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Glycolytic adjustments in tissues of frog Rana ridibunda and land snail Helix lucorum during seasonal hibernation

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Glycolytic adjustments in tissues of frog Rana ridibunda and land snail Helix lucorum during seasonal hibernation
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  Glycolytic adjustments in tissues of frog  Rana ridibunda  and land snail  Helix lucorum during seasonal hibernation Basile Michaelidis a, ⁎ , Pasqualina Kyriakopoulou-Sklavounou b , Alexandra Staikou b ,Ioanna Papathanasiou b , Kiriaki Konstantinou a a Laboratory of Animal Physiology, Department of Zoology, Faculty of Science, School of Biology, University of Thessaloniki, GR-54124 Thessaloniki, Greece b Laboratory of Zoology, Department of Zoology, Faculty of Science, School of Biology, University of Thessaloniki, GR-54124 Thessaloniki, Greece a b s t r a c ta r t i c l e i n f o  Article history: Received 14 April 2008Received in revised form 13 July 2008Accepted 14 July 2008Available online 18 July 2008 Keywords: Glycolysis Helix lucorum Land snails Rana ridibunda Seasonal hibernationWater frogs The present work aimed to contribute to the understanding of the adaptation of the glycolytic pathway intissues of frog  Rana ridibunda  and land snail species  Helix lucorum  during seasonal hibernation. Moreoverresponses of glycolytic enzymes from cold acclimated  R. ridibunda  and  H. lucorum  were studied as well. Thedrop in Po 2  in the blood of hibernated frogs and land snails indicated lower oxygen consumption and adecrease in their metabolic rate. The activities of glycolytic enzymes indicated that hibernation had adifferential effect on the glycolyis in the two species studied and also in the tissues of the same species. Theactivity of   L  -LDH decreased signi 󿬁 cantly in the skeletal muscle and heart of hibernated  R. ridibunda  indicatinga low glycolytic potential. Similar biochemical responses were observed in the same tissues during coldacclimation. The continuous increase in the activities of glycolytic enzymes studied, except for HK, mightindicate a compensation for the impacts of low temperature on the enzymatic activities. In contrast to R. ridibunda , the activities of the enzymes increased and remained at higher levels than those of theprehibernation controls indicating maintenance of glycolytic potential in the tissues of hibernating landsnails.© 2008 Elsevier Inc. All rights reserved. 1. Introduction Duringwinterintemperateregions,hibernatorsaswaterfrogsandsnails have to deal with low and sometimes subzero temperatures.Metabolic depression is a common response of water frogs and landsnails tolow temperaturein general and as well as during hibernation(Storey and Storey, 1990; Boutilier et al., 1997; Guppy and Withers,1999). The biochemical and physiological responses leading to ahypometabolic state have been studied extensively in freezingtolerant frog species (Storey and Storey, 1986, 1987, 1988). It hasbeen shown recently that the metabolic depression in frogs hibernat-ing under hypoxic water is accompanied by a signi 󿬁 cant decrease inthe aerobic capacity of their skeletal muscle, as indicated by areduction in the activity of key enzymes of the TCA cycle and of theelectron-transport chain (St-Pierre and Boutilier, 2001). The LDHactivity of the skeletal muscle of overwintering frogs was also muchlower than in prehibernation controls, supporting the idea of adecreased  󿬂 ux through the glycolytic pathway during hypoxichibernation (i.e. the so-called  ‘ reversed ’  Pasteur effect; Hochachkaand Somero, 2002; Donohoe and Boutilier, 1998). Similarly, hiberna-tioninlandsnailsduringwinterischaracterizedbyamarkeddecreasein their metabolic rate. However, compared with frogs, little is knownabout the biochemical and physiological responses of   Helix lucorum  tohibernation.Several works have emphasized that metabolic depression canhavequiteseparate anddistinctly differenteffectsondifferent tissues.As reported by Flanigan et al. (1991), metabolic depression at the whole-animal level might not be re 󿬂 ected equally in all tissues. Forexample while the  in vitro  respiration rate of intact skeletal musclefrom aestivating frogs is reduced compared with controls, no suchreduction occurs in the intestine, liver, skin or fat (Flanigan et al.,1991).Moreover,areductionintheactivitiesofglycolyticenzymeshasbeen observed during long-term aestivation in several species of terrestrial snails and the frog  Neobatrachus pelobatoides . The activitiesof hexokinase, phosphofructokinase, glyceraldehyde-3-phosphatedehydrogenase, phosphoglycerate kinase and lactate dehydrogenase(LDH) were reduced in the foot muscle of aestivating snails comparedwith controls (Brooks and Storey, 1990). Conversely, in the kidney,heart and hepatopancreas of aestivating snails no reduction in theactivityof LDH was found (Stuartet al.,1998b). The liver of aestivatingfrogs hadloweractivitiesof aldolase andglyceraldehyde-3-phosphatedehydrogenase compared with controls, but the activities of both Comparative Biochemistry and Physiology, Part A 151 (2008) 582 – 589 ⁎  Corresponding author. Laboratory of Animal Physiology, Department of Zoology,School of Biology, Faculty of Science, Aristotle University of Thessaloniki, Thessaloniki54124, Greece. Tel.: +30 2310 998 401; fax: +30 2310 998 269. E-mail address:  michaeli@bio.auth.gr (B. Michaelidis).1095-6433/$  –  see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpa.2008.07.017 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part A  journal homepage: www.elsevier.com/locate/cbpa  these enzymes remained unchanged during aestivation in the ven-tricle, gastrocnemius and brain (Flanigan et al., 1990).In the present study, we sought to determine whether changes inthe activities of several key enzymes of the glycolytic pathway aredown-regulated during hibernation in  Rana ridibunda  and  H. lucorum .In the central and south of Europe the  Rana esculenta  complex occurs(Graf and Polls-Pelaz, 1989). The latter involves the parental species R. ridibunda  Pallas 1771,  Rana lessonae  Camerano 1882 and thehybridogen  R. esculenta  Linnaeus 1758 (Berger,1967,1968). Comparedtothe other twofrogspecies,  R. ridibunda is poorly tolerant tohypoxia(Turner and Nopp, 1979; Plenet et al., 2000a,b), less tolerant tofreezing, while it has no cryoprotective system (Voituron et al., 2003,2005). Despite the differences in the habitat and wintering behavioramong the three frog species mentioned above little is known abouttheir physiological and biochemical responses during seasonalhibernation.In the present work we studiedthe glycolytic adjustmentof   R. ridibunda . Additionally we studied the glycolytic adjustment of land snail species. The species used,  H. lucorum , is found throughoutthe mainland of Greece especially in the northern regions where itsecology and biology have been studied (Staikou et al., 1988). In thenorth of Greece snails of this species hibernate during winter.Hibernation starts at the beginning of November and is terminatedby the end of March. 2. Materials and methods  2.1. Animals and experimental design Adult  R. ridibunda  frogs were collected from the surroundings of Thessaloniki in October 2006. They were transferred to the laboratorywithin the same day and they were put into plastic tanks measuring50 cm height, 80 cm wide and 1.50 m length. The plastic tankscontained soil and water in order to simulate the ponds where thefrogs were collected from. The plastic tanks were placed outdoors sothat frogs were exposed to natural conditions of light and tempera-ture. Tanks were covered to prevent  󿬂 ooding and animals drowningduring rainy periods. Frogs were fed with larvae of   Tenebrio molitor  everydayuntilthe 󿬁 rst5 – 7daysofNovember.Feedingwasstoppedasfrogs started to be less active and remained longer in the water. Fromthe  󿬁 rst days of November (2006) until the middle of March (2007)animals were removed at regular periods and were killed by doublepithing. Animals were rapidly dissected open and a blood sample wasremovedasdescribedbyStewartet al.(2004)forthedeterminationof partial pressure of oxygen (Po 2 ) (see below). Then the heart andskeletal muscle (gastrocnemius) tissues were dissected, freeze-clamped between aluminum tongs cooled in liquid nitrogen andground under liquid nitrogen. Tissue powders were stored at  − 80 °Cuntil measurements of enzymatic activities and the levels of   L  -lactate.To examine the effects of ambient temperature on the activities of glycolytic enzymes from active frogs, a plastic tank containing anumber of animals was put into a cool room at 5 °C and maintainedunder these conditions for 15 days. Individuals were drawn after 1, 5,10 and 15 days of acclimation at 5 °C, tissues were dissected andtreated for the measurements of enzymatic activities as describedabove.The snails ( H. lucorum ) used for the experiments described in thispaperwerecollectedinOctober2006fromapopulationinthevicinityof Edessa, in northern Greece. Adult snails (35 – 42 mm) weretransferred to the laboratory where they were put in large glassboxes,  󿬁 lled with soil derived from the area from where snails werecollected.Theboxeswereplacedoutdoorssothatsnailswereexposedto natural conditions of light and temperature. The boxes werecovered to prevent  󿬂 ooding and animals drowning during rainyperiods. Water was added periodically to ensure that soil was keptmoist and the ambient humidity was maintained above 80%. Snailswere fed fresh lettuce leaves everyday by the  󿬁 rst 5 – 7 days of November. Feeding was stopped as soon as snails started to burrowinto the ground. From the  󿬁 rst days of November (2006) until themiddle of March (2007) snails were removed at regular periods andimmediately their haemolymph was sampled as described by Pedleretal.(1996)forthedeterminationofPo 2 .Thenfootmuscleandmantletissues were dissected, freeze-clamped between aluminum tongscooled in liquid nitrogen and ground under liquid nitrogen. Tissuepowders were stored at  − 80 °C until measurements of enzymaticactivities and the levels of   D -lactate.To examine the effects of ambient temperature on the activities of glycolyticenzymesfromactivesnailsanumberofsnailsmaintainedat20 °C were put into a cool room at 5 °C and maintained under theseconditions for 15 days. Individuals were drawn after 1, 5, 10 and15 days of acclimation at 5 °C, tissues were dissected and treated forthe measurements of enzymatic activities as described above.Frogs and snails were sampled in the middle of the day andambient temperature was recorded at the day of sampling.  2.2. Collection of blood and haemolymph and determination of Po  2 Bloodsamplingfromfrogscloselyfollowedthemethodsof Stewartet al. (2004). Brie 󿬂 y, each frog was double-pithed and the heartexposed via aventral midline incision. A heparinized (100,000 units/L ammonium heparin in Ringer's solution) tuberculin syringe was usedtowithdrawa 0.4-ml blood sample anaerobically for determination of Po 2 .HaemolymphcollectionfromsnailswasperformedasdescribedbyPedler et al. (1996). In brief, after removing a small section of shell toexpose the pericardium, haemolymph was collected after puncturingtheheartwithaneedle 󿬁 ttedtoasyringe,previouslyequilibratedwithpure nitrogen. The Po 2  was determined in snails' haemolymph andfrogs'bloodbyuseofaClarketypeoxygenelectrode(E5047)inthegascuvette of the BMS3/MK2 Blood Micro System. All determinationswere performed at temperatures corresponding to those recorded atsampling occasions.  2.3. Preparation of tissues homogenates for the determination of enzymatic activities Glycolytic enzyme assays were adapted from those described byStorey and Storey (1984) and Stuart et al. (1998a). Brie 󿬂 y, samples of frozen tissue powders (200 – 500 mg) were rapidly weighed andhomogenized (1:5, wt/vol) in ice-cold 50 mM imidazole – HCl (pH 7.0)containing 100 mM sodium  󿬂 uoride (NaF), 5 mM EDTA, 5 mM EGTA,15 mM 2-mercaptoethanol and 0.1 mM PMSF added just prior tohomogenization, using a Polytron PT10 homogenizer (3 periods, 20 seach time). After centrifugation (15,000  g  , 4 min, 4 °C), thesupernatant was removed and passed through a 5-ml column of Sephadex G-25 equilibrated in 40 mM imidazole – HCl buffer (pH 7.0)containing 5 mM EDTA, 15 mM 2-mercaptoethanol, and 20% glycerolto remove metabolites of low molecular mass (Helmerhorst andStrokes,1980). The column was centrifuged in a desktop centrifuge at2000  g   for 1 min, and the supernatant was used for the determinationof enzyme activity. Enzyme activities (expressed as µmol/min g wetweight) were determined at 20 °C using a Hitachi 150-20 recordingspectrophotometer with water-jacketed cell. Assays were carried outin duplicate and rates of reactions involving NAD or NADH werefollowed at 340 nm (millimolar extinction coef  󿬁 cient  ε  340 =6.22). Inaddition, to examine the effects of low temperature on enzymaticactivities from hibernated frogs and snails, samples of frozen tissuepowders from animals collected at the end of December 2006 weretreatedas describedabove and the enzymatic activities determined at5 °C, which corresponds to the ambient temperature measured at thesame periods of animal collection. All enzymes were assayed for10 minin50mMimidazole – HClpH7.0ina 󿬁 nalassayvolumeof 1 ml.Speci 󿬁 c assays conditions were as follows. 583 B. Michaelidis et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 582 – 589   2.3.1. Hexokinase (HK) (ATP:  D -hexose-6-phospho-transferase; E.C. 2.7.1.1),10 mM MgCl 2 ,0.15 mM NAD  + , 5 U/ml NAD + -dependent glucose-6-phosphatedehydrogenase (from  Leuconostoe mesenteroides ), 10 mM glucose.The assay was started by the addition of 1 mM adenosine 5 ′ -triphosphate(ATP).Insamplesfromthemantletissue,HKactivitywascalculated by subtraction of the baseline activity due to endogenousglucose dehydrogenase.  2.3.2. Phosphofructokinase (PFK) (ATP:  D -fructose-6-phosphate 1-phospho-transferase; E.C.2.7.1.11), 1 mM dithiothreitol, 0.15 mM NADH, 5 mM MgCl 2 , 50 mMKCl, 5 U/ml aldolase, 50 U/ml triose phosphate isomerase, 5 U/mlglycerol-3-Pdehydrogenase,2.5mMATP.Theassaywasstartedbytheaddition of 2 mM fructose-6-phosphate (F-6-P).  2.3.3. Aldolase (Ald) ( D -Fructose-1,6-bisphosphate  D -glyceraldehyde-3-phosphate lyase;E.C. 4.1.2.13), 0.15 mM NADH, 50 U/ml triose phosphate isomerase, 5 U/ml  α -glycerophosphate dehydrogenase. The assay was started by theaddition of 3 mM fructose-1,6-bisphosphate (F-1,6-P 2 ).  2.3.4. Phosphoglycerokinase (PGK) (ATP: 3-phospho- D -glycerate 1-phospho-transferase; E.C. 2.7.2.3),1 mM EDTA, 5 mM MgCl 2 , 0.15 mM NADH, 1 mM ATP, 2.5 mlglyceraldehyde-3-phosphatedehydrogenase.Theassaywasstartedbythe addition of 40 mM 3-phosphoglyceric acid (3PGA).  2.3.5. Pyruvate kinase (PK) (E.C. 2.7.1.40): 10 mM MgCl 2 , 100 mM KCl, 0.15 mM NADH, 2 mMADP, 2 units LDH, 5 mM phophoenolpyruvate (omitted for control).  2.3.6.  L - or   D -lactate dehydrogenase (LDH) (E.C.1.1.1.27): 0.4 mM NADH,1 mM pyruvate (omitted for control).  2.4. Determination of   L - and  D -Lactate levels in tissue homogenates from frogs and snails Forthedeterminationoflactate,frozentissueswerehomogenizedin9 volumes of ice-cold 6% (w/v) perchloric acid containing 1 m Μ  EDTAusing a Polytron homogenizer. The homogenization tube and sampleswerekeptat4°Cinanicebath.Thehomogenateswerethencentrifugedat10,000  g  for20minat 4 °C and thesupernatantwasneutralized with5m Μ KOHcontaining50m Μ triethanolamine.Theneutralizedextractswerekeptonicefor15minandcentrifugedasdescribedabove. L  -Lactateand  D -lactate levels in the tissues of frogs and snails respectively weredetermined enzymatically according to the methods of  Lowry andPassonneau (1972) by using commercial  L  -LDH or  D -LDH respectively.  2.5. Statistical analysis Changes over time were tested for signi 󿬁 cance at the 5% level byusing one-way analysis of variance (ANOVA) and by performingBonferronipost-hoctestsforgroupcomparisons.Valuesarepresentedas means±S.E.M. Animals sampled at the beginning of November2006 were used as prehibernation controls. 3. Results  3.1. R. ridibunda The Po 2  of the blood of   R. ridibunda  decreased about 75% by thebeginning of December 2006 and it remained at low levels until theend of February 2007. Thereafter, Po 2  increased gradually until earlyMarch2007.Speci 󿬁 callyitincreasedfrom7.89±1.34mmHgto16.23±2.45 mm Hg (Fig.1).Changes in the activities of glycolytic enzymes in the tissues of   R.ridibunda areshowninFig.2.Asshown,theactivityofHKremainedatabout the same levels in the tissues of hibernating frogs. On thecontrary, the activity of PFK showed a differential pattern of changesin the tissues of   R. ridibunda . Speci 󿬁 cally, it remained at the samelevels in the skeletalmuscle, whileit increasedin the heart duringthehibernation period. In the skeletal muscle of hibernating  R. ridibunda ,after an initial increase within the 󿬁 rst month of hibernation, aldolaseactivity decreased and it recovered at control levels. Similarly, in theheart,aldolaseactivityincreasedsigni 󿬁 cantlybythemiddleofJanuaryandreturnedtocontrollevels.Thepatternofchangesintheactivityof PGK was similar to those observed in aldolase activity in all tissuesstudied. In the skeletal muscle of   R. ridibunda , the activity of PK, afteran initial signi 󿬁 cant decrease by the beginning of December itreturned to control levels. In the heart, the activity of the enzymeincreased about three folds by the middle of January and returned tocontrol levels. On the contrary, the activity of   L  -LDH decreasedsigni 󿬁 cantly in the skeletal muscle and heart of hibernated  R.ridibunda . After the end of January, however, the enzyme activityshowed a gradual increase in the heart.The pattern of changes in the levels of   L  -lactate in the tissues of hibernated  R. ridibunda  followed thoseobserved for the  L  -LDH (Fig.4).The data obtained after cold acclimation of   R. ridibunda  showedthat acute changes in ambient temperature affected some glycolyticenzymes differently depending on the tissues from which they wereextracted.ExceptforHKand L  -LDH,theactivitiesoftheotherenzymesstudied increased in the heart of   R. ridibunda  during cold acclimation.The activity of HK maintained at the same levels, while that of   L  -LDHdecreased signi 󿬁 cantly within the  󿬁 rst 2 days of cold acclimation. Incontrast to the heart, the activities of all glycolytic enzymes examineddecreased in the skeletal muscle by the 15 day of cold acclimation(Fig. 6).  3.2. H. lucorum The changes in Po 2  in the haemolymph of hibernating  H. lucorum are shown in Fig. 1. The mean Po 2  of   H. lucorum  haemolymphdecreased by about 70% by the beginning of December 2006.Thereafter it remained at lowlevels until the 󿬁 rst days of March 2007.Changes in the activities of glycolytic enzymes in the tissues of  H. lucorum  are shown in Fig. 3. The pattern of changes in the activities Fig.1.  Effects of hibernation on the Po 2  in the haemolymph of   H. lucorum  and blood of  R. ridibunda . Values are given as means±S.E.M.,  N  =15 determinations on separatepreparations from different animals. Asterisks indicate the values, which aresigni 󿬁 cantly different from the control value (prehibernation animals sampled at thebeginning of November 2006);  ⁎  p b 0.05. Ambient temperature is given on the right  y  axis.584  B. Michaelidis et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 582 – 589  of glycolytic enzymes examined indicated that hibernation had adifferential effect on each enzyme. As shown, the activity of HKwas  󿬂 uctuating in the tissues of   H. lucorum  during hibernation. Onthe contrary, the activity of PFK showed a gradual increase ashibernation proceeded and remained at higher levels than those of the control by the  󿬁 rst days of March. Similar to PFK, the activity of aldolase increased in the foot muscle and mantle of hibernating H. lucorum . The pattern of changes in the activity of PGK was similarto those observed in aldolase activity in all tissues studied. The ac-tivity of PK, after a gradual increase in the tissues of hibernating H.lucorum bythemiddleofJanuarythereafterdecreasedandreturnedto control levels. Similar to HK, the activity of   D -LDH  󿬂 uctuated in thetissues of   H. lucorum  during hibernation. Nevertheless, the activity of the enzyme increased and remained at higher levels than those of thecontrol.The pattern of changes in the levels of   D -lactate in the tissuesof hibernated  H. lucorum  followed those observed for the  D -LDH(Fig. 4). Fig.2. Effectsofhibernationontheactivityofglycolyticenzymesinthetissuesof  R. ridibunda .Valuesaregivenasmeans±S.E.M., N  =15determinationsonseparatepreparationsfromdifferent animals. Asterisks indicate the values, which are signi 󿬁 cantly different from the control value (prehibernation animals sampled at the beginning of November 2006); ⁎  p b 0.05. Ambient temperature is given on the right  y  axis.585 B. Michaelidis et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 582 – 589  Thedataobtainedaftercoldacclimationof   H. lucorum showedthatacutechangesintheambienttemperaturecorrelatedwithanincreasein the activities of glycolytic enzymes (Fig. 5). 4. Discussion 4.1. Glycolytic adjustments in hiberating R. ridibunda In Greece, the frog  R. ridibunda  usually enters hibernation in thesecond half of November and its hibernation period lasts about100 days (Kyriakopoulou-Sklavounou and Katoulas, 1990). Manyaquatic ectothermic vertebrates respond to the rigors of the over-wintering environment (Bradford, 1983; Pinder et al., 1992) byentering into quiescent states, which are referred to as dormancy,torpor,orhibernation.ThedropinPo 2 inthebloodofhibernatedfrogs(Fig. 1) indicated lower oxygen consumption and a decrease in theirmetabolic rate. Metabolic depression in frogs hibernating underhypoxic conditions is accompanied by a signi 󿬁 cant decrease in theaerobiccapacityof theirskeletalmuscle(DonohoeandBoutilier,1998;St-Pierre and Boutilier, 2001), as indicated by the reduction in the Fig.3.  Effects of hibernation on the activityof glycolytic enzymes in the tissues of   H. lucorum .Values aregiven as means±S.E.M.,  N  =15 determinations on separate preparations fromdifferent animals. Asterisks indicate the values, which are signi 󿬁 cantly different from the control value (prehibernation animals sampled at the beginning of November 2006); ⁎  p b 0.05. Ambient temperature is given on the right  y  axis.586  B. Michaelidis et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 582 – 589

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