- pacities (Loughna and Goldspink, 1984). Thermal change modifies tissue metabolic capacities in many fish species, with cold acclimation/acclimatiza- tion leading to increases in tissue aerobic capacity. Within fish species, increases in size typically enhance the glycolytic capacity of white muscle
while tissue aerobic capacity decreases with increases in size (Somero and Childress, 1980, 1990). For white and red muscle, the allometric patterns vary with the longitudinal position (Martinez et al., 2000). Therefore the metabolic capacities of tissues and organs in fish are dynamic, changing with the functional requirements and habitat conditions faced by the fish.
In many situations it is desirable to know the energetic status of a cell or tissue and many indicators have been proposed. As the adenylates are involved in the vast majority of energy-producing and energy-dependent reactions, Atkinson (1977) proposed the use of the energy charge, i.e., the proportion of the total adenylate pool which is available in the form of ATP, as a means of assessing energetic status. Examination of the changes in intracellular metabolites during major shifts in ATP use and production indicates that the levels of ATP and free ADP undergo only limited changes (Hochachka and McClelland, 1995). Although these findings underscore one of Atkinson’s central tenants, that of the central importance of the maintenance of relative adenylate levels, only extreme decreases in the en- ergetic status of fish and mammalian tissues are reflected in the energy charge. In tissues, such as fast glycolytic muscle, that use phosphocreatine to fuel initial contractile activity, phosphocreatine levels are linearly related to tissue energetic status (Arthuret al.,1992). More comprehensive param- eters, such as measures of tissue VO2, heat production, mechanical work, or ion pumping, would provide a clearer indication of a tissue’s capacity for energetic expenditures.
In the following sections we examine the metabolism of carbohydrates and proteins. The metabolism of lipids is covered in Chapter 4, by Sargent et al.
6.2