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1 Introduction: Metabolic Circuitry and Control Mechanisms

1 Introduction: Metabolic Circuitry and Control Mechanisms

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6. Intermediary Metabolism


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 intracellular binding of enzymes may modify their catalytic activity or the efficiency

of transfer of substrates between enzymes. Phosphorylation–dephosphorylation 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 modifications.

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 participate (Newsholme and Crabtree, 1986). Whereas the complex allosteric and

posttranslational control mechanisms regulating these enzymes certainly


Dabrowski and Guderley

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 (Suarez et 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-equilibrium reactions. Such reactions are typically catalyzed by enzymes with

maximal capacities 2–3 orders of magnitude higher than the netflux 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

the Fundulus heteroclitus system on the eastern seaboard of the United States

(Powers and Schulte, 1998). Application of the Haldane equation to phosphoglucoisomerase 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 maximum 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 (Brand et 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 longeracting 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 capacities (Loughna and Goldspink, 1984). Thermal change modifies tissue

metabolic capacities in many fish species, with cold acclimation/acclimatization leading to increases in tissue aerobic capacity. Within fish species,

increases in size typically enhance the glycolytic capacity of white muscle

6. Intermediary Metabolism


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 energetic 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 (Arthur et al., 1992). More comprehensive parameters, 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.


Carbohydrate Metabolism

6.2.1. Digestibility

Carbohydrates are excellent sources of energy and carbon, one of the major elements of which living organisms are composed. The breakdown of carbohydrates is the primary means by which animal tissues obtain their chemical energy. However, dietary carbohydrates are not the principal source of

energy or carbon for most fish. Polysaccharides tend to be repeating polymers of simple sugars, making the conformation of links between monomers

fairly easy to predict. Proteins are composed of ∼20 types of amino acids of

markedly differing structures and specific proteases are needed to recognize

the bonds between specific amino acids. Therefore the digestive breakdown

and absorption of carbohydrates are simpler than those of proteins. Despite


Dabrowski and Guderley

the apparent simplicity of carbohydrate degradation, animals typically can

only digest glycogen and starches with endogenous enzymes and lack the

enzymes required to degrade cellulose, chitin, and lignin. The strategy for

overcoming such deficiencies is illustrated by the ruminants that have been

famously successful in exploiting diets composed virtually only of plants

rich in cellulose but poor in other nutrients. The success of ruminants

is due to a symbiosis with microorganisms which digest and ferment the

plant material, and not to the ruminant’s capacity to produce cellulase. As

a major obstacle in breaking down plant material is mechanical, herbivory

requires structural adaptations for grinding plant material. Fish are no exceptions to the general rule that animals are best at breaking down starches

and glycogen and that digestion of cellulose and chitin generally requires

microbial assistance and specific mechanical adaptations to break down

plant structures.

Because most fish are primarily carnivorous or omnivorous, carbohydrates are not the major components of their diets. Nonetheless, some

fish species, in both freshwater and marine environments, have specialized

for herbivory and many species, including species of interest for aquaculture, are able to ingest and digest significant quantities of plant material.

Amylase, disaccharidases, cellulase, and chitinase have been extracted from

fish stomachs ( Jobling, 1995), but considerable species differences occur in

the capacities of fish to digest and absorb polysaccharides (Stickney, 1994).

Thus, channel catfish handle starch well and their diets can contain up to

40% starch (Wilson and Poe, 1987), whereas trout have only a limited ability

to digest starch (Hilton et al., 1983). The intestines of herbivorous fish

are longer relative to organismal size than are those of omnivorous or

carnivorous fish. On the intraspecific level, carp, Cyprinus carpio, roach,

Rutilus rutilus, and grass carp, Ctenopharyngodon idella, fed large quantities of

indigestible fiber have longer intestines than their conspecifics fed animal

prey ( Jobling, 1995).

The fish that opt to feed at a lower trophic level have several strategies to

facilitate the digestion of plant materials that have their parallels in other

vertebrates. The creation of a highly acidic environment in a thin-walled

stomach allows the lysis of plant cell walls. Use of this strategy is facilitated

by selective ingestion of plant food (i.e., separated from inorganic material).

A thick-walled muscular stomach or a pharyngeal mill can be used to grind

and rupture the plant cell walls. When species, such as parrotfish and mullets

ingest sediment particles with algal food, grinding the inert particles with the

food particles facilitates the mechanical breakdown of algal cells. Typically

these fish need to feed copiously to obtain sufficient food from the mixture

of sediment and algae. Alternately, microbial fermentation in a cecum of

the hindgut can be employed to digest plant material. Such strategies are

6. Intermediary Metabolism


likely central in the particular feeding habits such as those of wood-eating

fish. In the wood-eating catfishes of the genus Panaque, a consortium of

microorganisms seems to be required for cellulose breakdown (Nelson et al.,

1999). Whereas the digestibility of carbohydrates for herbivorous fish poses

no problem, more carnivorous fish, in particular, salmonids, are less able to

benefit from the nutritional value of carbohydrates.

The carbohydrates typically found in fish diets include chitin (from crustacean and insect exoskeletons), cellulose (plant material), glycogen (animal

tissues), and starch (plant materials). The ability to digest these products

parallels their presence in the diet, although in many cases it is not clear

whether the digestive enzymes arise in the fish tissues or in microorganisms in the fish intestine. Chitinases and cellulases most likely originate in

the intestinal flora (Stickney and Shumway, 1974; Fagbenro, 1990; Nelson

et al., 1999), whereas amylases are pancreatic in origin. The breakdown of

carbohydrates to monosaccharides is completed by enzymes located in the

brush border. Enzymes required for the breakdown of the specific linkages

present in algal storage carbohydrates have been isolated from herbivorous

marine fish. The capacity for carbohydrate digestion shows a certain plasticity, particularly in omnivorous fish. Thus, both cyprinids and tilapia modify their secretion of digestive enzymes when their diet is varied ( Jobling,

1995). The capacity for absorption of the sugar monomers also parallels the

relative importance of carbohydrates in the diet and changes with ontogenetic switches in dietary preferences.

6.2.2. Carbohydrate Storage and Breakdown Responses to Starvation and Refeeding

Glycogen is the major carbohydrate storage form in fish and major deposits typically occur in the liver and muscle. Both oxidative (red) and glycolytic (white) muscle contain significant concentrations of glycogen, but

given that white muscle makes up the bulk of the musculature, it stores most

of the body’s glycogen. The reliance on tissue glycogen stores during periods

of food limitation differs from the patterns observed in mammals, in which

starvation quickly leads to breakdown of liver glycogen so that blood glucose

levels remain constant. In cod (Gadus morhua), carp (Cyprinus carpio), and

roach (Rutilus rutilus), if hepatic lipids are present in significant amounts,

they are the first reserves used during starvation (Black and Love, 1986; Lim

and Ip, 1989; Blasco et al., 1992; M´endez and Wieser, 1993; Băohm et al., 1994).

Muscle lipids are next to be used, followed by liver and muscle glycogen.

In these species, muscle protein is the last “reserve” to be mobilized during starvation. In contrast, during the long spawning migrations of Pacific


Dabrowski and Guderley

salmon, muscle protein is degraded, while hepatic glycogen is conserved

as a fuel for spawning itself (Mommsen et al., 1980). During the salmon’s

migration, muscle protein seems to serve both as a fuel and as a source

of carbon skeletons required for the maintenance of hepatic glycogen levels. Similarly, the mudskipper (Boleophthalmus boddaerti) favors muscle over

liver glycogen during starvation (Lim and Ip, 1989). These changes in tissue

glycogen levels with starvation and refeeding are particularly pronounced

when tissue size is taken into account.

Feeding after a period of starvation leads to rapid recovery and particularly high growth rates (compensatory growth). During the beginning

of feeding after starvation, liver and muscle glycogen levels in cod, roach

carp, and mudskipper are quickly replenished (Black and Love, 1986; Lim

and Ip, 1989; Blasco et al., 1992; Mendez and Wieser, 1993; Băohm et al.,

1994). Recovery of tissue protein stores occurs more gradually, and once

this process is well advanced, lipid reserves begin to be deposited (Black

and Love, 1986; Băohm et al., 1994). Hepatic Glycogen Metabolism and Its Hormonal Control Glycolysis. Hepatic glycogen is broken down both to provide

glucose for export to the blood and to channel glucose into glycolysis,

oxidative phosphorylation, or other ATP-yielding metabolic conversions

(Fig. 6.2). Most glycogen mobilization is accomplished by glycogen phosphorylase. Glycolysis involves the gradual oxidation of glucose derived from

glycogen or from the blood. It proceeds via two initial phosphorylation steps

(catalyzed by hexokinase and phosphofructokinase), which serve primarily

to increase the equilibrium constant of the glycolytic pathway and to commit the carbon skeletons to their breakdown via this pathway. Subsequently,

the six-carbon sugar is broken into two triose molecules, which are then

oxidized before two-substrate level phosphorylation reactions (at phosphoglycerate kinase and pyruvate kinase) provide the limited ATP yield which

can be obtained in the absence of oxygen. During the oxidation of the sugar,

an NAD is reduced to an NADH. As NAD concentrations are no higher than

0.5 mM, the NAD supply must be renewed for glycolysis to continue. In fish

and mammalian muscle, this is typically the role of lactate dehydrogenase

that converts pyruvate into lactate while oxidizing the NADH to produce

the required NAD. As liver typically functions in aerobic mode, it does not

need to form lactate to maintain redox balance. Rather, the NADH produced during glycolysis is oxidized in mitochondria, through the action of

the electron transport system that allows the generation of a proton gradient

which can then be used for oxidative phosphorylation.

6. Intermediary Metabolism


FIG. 6.2

Glycolysis and gluconeogenesis: two opposite pathways sharing all but two enzymes.

Bypass reactions, with greater effective ATP investments, allow gluconeogenesis to be

thermodynamically favorable without radically altering intracellular metabolite

concentrations. Pentose Phosphate Pathway. This metabolic sequence is initiated

at the level of glucose 6-phosphate and has two major physiological roles:

the primary role is the production of NADPH for biosynthetic purposes

(Fig. 6.1); a secondary role is the production of the ribose sugar moiety

required for nucleotide synthesis. Beyond its role in reductive biosynthesis,

NADPH also protects cells against damage from oxygen radicals (Pelster

and Scheid, 1991, 1992). The swim bladder of fish can contain high levels of

oxygen, leading to considerable potential for free radical damage to its cells.

Flux through the pentose phosphate pathway in the swim bladder of the

toadfish virtually doubled under hyperoxic conditions, strongly suggesting


Dabrowski and Guderley

FIG. 6.3

The organization of the Krebs cycle, showing sites of NADH, FADH, and GTP


that the high levels of the pentose phosphate shunt enzymes in this tissue

are related to the protection of the tissue from free radical damage (Walsh

and Milligan, 1993). Krebs Cycle and Oxidative Phosphorylation. The pyruvate produced by glycolysis is fully oxidized to CO2 and H2 O in the mitochondria

through the combined action of Krebs cycle and the electron transport

system (ETS) (Figs. 6.3 and 6.4). Again, the basic principles of the functioning of mitochondrial substrate oxidation and oxidative phosphorylation are

similar to those in mammals, although the specific conditions under which

fish function have led mitochondrial design to diverge somewhat from the

mammalian model. Pyruvate is first converted into acetyl-CoA, through

the action of pyruvate dehydrogenase. The acetyl-CoA is condensed with

oxaloacetate through the action of citrate synthase and, thereby, enters into

the Krebs cycle; the functioning of the cycle is fairly straightforward. Essentially the two carbons of the acetyl-CoA are gradually split off (as CO2 ),

while the six-carbon compound formed at the start of the cycle is gradually

6. Intermediary Metabolism


FIG. 6.4

Mitochondrial electron transport, showing sites of proton flow across the inner

mitochondrial membrane as well as the cytochromes associated with the different

inner membrane complexes.

oxidized, allowing the formation of three NADHs, one FADH, and one GTP

per acetyl-CoA which enters the cycle. The formation of acetyl-CoA from

pyruvate forms an additional NADH. The NADH is converted to NAD by

NADH dehydrogenase, the first step in the electron transport chain. As the

electrons are passed along the different cytochromes and electron carriers

in the ETS (complexes 1–4 in Fig. 6.4), protons cross the inner mitochondrial membrane, creating a proton and an electrical gradient between the

inner mitochondrial matrix and the intermembrane and cytosolic compartments. This electrochemical gradient, i.e., protonmotive force, provides the

energy for the phosphorylation of ADP into ATP by the F1 -ATPase situated

in the inner mitochondrial membrane. Effectively, protons are thought to

pass through this membrane-spanning enzyme, providing it with the conformational energy required to transform ADP + Pi into ATP.

There is not an obligate stoichiometry between the number of electrons

which are passed among the cytochromes (or protons which are translocated across the membrane) and the number of ATP molecules produced.

Textbooks typically suggest that three ATP molecules are synthesized for

each NADH molecule oxidized by the NADH dehydrogenase. However,


Dabrowski and Guderley

not all proton flow from the outside to the inside of the inner mitochondrial membrane is linked to oxidative phosphorylation. Proton leak across

this membrane is considerable and reduces the coupling between oxygen

consumption and ATP synthesis. Thus, electron transport can occur with no

concomitant ATP synthesis. Current estimates for the stoichiometry between

mitochondrial electron transport and oxidative phosphorylation range from

1.4 to 2.5 ATP per oxygen consumed and are all considerably lower than

the theoretical value of 3 (Brand et al., 1993). The genes for UCP-2 (uncoupling protein 2) have been sequenced in carp and zebrafish, indicating

that the proteins implicated in this proton leak are as present in fish as

in other vertebrates (Stuart et al., 1999). Thus, while the efficiency of carbohydrate oxidation is higher than that of anaerobic glycolysis, it is lower

than the 36 molecules of ATP per molecule of glucose that is traditionally

presented. Glycogen Synthesis and Gluconeogenesis. Hepatic glycogen synthesis is based both on the incorporation of bloodborne glucose into glycogen

and on gluconeogenesis from lactate and amino acids. Glucose incorporation into glycogen occurs via production of UDP glucose from glucose

1-phosphate via the glycogen synthase reaction. Gluconeogenesis from lactate or amino acids requires the reversal of many glycolytic reactions (Fig. 6.2)

and follows enzymatic bypasses for the pyruvate kinase (PK) and phosphofructokinase (PFK) reactions. The bypass for PK requires two enzymes. The

first reaction is catalyzed by pyruvate carboxylase, which converts pyruvate

into oxaloacetate (the functionally equivalent reaction can be catalyzed by

malic enzyme, which converts pyruvate into malate, which can then be converted to oxaloacetate via the malate dehydrogenase reaction). Next the oxaloacetate is converted into phosphoenol pyruvate by phosphoenol pyruvate

carboxykinase. The complete PK bypass requires two ATP equivalents. The

bypass enzymes may be located either in the cytosol or in the mitochondria.

The precise location influences the regulation of the reactions. The second bypass reaction requires fructose bisphosphatase (FBPase), which converts fructose 1,6-bisphosphate (F1,6BP) into fructose 6-phosphate (F6P)

(Fig. 6.2). An additional enzyme produces fructose 2,6-bisphosphate from

fructose 6-phosphate (Fig. 6.5). The sole apparent role of this compound is

to stimulate the activity of PFK and inhibit that of FBPase. Hormonal Control Mechanisms. As in mammals, the enzymes

involved in glycogen metabolism are sensitive to hormonal controls, via

phosphorylation and dephosphorylation reactions, as well as responding

to intracellular metabolite signals. In general, the hormones that regulate

glycogen mobilization and storage are similar to those that are active in

6. Intermediary Metabolism


FIG. 6.5

The intermediates involved in the phosphofructokinase (PFK) and

fructose-1,6-bisphosphatase (FB Pase) reactions.

mammals, although the precise regulatory patterns found in mammals are

not necessarily present in fish (Fig. 6.6). This may be partly because the

well-studied fish (trout) tend to be more carnivorous than the well-studied

mammals (rat). Accordingly, gluconeogenesis occurs at considerable rates,

even in fed fish, possibly reflecting the paucity of carbohydrate in the normal

piscine diet.

Catecholamines, glucagon, and glucagon-like peptides and glucocorticoids are the major hormones stimulating glucose liberation from glycogen,

while insulin and the insulin-like growth factors are the major hormones

stimulating glycogen storage (Fig. 6.6). Glucagon, glucagon-like peptides,

FIG. 6.6

Hormones implicated in the control of glucose liberation and uptake by fish


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