6.2.2. Carbohydrate Storage and Breakdown
6.2.2.2. Hepatic Glycogen Metabolism and Its Hormonal Control
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.
6.2.2.2.2. 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
FIG. 6.3
The organization of the Krebs cycle, showing sites of NADH, FADH, and GTP generation.
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).
6.2.2.2.3. Krebs Cycle and Oxidative Phosphorylation. The pyruvate pro- duced by glycolysis is fully oxidized to CO2 and H2O 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 function- ing 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. Es- sentially 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
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 mitochon- drial membrane, creating a proton and an electrical gradient between the inner mitochondrial matrix and the intermembrane and cytosolic compart- ments. 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 con- formational energy required to transform ADP+Piinto ATP.
There is not an obligate stoichiometry between the number of electrons which are passed among the cytochromes (or protons which are translo- cated 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,
not all proton flow from the outside to the inside of the inner mitochon- drial 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 (un- coupling 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 (Stuartet al.,1999). Thus, while the efficiency of car- bohydrate 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.
6.2.2.2.4. Glycogen Synthesis and Gluconeogenesis. Hepatic glycogen synth- esis is based both on the incorporation of bloodborne glucose into glycogen and on gluconeogenesis from lactate and amino acids. Glucose incorpo- ration into glycogen occurs via production of UDP glucose from glucose 1-phosphate via the glycogen synthase reaction. Gluconeogenesis from lac- tate or amino acids requires the reversal of many glycolytic reactions (Fig. 6.2) and follows enzymatic bypasses for the pyruvate kinase (PK) and phospho- fructokinase (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 con- verted to oxaloacetate via the malate dehydrogenase reaction). Next the ox- aloacetate is converted into phosphoenolpyruvate by phosphoenolpyruvate 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 sec- ond bypass reaction requires fructose bisphosphatase (FBPase), which con- verts 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.
6.2.2.2.5. 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
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 glucocorti- coids 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 hepatocytes.
FIG. 6.7
Amplification of metabolic signaling allowed by the activation of glycogen breakdown by sequential phosphorylation reactions.
and cortisol can also stimulate hepatic gluconeogenesis from amino acids.
The glucoregulatory hormones affect hepatic enzyme activities both through short-term modification of kinetic properties and through changes in gene expression. The regulation of glycogen phosphorylase and synthase is con- trolled largely by phosphorylation–dephosphorylation reactions (Fig. 6.7).
The hormonal activation of hepatic gluconeogenesis by glucagon and epinephrine occurs via phosphorylation of PK and of PFK2, the enzyme that produces fructose 2,6-bisphosphate. Several enzymes in glycolysis and gluconeogenesis show changes in their kinetic properties shortly after expo- sure to insulin or glucagon (Wrightet al.,1989; Foster and Moon, 1990). The reader is referred to some excellent, recent reviews on insulin, glucagon, and glucagon-like peptides for details concerning the specific peptides and their actions (Mommsen and Plisetskaya, 1991; Duguay and Mommsen, 1994;
Plisetskaya and Mommsen, 1996).
Glycogen synthesis in trout hepatocytes occurs at least partly via direct incorporation of glucose into glycogen (i.e., glucose→glucose 6-phosphate
→glucose 1-phosphate→UDP-glucose→glycogen) (Pereiraet al.,1995) and does not necessarily involve a partial glycolytic breakdown of glucose and subsequent gluconeogenesis, as occurs in mammals. The incorpora- tion of glucose into glycogen is influenced by the relative activities of phos- phorylase and synthetase. When isolated hypatocytes were incubated with insulin, glycogen phosphorylase activities decreased, whereas glycogen syn- thase activities did not increase. However, when both insulin and higher glucose levels (10 mM) were present in the incubation medium, glyco- gen synthase activities increased. Thus, as both glycogen phosphorylase and
synthetase activities seem to function simultaneously in the fish hepatocyte, shifts in the ratio of their activities are likely to dictate cellular glycogen contents.
6.2.2.2.6. Receptors and Signaling Systems. During the last decade, consid- erable effort has been directed toward elucidating the receptors and signal- ing systems involved in cellular responses to glucoregulatory hormones in fish. Moon and co-workers have made considerable progress in identifying the signals implicated in cellular responses to the hormones stimulating glycogen breakdown. Thus, glucagon acts on eel and bullhead hepato- cytes through both the cAMP and the inositol triphosphate (IP3) signaling pathways (Moon et al., 1997). Epinephrine actions on hepatocytes from these species also involve both the cAMP and the IP3pathways (Fabbriet al., 1995). Changes in intracellular Ca2+ levels caused by epinephrine may be mediated by its effects onα-adrenergic receptors as demonstrated for catfish, Ictalurus melas(Zhanget al.,1993), bullhead, trout (Oncorhynchus mykiss), and eel (Moonet al.,1993). Species differences can exist in the receptor types implicated and in the strength of the responses to a given hormone. The va- soactive peptides, vasotocin and mesotocin, stimulate gluconeogenesis only in eel hepatocytes, where they act via a V2-type receptor (Moon and Momm- sen, 1990). Chronic increases in cortisol levels in trout raised hepatocyte β2-adrenoceptors, suggesting an enhanced sensitivity to adrenergic stimu- lation, which could facilitate hepatic glucose production during periods of chronic stress (Reid et al.,1992). Guti´errez and co-workers have detected the receptors for insulin and insulin-like growth factor in fish liver, glycolytic and oxidative muscle, heart, ovary, and adipose tissue (Planaset al.,2000).
The receptors are similar to those present in mammals and change in their cellular levels with physiological status (Navarroet al.,1999).
Molecular techniques are being increasingly used to assess how the regu- lation of the expression of hepatic enzymes is as modified. Feeding trout with carbohydrates did not modify the expression of phosphoenolpyruvate carboxykinase, which catalyzes the conversion of oxaloacetate into phos- phoenolpyruvate (Fig. 6.2) (Panserat et al., 2001), or that of glucose-6- phosphatase (Panserat et al., 2000a). Similarly, carbohydrate feeding did not change the expression of fructose bisphosphatase in Atlantic salmon (Salmo salar) and Eurasian perch (Perca fluviatilis) (Tranulis et al., 1996;
Borreback and Christophersen, 2000). On the other hand, dietary carbohy- drate induces the expression of the glucokinase gene in trout, carp, and sea bream (Panseratet al.,2000b). Given that the natural diet of these fish is low in carbohydrate, they may lack mechanisms by which dietary carbohydrates decrease the expression of gluconeogenic enzymes.
6.2.2.2.7. Glucosidic Pathways of Glycogen Breakdown. In animal tissues, glycogenolysis can occur via both phosphorolytic (producing glucose 1-phosphate) and glucosidic (producing glucose) pathways. A variety of en- zymes cleaves glucose units off glycogen; the forms with acidic pH optima are thought to function in lysosomal recycling of the products of cellular autophagy. Whereas the physiological significance of the phosphorolytic pathway is well established, little is known of the physiological regulation of the glucosidic pathways. In mammals, defects in the glucosidic pathway lead to massive accumulation of glycogen in the lysosomes (type II glycogenosis).
In frogs, the glucosidase pathways liberate significant amounts of glucose from muscle glycogen after exhaustive exercise (Fournier and Guderley, 1992), leading to a marked postexercise hyperglycemia. Whereas few studies have examined the roles of the glucosidic pathways in fish, three forms of α-glucosidase were found in trout liver, and one of these differed in kinetic properties with the exercise status of the trout (Mehrani and Storey, 1993).
6.2.2.2.8. Regulation of Blood Glucose. A major difference between mam- mals and fish, which may extend to all ectothermal vertebrates, is the con- siderably greater tolerance which fish show to wide fluctuations of blood glucose levels. Thus, many fish species survive periods, caused by either nat- ural or experimental treatments, during which blood glucose levels are un- detectable (as reviewed by Mommsen and Plisetskaya, 1991). Nonetheless, under most physiological conditions, blood glucose levels are fairly stable and respond to hormonal treatments: rising with increases in cortisol, cate- cholamines, and glucagon and decreasing with insulin administration.
Because fish erthryocytes are nucleated and possess mitochondria, they can oxidize their fuels. Blood glucose is their most probable fuel, but the actual fuel use by blood cells is not entirely clear. Glycolytic and Krebs cycle enzymes are present in fish blood cells (Sephtonet al.,1991; Ferguson and Storey, 1991). When the oxidation of [6-14C]glucose is followed, the calcu- lated rates are 1000-fold lower than those of glucose disappearance, as such (Sephtonet al.,1991). Lack of knowledge about the exact fuels which are available in plasma complicates the analysis of fuel use by fish blood cells (Guppyet al.,1999).