Metabolic reorganization and signal transduction during estivation in the spadefoot toad

The maximal activities of 28 enzymes, representing multiple pathways of intermediary metabolism, were quantified in the brain, liver and skeletal muscle of spadefoot toads Scaphiopus couchii, comparing control toads with animals that had estivated for 2 months. Estivation-induced changes in brain enzyme activities were consistent with suppressed glycolysis and increased ketone body and amino acid catabolism. In liver, estivation resulted in reduced activities of eight enzymes representing carbohydrate, amino acid, ketone body and phosphagen metabolism, but the maximal activity of malic enzyme increased by 2.4-fold. Estivation led to a large-scale reorganization of skeletal muscle affecting most of the enzymes analyzed. Activities of enzymes of carbohydrate catabolism were generally elevated except for glycogen phosphorylase and hexokinase, whereas those of enzymes of fatty acid synthesis and ketone body metabolism were reduced. Increased glutamate dehydrogenase activities in both brain and muscle, as well as activities of other amino-acid-catabolizing enzymes in muscle, correlated with specific changes in the free amino acids pools in those tissues (reduced glutamine activity, increased glutamate, alanine and valine activities) that appear to be related to protein catabolism, for the purposes of elevating urea levels. The effects of estivation on signal transduction systems were also assessed. Total activities of protein kinases A and C (PKA and PKC) were largely unaltered in toad tissues during estivation (except for a 57% reduction in liver total PKC), but in seven organs there were strong reductions in the percentage of PKA present as the active catalytic subunit in estivating animals, and three contained a much lower percentage of membrane-bound active PKC during estivation. Activities of protein phosphatase types 1, 2A, 2B, and 2C were also frequently reduced during estivation. Overall, these results suggest that anuran estivation involves metabolic reorganization, including changing the maximal activities of key enzymes of intermediary metabolism as well as depressing the metabolic rate by suppressing signal transducing enzymes.     

Urea and KCl have differential effects on enzyme activities in liver and muscle of estivating versus nonestivating species.

The effects of 300 mM urea or 300 mM KCl on the maximal activities of 25 enzymes of intermediary metabolism were assessed in extracts of liver and muscle from spadefoot toads (Scaphiopus couchii), leopard frogs (Rana pipiens), and rats to assess their sensitivity to these osmolytes. During estivation, toads can lose -50% of total body water, and urea, which is known for its action as a protein denaturant, accumulates to 200-300 mM. The data show that the maximal activities of toad liver enzymes were not affected when assayed in the presence of 300 mM urea in vitro whereas urea inhibited the activities of seven enzymes in frog and 11 enzymes in rat liver. High KCl affected 12 or 13 enzymes in liver of each species causing inhibition in eight or nine cases each, and for frog and rat enzymes, inhibition was frequently more pronounced than for urea. Both urea and KCl affected enzyme activities in muscle extracts of all three species, but whereas their effects were largely negative for frog and rat enzymes, the enzymes affected by urea or KCl in toad muscle were primarily activated by these osmolytes (six out of nine enzymes affected by urea and eight of 15 enzymes affected by KCl). Urea, KCl, and polyethylene glycol (a protein crowding agent) also had species-specific effects on the dissociation constant (Ka) for cAMP of protein kinase A. The data suggest that the accumulation of urea by water-stressed anurans not only contributes to minimizing cell volume reduction, but by doing so also limits the increase in intracellular ionic strength that occurs and thereby helps to minimize the potential inhibitory effects of high salts on metabolic enzymes.     

Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation

For many animals, the best defense against harsh environmental conditions is an escape to a hypometabolic or dormant state. Facultative metabolic rate depression is the common adaptive strategy of anaerobiosis, hibernation, and estivation, as well as a number of other arrested states. By reducing metabolic rate by a factor ranging from 5 to 100 fold or more, animals gain a comparable extension of survival time that can support months or even years of dormancy. The present review focuses on the molecular control mechanisms that regulate and coordinate cellular metabolism for the transition into dormancy. These include reversible control over the activity state of enzymes via protein phosphorylation or dephosphorylation reactions, pathway regulation via the association or dissociation of particle-bound enzyme complexes, and fructose-2,6-bisphosphate regulation of the use of carbohydrate reserves for biosynthetic purposes. These mechanisms, their interactions, and the regulatory signals (e.g., second messenger molecules, pH) that coordinate them form a common molecular basis for metabolic depression in anoxia-tolerant vertebrates (goldfish, turtles) and invertebrates (marine molluscs), hibernation in small mammals, and estivation in land snails and terrestrial toads.     

Heat shock proteins (Hsp70) and water content in the estivating Mediterranean Grunt Snail (Cantareus apertus)

Pulmonate land snails often are able to estivate to survive dry hot seasons were water and food are scarce. The aperture of the shell is closed with an epiphragm, and metabolism is depressed to approximately one fourth of basal metabolism. We investigated a molecular aspect of estivation focussing on the heat shock protein 70 (Hsp70) stress response during estivation in the Mediterranean Grunt Snail Cantareus apertus. Sequences of a new inducible hsp70 and of actin are presented and expression of the hsp70 gene as well as Hsp70 protein content was measured in estivating animals. Both Hsp70 protein and mRNA do not show a significant change from the control, although there is a trend that hsp70 mRNA is less abundant in estivating specimens. After heat shock, the expression of hsp70 increased and a higher Hsp70 protein content was detected. Water relations were also investigated. After a period of 6 months in the dormant state, the snails contained 14% less water than active ones, implying a constricted protection against desiccation, compared to the desert snail Sphincterochila zonata, and a Mediterranean-type water economy.     

The regulation of thapsigargin-sensitive sarcoendoplasmic reticulum Ca<Superscript>2+</Superscript>-ATPase activity in estivation

Estivation (aerobic dormancy) is characterized by sustained metabolic rate depression, which is crucial to survival in the face of unfavorable environmental conditions and enables the preservation of endogenous fuel reserves. Ion pumping is one of the most energetically taxing physiological processes in cells, and ion motive ATPases are likely loci to be differentially regulated in models of metabolic arrest. We proposed that the sarcoendoplasmic reticulum (SER) calcium-ATPase (SERCA) would be deactivated in the estivating desert snail Otala lactea, potentially contributing to the overall suppression of metabolism. SERCA kinetic parameters [decreased maximal velocities, increased substrate K _m values, increased Arrhenius activation energy (E _a)] were indicative of a less active enzyme in the estivated state. Interestingly, the less active SERCA population in dormant snails featured greater kinetic (K _m Mg.ATP versus temperature) and conformational (resistance to urea denaturation) stability than that in active snails. Western blotting confirmed that SERCA protein content did not change during estivation. In light of this observation, we proposed that estivation-dependent changes in SERCA activity was due to changes in SERCA phosphorylation state. In vitro studies promoting specific kinase or phosphatase action indicated that decreased SERCA activity in estivation was linked with endogenous kinase activity whereas reactivation of SERCA was facilitated by endogenous protein phosphatases (PP).     

Metabolic responses of the South American ornate horned frog (Ceratophrys ornata) to estivation

We examined the metabolic responses of the South American frog, Ceratophrys ornata, to laboratory-induced estivation. Whole-animal and mass-specific oxygen consumption rates (VO₂) did not change during fasting or 56 days of estivation, despite observing significant decreases in body mass. The maintenance of mass-specific metabolic rate at routine levels during estivation suggests that metabolic rate suppression is not a major response to estivation in this species. There was a significant decline in liver glycogen and a loss of adipose tissue mass during estivation, suggesting that both carbohydrate and lipid pathways are used to fuel metabolism during estivation. The activity of pyruvate dehydrogenase, an important regulator of carbohydrate oxidation, and carnitine palmitoyltransferase and 3-hydroxyacyl-CoA dehydrogenase, regulators of lipid oxidation, showed no significant change in activity in liver, heart, and muscle between estivating and active frogs. There was an increase in plasma osmolality, which is characteristic of estivating animals. Overall, our metabolic analysis of estivation in C. ornata indicates that this species does not employ a dramatic suppression metabolic rate to survive dehydration stress and that both endogenous carbohydrates and lipids are used as metabolic fuels.     

Differential gene expression in the liver of the African lungfish, <Emphasis Type="Italic">Protopterus annectens</Emphasis>, after 6 days of estivation in air

This study aimed to identify estivation-specific gene clusters through the determination of differential gene expressions in the liver of Protopterus annectens after 6 days of estivation in a mucus cocoon in air (normoxia) using suppression subtractive hybridization polymerase chain reaction. Our results demonstrated that 6 days of estivation in normoxia led to up-regulation of mRNA expressions of several genes related to urea synthesis, including carbamoyl phosphate synthetase (Cps), argininosuccinate synthetase and glutamine synthetase. They indicate that increased urea synthesis, despite being energy-intensive, is an important adaptive response of estivation. They also offer indirect support to the proposition that urea synthesis in this lungfish involved a Cps that uses glutamine as a substrate. In addition, up- or down-regulation of several gene clusters occurred in the liver of P. annectens after 6 days of estivation in normoxia. These estivation-specific genes were involved in the prevention of clot formation, activation of the lectin pathway for complement activation, conservation of minerals (e.g. iron and copper) and increased production of hemoglobin beta. Since there were up- and down-regulation of mRNA expressions of genes related to ribosomal proteins and translational elongation factors, there could be simultaneous increases in protein degradation and protein synthesis during the first 6 days (the induction phase) of estivation, confirming the importance of reconstruction of protein structures in preparation for the maintenance phase of estivation.     

Akt and its downstream targets play key roles in mediating dormancy in land snails

Estivation, a state of aerobic dormancy, facilitates survival during adverse environmental conditions and is characterized at the molecular level by regulatory protein phosphorylation. The Akt (protein kinase B) signaling pathway regulates diverse responses in cells and the present study analyzes its role in the estivating desert snail Otala lactea. Kinetic analysis (maximal velocity, substrate affinities) determined that Akt was activated in tissues of estivating snails and Western blotting and in vitro incubations promoting changes to Akt phosphorylation state both confirmed that higher amounts of active (phosphorylated Ser473) Akt were present during estivation. Akt protein stability was also enhanced during estivation as assessed from urea denaturation studies. Multiple downstream targets of Akt were differentially regulated during estivation. Estivating animals showed elevated levels of phosphorylated FOXO3a (Ser253) and BAD (Ser136), no change in mTOR (Ser2481 and Ser2448), and reduced amounts of phosphorylated glycogen synthase kinase-3 (GSK-3) beta subunit (Ser9). Kinetic analysis of GSK-3 showed 1.5-1.7 fold higher activities in estivating snails coupled with increased GSK-3 substrate affinities in hepatopancreas. The data suggest an active role for Akt signaling during estivation emphasizing anti-apoptotic actions but uncoupling growth/proliferation actions to help achieve life extension on a limited energy budget.     

Urea production and transport in teleost fishes

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.     

Urea and salt effects on enzymes from estivating and non-estivating amphibians

The effects of urea, cations (K⁺, NH₄, Na⁺, Cs⁺, Li⁺), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle, were analyzed in two anuran amphibians, an estivating species, the spadefoot toadScaphiopus couchii, and a semi-aquatic species, the leopard frogRana pipiens. Urea, which accumulates naturally to levels of 200–300 mM during estivation in toads, had only minor effects on the V_max, kinetic constants and pH curves of PK from either species and no effects on PFK V_max or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the V_max of toad PFK and of PK from both species and altered the K_m values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the V_max of PK from either species was reduced by more than 80% by the addition of 200 mM NH₄Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the K_m values for substrates of toad lactate dehydrogenase and strongly reduced the V_max of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes. Rather, we found that urea is largely a non-perturbing solute for anuran enzymes (I₅₀ values were>1 M for both PK and PFK in both species) and we propose that its accumulation in high concentrations during estivation helps to minimize the increase in cellular ionic strength that would otherwise occur during desiccation and to alleviate the accompanying negative effects of high salt on individual enzyme activities and overall metabolic regulation.Abbreviations PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Overproduction of microbial products--facts and ideas

Overproduction of microbial metabolites is related to developmental phases of microorganisms. Inducers, effectors, inhibitors and various signal molecules play a role in different types of overproduction. Primary and secondary metabolism are interconnected. Biosynthesis of enzymes catalyzing metabolic reactions in microbial cells is controlled by well-known positive and negative mechanisms, e.g. induction, repression, catabolite repression, mechanisms controlling enzyme activity include isosteric and allosteric interactions, e.g. competitive and non-competitive inhibition, allosteric effects, molecular conversion etc. Biosynthesis of secondary metabolites is catalyzed by unaltered enzymes of primary metabolism, by altered enzymes of primary metabolism and by specific enzymes of secondary metabolism. In addition to classical mutagenesis and selection of suitable microbial cells, methods of molecular genetics are used in the overproduction of microbial products.     

Antioxidant defenses and lipid peroxidation damage in estivating toads, Scaphiopus couchii

Tissue-specific changes in antioxidant defenses and lipid peroxidation damage were analyzed in spadefoot toads, Scaphiopus couchii, to determine how these responded during estivation, a state of suppressed oxygen consumption. Maximal activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase were measured in six organs from 2-month-estivated toads and compared with activities in animals awakened for 10 days after estivation. Activities of many enzymes, particularly the glutathione-linked enzymes, were significantly lower in tissues of estivating toads than in awake toads. This indicates that enzymatic antioxidant defenses are probably modulated in response to the rate of reactive oxygen species generation in tissues, which is proportional to oxygen consumption. Antioxidant enzyme activities were largely insensitive to high urea, which accumulates during estivation, but were inhibited by elevated KCl. Levels of reduced glutathione were also significantly lower in three organs during estivation and all organs, except skeletal muscle, exhibited a higher oxidized/reduced glutathione ratio, indicating a more oxidized state during estivation. Products of lipid peroxidation (conjugated dienes, lipid hydroperoxides) were higher in tissues of estivated than control toads, suggesting accumulated oxidative damage to lipids during dormancy. One enzymatic source of free radical generation, xanthine oxidase, appeared to have little impact because its activity was detectable only in liver and was significantly lower in estivated toads. The data indicate that both enzymatic and metabolite antioxidant defenses in toads are adaptable systems that are modulated in estivating versus awake states. Accepted: 21 October 1997     

Studies on the extra- and intracellular acid-base status and its role on metabolic depression in the land snail <Emphasis Type="Italic">Helix lucorum</Emphasis> (L.) during estivation

The aim of the present study was to examine the acid-base status of extra- and intracellular fluids and its possible role on the regulation of the metabolic rate of Helix lucorum during prolonged estivation. For this purpose, the rate of oxygen consumption for active and estivating snails was determined. The acid-base status was also examined in the hemolymph and tissues from active and estivating snails acclimated at 25 degrees C. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether there is a change in the snails during estivation. The rate of oxygen consumption decreased significantly within the 1st 10 days of estivation from 122.51+/-10 microl.g(-1).h(-1) to 25.86+/-5.2 microl.g(-1).h(-1), indicating a marked decrease in metabolic rate. P(CO2)increased within the 1st 20 days of estivation from 13.52+/-0.68 mmHg to 25.09+/-2.05 mmHg, while the pH of hemolymph (pH(e)) decreased from 7.72+/-0.04 to 7.44+/-0.06. The level of bicarbonates decreased in the hemolymph of estivating snails, indicating a metabolic acidosis, which was moderate in extracellular fluids. In contrast to pH(e), the intracellular pH (pH(i)) was maintained in the tissues of estivating H. lucorum, indicating a regulation of pH(i) despite the developed hypercapnia. According to the results presented here, it seems that the timing of pH(e) changes does not correlate with the timing of metabolic rate reduction in estivating H. lucorum.     

Nitrogen metabolism and excretion in Allenbatrachus grunniens(L): effects of variable salinity, confinement, high pH and ammonia loading

The nitrogen metabolism and excretion patterns of the grunting toadfish Allenbatrachus grunniens and the effects of salinity on these processes were examined. Individuals of A. grunniens were subjected to several experimental treatments, including variable salinity (2 to 30), high pH (8·5 compared to 7·0 for controls), high environmental ammonia (10 mM) and confinement to small water volumes, and measurements were made of activities of selected enzymes of nitrogen metabolism, ammonia and urea excretion rates, and tissue and plasma contents of ammonia, urea and amino acids. Activities of key ornithine‐urea cycle enzymes were rather low ( e.g . liver carbamoyl phosphate synthetase III activity was 0·001 μmols min⁻¹ g⁻¹), and A. grunniens consistently demonstrated a low capacity for urea excretion despite significant elevations of plasma and tissue ammonia contents by the high pH and high ammonia treatments. This species could thus be categorized as ammoniotelic. Total free amino acid contents in plasma and tissues were increased by the high pH and high ammonia treatments, but no patterns were discerned in individual amino acids that would indicate any preferential accumulation ( e.g . alanine and glutamine) as has been noted previously in several semi‐terrestrial fish species. Thus, it appeared that A. grunniens was not unusual in its patterns of nitrogen metabolism and excretion in comparison to other 'typical' teleosts. Furthermore, manipulation of salinity had no major effects on nitrogen excretion in either this species or in comparative studies with the ureotelic gulf toadfish Opsanus beta . The results are discussed in the context of the broader pattern of nitrogen metabolism and excretion in the Batrachoididae.     

Metabolic engineering: techniques for analysis of targets for genetic manipulations

Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement-random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied: (1) detailed physiological studies, (2) metabolic flux analysis (MFA), (3) metabolic control analysis (MCA), (4) thermodynamic analysis of pathways, and (5) kinetic modeling. In this article, these different techniques are discussed and their applications to the analysis of different processes are illustrated.     

The regulation of AMPK signaling in a natural state of profound metabolic rate depression

In response to energy stress (and elevated AMP), the AMP-activated protein kinase (AMPK) coordinates the restoration of energy homeostasis. We determined that AMPK is activated in a model system (desert snail Otala lactea) during a physiological state of profound metabolic rate depression (estivation) in the absence of a rise in AMP. Kinetic characterization indicated a strong increase in AMPK activity and phosphorylation in estivation, consistent with an increase in P-Ser428 LKB, an established regulator of AMPK. Accordingly, ~2-fold increases in AMPKα1 protein and activity were observed with LKB1 immunoprecipitates from estivating snails. In vitro studies determined that AMPK in crude extracts was activated in the presence of cGMP and deactivated in conditions that permitted protein phosphatase type-2A (PP2A) activity. Furthermore, AMPKα1 protein and activity increased in PKG immunoprecipitates from estivating tissues, suggesting a novel role for PKG in the regulation of AMPK in vivo. We evaluated several downstream targets of AMPK. Acetyl-CoA carboxylase (ACC) activity was strongly inhibited in estivation, consistent with increased P-Ser79 content, and in vitro stimulation of AMPK negated citrate’s ability to stimulate ACC aggregation. Analysis of other targets revealed a strong decrease in PPARγ-coactivator 1α expression in both tissues, which was related to decreased gluconeogenic protein expression in hepatic tissue, but no changes in mitochondrial biogenesis markers in muscle. We concluded that AMPK activation in O. lactea aids in facilitating the suppression of anabolic pathways, without necessarily activating ATP-generating catabolism.     

Seasonal modulation of free radical metabolism in estivating land snails Helix aspersa

We investigated the regulation of free radical metabolism in Helix aspersa snails during a cycle of 20-day estivation and 24-h arousal in summer in comparison with estivation/arousal in winter-snails. In winter-snails (J. Exp. Biol. 206, 675-685, 2003), we had already observed an increase in the selenium-dependent glutathione-peroxidase (Se-GPX) activity in foot muscle and hepatopancreas and in the contents of hepatopancreas GSH-equivalents (GSH-eq=GSH+2 GSSG) during estivation compared with 24-h aroused snails. Summer-estivation prompted a 3.6-fold increase in Se-GPX activity in hepatopancreas, though not in foot muscle. Total-superoxide dismutase and catalase activities in hepatopancreas decreased (by 30-40%) during summer-estivation; however, no changes occurred in the activities of glutathione reductase, glutathione S-transferase and glucose-6-phosphate dehydrogenase in the two organs. GSH-eq levels were increased (by 54%) in foot muscle during estivation, but were unchanged in hepatopancreas. In contrast with winter-snails, oxidative stress markers (lipid peroxidation, carbonyl protein, and the GSSG/GSH-eq ratio) were unaltered during estivation/arousal in summer. These results demonstrate that seasonality modulates not only the absolute activities/levels of antioxidants (enzymes and GSH-eq) in H. aspersa, but also the regulatory process that controls the snail's antioxidant capacity during estivation/arousal. These results suggest that H. aspersa has an "internal clock" controlling the regulation of free radical metabolism in the different seasons.     

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad,Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.     

Jaws: The Inside Story. The Metabolism of Elasmobranch Fishes

Elasmobranchs are of metabolic interest for several reasons, including their primitive evolutionary position, their osmotic strategy and their low incidence of neoplasia. Some aspects of the metabolism of elasmobranch fishes are unique when compared with those of the other vertebrates. Although many features of their metabolism can be attributed to their primitive evolutionary position (e.g., fewer isoforms of enzymes and other proteins), some unique features appear to be related to the unusual solute system (urea and methylamines) used by elasmobranchs. The solute system exerts widespread effects, which has an impact on the metabolism of lipids, ketone bodies and amino acids and the structure of proteins and membranes. Effects of urea on the transport of lipid may influence aspects of lipid metabolism, reducing extrahepatic lipid catabolism via effects on nonesterified fatty acid transport and enhancing a need for reliance on ketone bodies. Amino acid metabolism of elasmobranchs is also heavily influenced by the need for continuous synthesis of urea with glutamine as the nitrogen donor. These effects, in turn, may play a role in their low incidence of cancer. Specifically, the reduced availability of glutamine (an important nutrient for rapidly growing cells) coupled with the low levels of nonesterified fatty acids in the blood reduces the availability of molecules essential for tumor growth. This metabolic design may thus provide marine elasmobranchs with a “systemic” resistance to cancer.