Low levels of extracellular glucose limit cardiac anaerobic metabolism in some species of fish

Transcription

1 17. Pulished y The Compny of iologists Ltd Journl of Experimentl iology (17), doi:1.14/je RESEARCH ARTICLE Low levels of extrcellulr glucose limit crdic neroic metolism in some species of fish Kthy A. Clow, Connie E. Short nd Willim R. Driedzic* ASTRACT There is wide interspecific rnge in plsm glucose levels in teleosts from less thn.5 to greter thn 1 mmol l 1. Here we ssessed how glucose vilility influences glucose metolism in herts of Atlntic cod (Gdus morhu), rinow trout (Oncorhynchus mykiss), lumpfish (Cyclopterus lumpus) nd short-horned sculpin (Myoxocephlus scorpius) under normoxic nd hypoxic conditions. These species hd plsm glucose levels of 5.1, 4.8,.9 nd.5 mmol l 1, respectively. Rtes of glucose metolism nd lctte production were determined in isolted herts perfused with medium contining physiologicl levels of glucose. Under normoxic conditions there ws no significnt difference in rtes of either glucose metolism (verge 15 nmol g 1 min 1 ) or lctte production (verge 3 nmol g 1 min 1 ) cross species. Under hypoxi (1% of ir sturtion) there were significnt increses in rtes of glucose metolism nd lctte production in herts from Atlntic cod (glucose-13; lctte-663 nmol g 1 min 1 ) nd rinow trout (glucose-13; lctte-774 nmol g 1 min 1 ); however, there ws no chnge in rte of glucose metolism in herts from either lumpfish or short-horned sculpin nd only increses in lctte production to rtes much lower thn the other species. Furthermore, Atlntic cod herts perfused with medium contining low nonphysiologicl levels of glucose (.5 mmol l 1 ) hd the sme rtes of glucose metolism under normoxic nd hypoxic tretments. Aneroic metolism supported y extrcellulr glucose is compromised in fish with low levels of plsm glucose, which in turn my decrese performnce under oxygen-limiting conditions t the whole-niml level. KEY WORDS: Aneroic metolism, Glycolysis, Fish herts, Lctte production INTRODUCTION There is wide interspecific rnge in the level of lood glucose mongst teleost fishes. Some species exhiit levels in excess of 1 mmol l 1 while other species hve extremely low levels y verterte stndrds, often elow 1 mmol l 1 (Chvin nd Young, 197; Polkof et l., 1). How interspecific vriility influences the utiliztion of glucose y different tissues is only now eing ddressed in rigorous fshion. Fish red lood cells (RCs) hve mitochondri nd predominntly eroic respirtion (see Driedzic et l., 13 for references). The rte of glucose metolism ws determined in RCs Deprtment of Ocen Sciences, Ocen Sciences Centre, Memoril University of Newfoundlnd, St. John s, NL A1C 5S7, Cnd. *Author for correspondence (wdriedzic@mun.c) W.R.D., Received 16 Mrch 17; Accepted June 17 from Atlntic cod (Gdus morhu), Atlntic slmon (Slmo slr), cunner (Tutogolrus dspersus) nd short-horned sculpin (Myoxocephlus scorpius) tht hd plsm glucose levels of 4, 4,, nd.7 mmol l 1, respectively (Driedzic et l., 13). At physiologicl levels of extrcellulr glucose, the rte of glucose metolism ws significntly higher in RCs from Atlntic cod thn either cunner or short-horned sculpin, with Atlntic slmon RCs eing intermedite etween Atlntic cod nd the other two species (Driedzic et l., 13). A follow-up study involved Atlntic cod (plsm glucose, 3 mmol l 1 ) nd short-horned sculpin (plsm glucose,.3 mmol l 1 ) (Driedzic et l., 14). At physiologicl levels of extrcellulr glucose, the rte of oxygen consumption y RCs ws similr etween species, ut the rte of glucose metolism y Atlntic cod RCs ws sixfold higher thn in short-horned sculpin RCs. The shortfll in metolic fuel is likely mde up y the ctolism of on-ord mino cids (Driedzic et l., 14). These experiments were extended to isolted myocytes tht hve rtes of oxygen consumption.5- to 3-fold higher thn RCs. As with RCs, t physiologicl levels of extrcellulr glucose there ws no difference in the rte of oxygen consumption of isolted hert cells, ut the rte of glucose metolism ws fourfold higher in Atlntic cod thn shorthorned sculpin. In isolted hert preprtions, oxygen consumption nd power development re likely mintined y endogenous triglycerides in the sence of exogenous metolic fuels (Driedzic nd Hrt, 1984; Clow et l., 4, 16). Collectively, these dt suggest tht in species comprisons of RCs nd hert cells, operting t sl or su-sl levels of metolism under normoxic conditions, the higher the level of extrcellulr glucose the higher the rte of utiliztion of glucose. The present study ssesses the ove puttive reltionship in perfused herts from vrious species. Contrcting, whole herts were utilized in order to increse the level of energy demnd nd to provide more physiologicl setting thn isolted cells. In ddition to Atlntic cod (G. morhu Linneus 1758) nd short-horned sculpin [M. scorpius (Linneus 1758)], the experimentl mtrix included rinow trout [Oncorhynchus mykiss (Wlum 179)] nd lumpfish (Cyclopterus lumpus Linneus 1758), which hve plsm glucose levels of pproximtely 5 nd 1 mmol l 1, respectively. Furthermore, rtes of glucose metolism nd lctte production were ssessed under oth normoxic nd hypoxic conditions, s it is well recognised tht oxygen limittion increses rtes of neroic metolism in fish herts (Gmperl nd Driedzic, 9). In perfused herts from rinow trout receiving only trce mounts of rdiolelled glucose in the medium, cynide tretment resulted in incresed utiliztion of glucose (West et l., 1993). In ddition, hypoxi led to n increse in uptke of the nonmetolizle glucose nlog -deoxyglucose y isolted hert preprtions from Americn eel (Anguill rostrt) (Rodnick et l., 1997) nd Atlntic cod (Clow et l., 4). The impct of hypoxi on the rte of metolism of glucose t physiologiclly relevnt levels of glucose hs never een directly ssessed. Here we test the Journl of Experimentl iology 97

2 Journl of Experimentl iology (17), doi:1.14/je hypothesis tht under normoxic nd hypoxic conditions, species with high physiologicl levels of glucose hve higher rtes of glucose metolism thn species with low extrcellulr glucose levels. Rtes of glucose metolism were ssessed y the production of 3 H from [- 3 H] glucose (Driedzic et l., 14; Clow et l., 16). The most importnt finding is tht glucose metolism is compromised under hypoxi t low levels of extrcellulr glucose within the physiologicl rnge for some species. MATERIALS AND METHODS Animl collection nd husndry Atlntic cod, rinow trout, lumpfish nd short-horned sculpin were mintined in running sewter t temperture of 8 1 C nd kept on nturl photoperiod with fluorescent lights set y n outdoor photocell. Atlntic cod nd lumpfish were htchery rered t the Ocen Sciences Centre (OSC). Rinow trout were otined from the Mrine Institute of Memoril University of Newfoundlnd (MUN) nd trnsferred to the OSC. Short-horned sculpin were collected loclly s required y the field services unit (OSC) during the summer period. Atlntic cod, rinow trout nd lumpfish received Optiline Slmonid Feed (Skretting, St Andrews, N, Cnd) every dy. Short-horned sculpin were fed to stition weekly with chopped herring. ody msses nd smple sizes were s follows: Atlntic cod (98±68 g; N=3), rinow trout (565±8 g; N=1), shorthorned sculpin (86±78 g; N=) nd lumpfish (93±16 g; N=1). Reltive ventricle msses [expressed s % ventricle mss (g)/ody mss (g)] were s follows: Atlntic cod (.8±.3), rinow trout (.7±.3), short-horned sculpin (.7±.4) nd lumpfish (.6±.4). lood ws collected for glucose nlysis from the cudl vessel with heprinized syringe prior to removl of the hert. lood ws centrifuged t 15 g for 5 min nd plsm ws frozen for future nlysis. Animl protocols were pproved y the Animl Cre Committee t the Memoril University of Newfoundlnd. Hert perfusions The sic perfusion/incution medium consisted of (in mmol l 1 ): 18 NCl, 5 KCl, 1.99 MgSO 4,.3 CCl, 7.33 TES se,.58 TES cid nd vrious concentrtions of glucose (ph 7.8 t 8 C). The hert perfusion pprtus ws similr to tht descried in Clow et l. (4). The system ws designed to recirculte 1 ml of perfusion medium t temperture of 8 C. Temperture of the input chmer ws mintined using recirculting wter th. A metl cnnul ttched to the input chmer ws plced just ove the hert, llowing the perfuste to fill the hert y grvity. The input cnnul ws inserted into the trium nd the hert ws perfused without recircultion for 3 s to llow wshout of lood. The ort ws then cnnulted with PE tuing in order to effectively collect the fluid into grduted cylinder for recircultion. The fluid in the cylinder ws pumped ck to the input chmer. Herts were electriclly pced using Grss Model SD9 squre wve genertor set t 5 V nd ms durtion. One electrode ws fused to the input cnnul nd second electrode, connected to smll hook, ws inserted into the ventricle. The contrction rte ws set to 3 35 ets min 1, typicl for mrine teleosts t the temperture of this study, nd only those herts tht showed visile contrctions were used in nlysis. Herts were perfused for 5 to 1 min with sic medium in order to llow contrction to stilize. Therefter, herts were perfused with recirculting medium contining [- 3 H]-glucose (7.4 kq; Americn Rdioleled Chemicls, urny, Cnd). Experimentl protocols Preliminry experiments were conducted with herts isolted from two Atlntic cod to ssess whether the preprtion ws responsive to chnge in oxygen level in the medium. In these studies, perfusion ws initited with medium gssed with ir nd fter 45 min of perfusion the gs ws switched to 1% nitrogen, resulting in drop of oxygen level. Oxygen redings were tken using fire-optic oxygen meter (model OXY-4 mini) nd preclirted dipping proe (PreSens). Rtes of glucose metolism nd lctte production were monitored for 1 min y collecting 15 µl of medium every 15 min for 6 min nd every 3 min therefter. Following the perfusion, herts were frozen nd lter nlyzed for lctte nd glycogen content. In the min series of experiments, one group of herts, herefter referred to s normoxic, were perfused under conditions of constnt ertion (oxygen level, 95±.7% ir sturtion, N=8). In prllel group, herts were initilly perfused with erted medium nd, once vigorous contrction ws oserved, switched to medium tht ws gssed continuously with nitrogen. This group is considered to e hypoxic (oxygen level, 1±.6% ir sturtion, N=3). All experiments lsted for 18 min. Smples for glucose metolism nd lctte production were collected s descried in the preliminry experiment. Experiments were conducted with ll four species with medium contining glucose t level in the physiologicl rnge. Levels were 5. mmol l 1 for Atlntic cod nd rinow trout, 1 mmol l 1 for lumpfish nd.5 mmol l 1 for short-horned sculpin. A further series of experiments ws conducted with Atlntic cod t non-physiologiclly low level of.5 mmol l 1 glucose nd with short-horned sculpin t non-physiologiclly high level of 5. mmol l 1 glucose. iochemicl nlysis The metolism of glucose ws determined y mesuring the rte of 3 H O production tht is formed from [- 3 H]-glucose vi wter exchnge in the rection ctlyzed y phosphoglucose isomerse (Hutton, 197; Driedzic et l., 13). A volume of 1 µl of smple medium t time, 15, 3, 45, 6, 9, 1 or 18 min ws dded to 1 ml of resin in polystyrene columns nd four 1 ml wter frctions contining the 3 H O were collected in scintilltion vils nd counted. Lctte ws mesured in the remining perfuste t ech time point. The medium ws deproteinized 1:1 with 6% PCA nd 5 5 μl of extrct ws dded to μl ssy medium contining glycine uffer (Sigm-Aldrich, G5418) nd.5 mmol l 1 NAD +, ph 9. t room temperture. Smples were red fter 1 min efore dding 3 IU ml 1 lctte dehydrogense. Asornces were red for nother 6 min or until stle. All sornces were determined with DTX 88 microplte reder (eckmn Coulter, Mississug, ON, Cnd). Lctte ws mesured in the hert y homogenizing the muscle in 6% PCA nd centrifuging t 1, g. The superntnt ws mesured s descried ove. Glycogen ws mesured in herts sed upon procedure modified from Keppler nd Decker (1974) using myloglucosidse (EC 3.1.3) to hydrolyze glycogen. riefly, herts were homogenized nd deproteinized in 6% PCA, centrifuged nd neutrlized with mol l 1 potssium icronte. The neutrlized homogente ws dded to 56 IU ml 1 myloglucosidse in.4 mol l 1 cette uffer, ph 4.8 nd heted t 4 C. Hydrolysis ws stopped fter h y dding 1% PCA. Glucose from these smples ws mesured s descried y Clow et l. (4). Free glucose from these smples without hydrolysis ws lso mesured nd sutrcted from the glucose produced from glycogen. Plsm glucose ws lso ssyed s descried y Clow et l. (4). Journl of Experimentl iology 971

3 Journl of Experimentl iology (17), doi:1.14/je Dt nlysis Vlues re expressed s mens±s.e.m. Sttisticl nlyses pplied re stted in the legends to figures. In ll cses, P.5 ws considered to e significnt. In the min experiment, rtes of glucose metolism nd lctte production were clculted from the initil liner rtes. Of the 5 dt sets presented, ll ut two (shorthorned sculpin, hert glycogen, normoxi; rinow trout, glucose metolism, normoxi) pssed normlity test. Given the roustness of ANOVA tests, we elected to ccept these nomlies in the nlyses. RESULTS As nticipted, plsm glucose levels were sustntilly nd significntly higher in Atlntic cod (5.1±.4 mmol l 1 ) nd rinow trout (4.8±.4 mmol l 1 ) thn in lumpfish (.89±.7 mmol l 1 ) or short-horned sculpin (.5±.9 mmol l 1 ). There ws no significnt difference in plsm glucose levels etween Atlntic cod nd rinow trout, or etween lumpfish nd short-horned sculpin (Fig. 1). Preliminry experiments were conducted to ssess whether herts responded to chnge in oxygen level in the perfusion medium (Fig. ). In the cse of the experiment with Atlntic cod specimen 1, the initil level of oxygen ws 9% ir sturtion. The medium ws gssed with N fter 45 min of perfusion, nd t the end of the experiment (1 min) the oxygen level ws 7% of ir sturtion. The slopes of ritrrily selected liner portions of the dt sets were clculted. The initil rte of glucose metolism ws 8.33 nmol g 1 min 1, which incresed to 79.1 nmol g 1 min 1 for the perfusion period etween 1 nd 1 min. Lctte production, determined from lctte ccumultion in the medium, followed similr pttern: the initil rte ws 4.4 nmol g 1 min 1 nd this incresed to 37 nmol g 1 min 1. The experiment with Atlntic cod specimen lso showed rte increses, lthough the liner portions of the curves were shifted. In this cse, the initil oxygen level ws 8% nd the finl level ws 14% of ir sturtion. The rte of glucose metolism incresed from n initil level of 3.3 nmol g 1 min 1 to 18 nmol g 1 min 1 for the period etween 6 nd 1 min. The rte of lctte production incresed from 3. to 397 nmol g 1 min 1. Although limited, the dt clerly show tht herts re responsive to decrese in oxygen vilility. The trnsition to hypoxic environment led to n verge sevenfold increse in glucose metolism tht ws ccompnied y n verge increse in lctte production y pproximtely 1-fold. These preliminry studies lso illustrte, t lest in the cse of Atlntic cod Glucose (mmol l 1 ) 6 4 Cod Trout Lumpfish Sculpin Fig. 1. Plsm glucose in Atlntic cod, rinow trout, lumpfish nd shorthorned sculpin. Dt re mens+s.e.m. N=1 for Atlntic cod; N=1 for rinow trout; N=11 for lumpfish; N= for short-horned sculpin. Dt were nlyzed with one-wy ANOVA. Vlues shring common letter re not significntly different. herts perfused with 5 mmol l 1 glucose in the medium, tht the rte of increse in the use of exogenous glucose under hypoxi is not sufficient to support lctte production. The reson for the vriility etween preprtions is not known ut could e relted to the time course of the decrese in oxygen level in the medium. In order to control for this, ll further experiments were conducted with individul herts exposed to either normoxic or hypoxic conditions from time zero. In the min series of experiments, ll hert preprtions were perfused for 18 min. Lctte nd glycogen levels following perfusion were determined in ll herts. Given tht we were only interested in ssessing whether hypoxi resulted in chnge in lctte or glycogen level, within species t specific level of extrcellulr glucose, the dt were nlyzed with t-test. There ws no significnt difference in hert lctte etween herts perfused under normoxic or hypoxic conditions for ny species under conditions of 5, 1 or.5 mmol l 1 glucose in the medium (Fig. 3A). ecuse lctte levels in the hert do not chnge, this mens tht ny lctte produced is relesed to the medium. Glycogen levels were significntly lower in herts from Atlntic cod perfused with 5 mmol l 1 in the medium under hypoxic thn normoxic conditions (Fig. 3). A similr decrese ws evident for herts from rinow trout. There ws no significnt difference etween normoxic nd hypoxic tretments for either lumpfish or short-horned sculpin. We cution, though, tht given the high individul vriility within short-horned sculpin herts, it would e difficult to revel ny chnge owing to tretment with the smll smple size. The reltionship etween glucose metolism or lctte production nd time for the four species studied is presented in Figs 4 7. The rw dt lone suggest numer of qulittive findings. Foremost, rtes of glucose metolism pper to e higher under hypoxic thn normoxic conditions for Atlntic cod (Fig. 4A,C) nd rinow trout (Fig. 5A), ut not lumpfish (Fig. 6A) or short-horned sculpin (Fig. 7A,C). Rtes of lctte production pper to e higher for Atlntic cod (Fig. 4,D) nd rinow trout (Fig. 5) under hypoxi thn normoxi. The rw dt lso revel tht in some cses glucose metolism (Fig. 4C) nd in mny cses lctte production (Figs 4,D, 5, 7,D) pper to lose linerity with respect to time nd plteus. On the sis of this finding, rtes of glucose metolism nd lctte production were clculted for the initil liner portion of the curves for ech individul experiment. The experiment designed to ssess rtes of glucose metolism nd lctte production t physiologicl levels of extrcellulr glucose involved eight conditions with different species, oxygen levels or extrcellulr glucose levels (Fig. 8). The dt were nlyzed with two-wy ANOVA including ll conditions, ut the figures re prtitioned out to etter visulize the relevnt physiologicl comprisons consistent with the hypotheses eing tested. Under conditions of normoxi nd physiologicl levels of glucose, there ws no difference in rtes of either glucose metolism or lctte production cross species (Fig. 8A,). The pttern chnged under hypoxi, where rtes of glucose metolism nd lctte production y herts from Atlntic cod nd rinow trout were significntly higher thn those y herts from lumpfish nd short-horned sculpin (Fig. 8C,D). Although of similr mgnitude, the rte of glucose metolism ws significntly higher in Atlntic cod thn rinow trout herts. There ws no difference in rtes of lctte production y herts from these two species. Lumpfish nd short-horned sculpin herts exhiited similr rtes of oth glucose metolism nd lctte production under hypoxi. Rtes of glucose metolism nd lctte production etween normoxic nd hypoxic Journl of Experimentl iology 97

4 Journl of Experimentl iology (17), doi:1.14/je Glucose metolism (µmol g 1 ) A N C 1 Lctte production (µmol g 1 ) N D 5 N N Time (min) Fig.. Glucose metolism nd lctte production y isolted herts from Atlntic cod following the trnsition from normoxic to hypoxic medium. Individul experiments (A,: specimen 1; C,D: specimen ) show the chnge in rtes of glucose metolism (A,C) nd lctte production (,D) following the trnsition from normoxic to hypoxic condition. Glucose metolism ws mesured s the ccumultion of 3 H O in medium contining 3 H-glucose. Liner regressions were ritrrily drwn to illustrte rte chnges ssocited with gssing the medium with N fter 45 min of perfusion. conditions my e ssessed y compring Fig. 8A with C nd with D. These rtes were significntly higher under hypoxic thn normoxic conditions for herts from oth Atlntic cod nd rinow trout. There ws no significnt difference for either glucose metolism or lctte production for lumpfish nd short-horned sculpin herts etween normoxic nd hypoxic conditions. However, the verge vlues for lctte production were pproximtely twofold higher for lumpfish nd 17-fold higher for short-horned sculpin under hypoxi thn normoxi, respectively. The ove dt suggest tht herts from Atlntic cod nd rinow trout respond to hypoxi with n increse in glucose metolism nd lctte production, ut lumpfish nd shorthorned sculpin herts hve no cpcity to increse glucose metolism under hypoxi nd t the very est limited cpcity to increse lctte production. This could e ecuse of the low extrcellulr glucose or other properties inherent to the hert. In n ttempt to tese out these possiilities, herts were perfused with medium contining non-physiologicl levels of extrcellulr glucose. More specificlly, Atlntic cod herts received.5 mmol l 1 glucose nd short-horned sculpin herts 5. mmol l 1 glucose in the medium. Rtes of glucose metolism nd lctte production re presented in Fig. 9 nd for comprtive Lctte (µmol g 1 ) 3 A Glucosyl units (µmol g 1 ) Cod Trout Lumpfish Sculpin Cod Trout Lumpfish Sculpin * * 5..5 Fig. 3. Hert lctte nd glycogen levels following 18 min of perfusion in Atlntic cod, rinow trout, lumpfish nd short-horned sculpin. (A) Lctte; () glycosyl units. Open rs, normoxic; solid rs, hypoxic. Vlues ove columns indicte the level of glucose (mmol l 1 ) in the medium. N=5 for ll conditions except Atlntic cod hypoxic (5 mmol l 1 ) nd trout hypoxic, where N=6. *Significnt difference etween hypoxic nd normoxic conditions when compred with Student s t-test (P<.5). 973 Journl of Experimentl iology

5 Journl of Experimentl iology (17), doi:1.14/je Glucose metolism (µmol g 1 ) 5 A 5 mmol l 1 glucose C.5 mmol l 1 glucose 1 Lctte production (µmol g 1 ) mmol l 1 glucose D.5 mmol l 1 glucose Time (min) Fig. 4. Glucose metolism nd lctte production y isolted herts from Atlntic cod under normoxic or hypoxic conditions. Herts were perfused with medium contining either 5 mmol l 1 (A,) or.5 mmol l 1 (C,D) extrcellulr glucose. (A,C) Glucose metolism. (,D) Lctte production. Circles, normoxi; squres, hypoxi. N=5 for ll experiments, except for A, where N=6 for oth conditions. purposes vlues t physiologicl levels of extrcellulr glucose re lso included. For Atlntic cod, there ws no significnt difference in rtes of glucose metolism under normoxic conditions etween conditions of.5 nd 5. mmol l 1 glucose in the medium, nor etween normoxi nd hypoxi t.5 mmol l 1 glucose (Fig. 9A). Lctte production ws significntly higher in Atlntic cod herts perfused with hypoxic thn normoxic medium with.5 mmol l 1 glucose in the medium (Fig. 9). For short-horned sculpin herts there ws no difference in rte of glucose metolism etween normoxi nd hypoxi with either 5. or.5 mmol l 1 glucose in the medium (Fig. 9C). The rte of glucose metolism ws significntly higher (pproximtely threefold) t 5. thn t.5 mmol l 1 glucose under normoxic conditions. Lctte production ws significntly higher under hypoxi thn normoxi t oth levels of extrcellulr glucose. DISCUSSION Experimentl pproch The rnge of plsm glucose for the four species under study ws s nticipted, with Atlntic cod nd rinow trout t pproximtely 5 mmol l 1, which is in the window for most teleosts, typiclly rnging from to 8 mmol l 1. Lumpfish nd short-horned sculpin hd similr levels of plsm glucose t less thn 1 mmol l 1. This is extremely low even y fish stndrds (Chvin nd Young, 197; Polkof et l., 1). As such, the selection of species ws suitle to ssess whether low plsm glucose compromised glucose utiliztion. Isolted, perfused herts were used s the experimentl preprtion. Herts received oxygen nd glucose directly from the medium eing pumped y the trium nd ventricle, s is the norml physiologicl condition for these herts tht hve either no or only poorly developed coronry rteries (Driedzic nd Gesser, 1994). Glucose metolism (µmol g 1 ) A Time (min) Fig. 5. Glucose metolism nd lctte production y isolted herts from rinow trout under normoxic or hypoxic conditions. Herts were perfused with medi contining 5 mmol l 1 extrcellulr glucose. (A) Glucose metolism. () Lctte production. Circles, normoxi; squres, hypoxi. N=6 for ll experiments. 974 Lctte production (µmol g 1 ) Journl of Experimentl iology

6 Journl of Experimentl iology (17), doi:1.14/je Glucose metolism (µmol g 1 ). A Lctte production (µmol g 1 ) Time (min) Fig. 6. Glucose metolism nd lctte production y isolted herts from lumpfish under normoxic or hypoxic conditions. Herts were perfused with medi contining 1 mmol l 1 extrcellulr glucose. (A) Glucose metolism. () Lctte production. Circles, normoxi; squres, hypoxi. N=5 for ll experiments. Herts were filled from pressure hed nd emptied ginst tmospheric pressure. Thus the level of mechnicl work, which is primry determinnt of energy demnd, ws similr for ll species nd considered to e su-sl. Herts contrcted during the tril period, lthough it ppered tht the strength of contrction decresed in some preprtions, especilly under hypoxic conditions, s evidenced y the force t which the perfuste ws expelled. The input oxygen level ws 1% of ir sturtion. sed on mesurements in myogloin-contining herts, oxygen consumption y Atlntic cod, rinow trout nd short-horned sculpin herts should e pproximtely % of normoxi, wheres myogloin-devoid lumpfish herts should e neroic (iley et l., 199). Glucose metolism ws determined y the rte of 8 A 6 5 mmol l 1 glucose 3 H O production from [- 3 H]-glucose. The virtue of this technique is tht the specific ctivity of extrcellulr glucose is close to tht of [- 3 H]-glucose 6-phosphte, the sustrte tht produces 3 H O (Neely et l., 197). In preliminry experiments, herts from Atlntic cod responded to decrese in oxygen level with n increse in rte of 3 H O production. Therefter, in experiments tht pplied normoxic or hypoxic conditions from time zero there ws liner rte of production of 3 H O tht lsted for 18 min under normoxi nd t lest 1 min under hypoxi in most preprtions. Rtes of glucose metolism were clculted from the initil liner period. Rtes of glucose metolism in Atlntic cod nd shorthorned sculpin were pproximtely twofold higher thn in isolted myocytes (Clow et l., 16). In summry, the experimentl mmol l 1 glucose 4 4 Glucose metolism (µmol g 1 ) C mmol l 1 glucose Lctte production (µmol g 1 ) D Time (min).5 mol l 1 glucose Fig. 7. Glucose metolism nd lctte production y isolted herts from short-horned sculpin under normoxic or hypoxic conditions. Herts were perfused with medi contining either 5 mmol l 1 (A,) or.5 mmol l 1 (C,D) extrcellulr glucose. (A,C) Glucose metolism. (,D) Lctte production. Circles, normoxi; squres, hypoxi. N=5 for ll experiments. 975 Journl of Experimentl iology

7 Journl of Experimentl iology (17), doi:1.14/je A Glucose metolism (nmol g 1 min 1 ) Cod Trout Lumpfish Sculpin Cod Trout Lumpfish C, *, * Lctte production (nmol g 1 min 1 ) D, 1 *, * Sculpin c c Cod Trout Lumpfish Sculpin Cod Trout Lumpfish Sculpin Fig. 8. Rtes of glucose metolism nd lctte production y isolted fish herts t physiologicl levels of extrcellulr glucose. (A,C) Glucose metolism. (,D) Lctte production. (A,) Normoxic conditions with physiologicl levels of extrcellulr glucose in the medium (Atlntic cod, 5 mmol l 1 ; rinow trout, 5 mmol l 1 ; lumpfish, 1 mmol l 1 ; short-horned sculpin,.5 mmol l 1 ). (C,D) Hypoxic conditions with physiologicl levels of extrcellulr glucose in the medium. Open rs, normoxi; closed rs, hypoxi. Dt for the eight experimentl conditions were compred with two-wy ANOVA followed y onferroni post hoc test. The dt re prtitioned to present physiologicl meningful comprisons. Vlues shring common letter within pnel re not significntly different. *Significnt difference within species etween normoxi nd hypoxi (A versus C or versus D; P<.5). Smple sizes re given in the legends to Figs 4 7. pproch used here of providing [- 3 H]-glucose to isolted herts ppers to e quite suitle to ddress the issue of species-specific rtes of glucose metolism under normoxic nd hypoxic conditions given the viility of the preprtion, the similrity of mechnicl work for ll species, the responsiveness to chnge in oxygen level, the linerity of 3 H O production, nd higher rtes of metolism compred with isolted myocytes. Metolism under normoxi There ws no significnt difference cross species in rtes of glucose metolism when considered t physiologicl levels of extrcellulr glucose. Under normoxi nd physiologicl levels of glucose, the level of extrcellulr glucose is not primry determinnt of glucose utiliztion, t lest in herts working t su-sl levels of energy demnd. Tht is, lumpfish nd short-horned sculpin operting with 1. nd.5 mmol l 1 extrcellulr glucose, respectively, re s effective in tking up nd metolizing glucose s Atlntic cod nd rinow trout with n vilility of 5 mmol l 1 extrcellulr glucose. Within Atlntic cod, the rte of glucose metolism ws the sme t oth 5 nd.5 mmol l 1 extrcellulr glucose, consistent with the interprettion tht extrcellulr glucose is not n importnt fctor in setting the rte of glucose metolism under normoxic susl working conditions. In short-horned sculpin herts there ws threefold increse in rte of glucose metolism etween.5 nd 5 mmol l 1 extrcellulr glucose. Although the tissue hs some cpcity to increse glucose metolism, this would not e chieved in vivo given tht extrcellulr glucose is never ny higher thn pproximtely.5 mmol l 1 (McCormck nd Driedzic, 7; Driedzic et l., 13, 14; Clow et l., 16). The current finding of sttisticlly similr rtes of glucose metolism in herts from Atlntic cod nd short-horned sculpin differs from results with isolted myocytes, where glucose metolism ws fourfold higher in Atlntic cod thn in shorthorned sculpin (Clow et l., 16). Contrctility pprently elevted glucose metolism reltively more in short-horned sculpin thn in Atlntic cod, thus eliminting the difference. In n erlier study with rinow trout herts, West et l. (1993), using the sme experimentl pproch s here, reported the sme rte of glucose metolism s currently oserved. In tht study, n increse in work from su-sl to resting levels of power output (1.75 mw g 1 ) resulted in fourfold increse in the rte of glucose metolism. It would e of interest to determine whether increses in power output to physiologicl levels re mtched with increses in glucose metolism in species with extremely low plsm glucose concentrtions. Under normoxic conditions, glycolysis leding to lctte production mkes only minor contriution, less thn 5%, to ATP turnover in perfused fish herts nd isolted myocytes (Driedzic et l., 1983; Arthur et l., 199; West et l., 1993; Lgue et l., 1; Clow et l., 16); however, low rtes of cytosolic genertion of ATP vi lctte production my e importnt in mintining resting tension (iley et l., ; Gesser, ). Journl of Experimentl iology 976

8 Journl of Experimentl iology (17), doi:1.14/je A Glucose metolism (nmol g 1 min 1 ) C , Lctte production (nmol g 1 min 1 ) D ,d c,d,c Glucose (mmol l 1 ) Fig. 9. Rtes of glucose metolism nd lctte production y isolted fish herts t physiologicl nd non-physiologicl levels of extrcellulr glucose. (A,) Atlntic cod. (C,D) Short-horned sculpin. (A,C) Glucose metolism. (,D) Lctte production. Dt for Atlntic cod t 5 mmol l 1 glucose nd short-horned sculpin t.5 mmol l 1 glucose re repeted from Fig. 8. Open rs, normoxi; closed rs, hypoxi. Dt within species were compred with one-wy ANOVA followed y Tukey post hoc test. Vlues shring common letter within pnel re not significntly different. Smple sizes re given in the legends to Figs 4 7. There ws no significnt difference in rtes of lctte production y herts from the four species in the present study, ut the high vriility should e noted. Agin, incresed crdic energy demnd might chnge this reltionship, s incresed work led to incresed rtes of lctte production in perfused herts from rinow trout (West et l., 1993) nd tilpi (Lgue et l., 1) ut not in se rven (Driedzic et l., 1983), scorpeniforme closely relted to short-horned sculpin. Metolism under hypoxi Glucose enhnces the hypoxic performnce of isolted hert preprtions nd lctte production is enhnced under hypoxi in oth hert preprtions nd t the whole-niml level (Gmperl nd Driedzic, 9; Lgue et l., 1; ecker et l., 13). However, the contriution of extrcellulr glucose to lctte production is poorly documented. In perfused rinow trout herts treted with cynide, the rte of glucose metolism nd lctte production incresed 4- nd -fold, respectively, reltive to control preprtions, implying tht the use of extrcellulr glucose ws insufficient to meet lctte production nd most likely the glycogen pool ws moilized (West et l., 1993). Experiments with rinow trout ventricle strips present different scenrio in tht severe hypoxi led to sixfold increse in lctte efflux ut there ws no chnge in the rte of uptke of -deoxyglucose (ecker et l., 13). Anlyzing differences in glucose trfficking etween these two experiments is eyond the scope of the present study, ut oth reports suggest moiliztion of the glycogen pool s ws shown in the current experiment. Crdic glycogen moiliztion, under oxygen limittion, hs lso een noted in preprtions from Americn eel (iley et l., ), Atlntic cod (Clow et l., 4) nd Pcific hgfish (Epttretus stoutii) (Gillis et l., 15). The prdigm tht emerges is tht under oxygen limittion, the fish hert metolizes on-ord glycogen nd extrcellulr glucose, which re utilized to produce lctte with the ssocited genertion of ATP tht, in turn, supports contrctility. However, none of the studies conducted to dte involve species with chroniclly low (<1 mol l 1 ) plsm glucose levels, such s lumpfish or shorthorned sculpin. The current experiments re the first to ssess glucose metolism under hypoxi in species with exceptionlly low plsm glucose levels y verterte stndrds. When considered t physiologicl levels of extrcellulr glucose, hypoxi resulted in increses in glucose metolism in herts from Atlntic cod (4.5-fold) nd trout (7.4-fold) ut no chnge in herts from lumpfish or short-horned sculpin. As consequence, under hypoxic chllenge the rte of glucose metolism ws higher in Atlntic cod nd rinow trout thn in lumpfish nd short-horned sculpin herts. Tht is, the species with high physiologicl levels of extrcellulr glucose hve greter cpcity to increse rtes of crdic glucose metolism under hypoxi. Low levels of extrcellulr glucose would result in low diffusion grdient from the extrcellulr to the intrcellulr spce nd therefore could limit glucose utiliztion. In Atlntic cod hert perfused with medium t the low level of extrcellulr glucose (.5 mmol l 1 ), hypoxi did not result in n increse in glucose metolism, finding tht supports the contention tht low extrcellulr glucose compromises the ility to increse glucose utiliztion. In herts from short- Journl of Experimentl iology 977

9 Journl of Experimentl iology (17), doi:1.14/je horned sculpin, non-physiologiclly high levels of glucose did not result in n enhnced cpcity to increse glucose metolism under hypoxi. For unknown resons, possily owing to reching the ceiling of trnsport or enzymtic cpcity, these herts simply do not hve the ility to ttin high rtes of glucose metolism, even when provided with extrcellulr glucose 1 times the norml physiologicl level. Rtes of lctte production responded in similr fshion to glucose metolism. At physiologicl levels of extrcellulr glucose, significnt increses in lctte production occurred under hypoxi in Atlntic cod (51-fold) nd rinow trout (18-fold) herts, ut not in herts from lumpfish nd short-horned sculpin. As with glucose metolism, under hypoxic chllenge the rte of lctte production ws higher in Atlntic cod nd rinow trout thn in lumpfish nd short-horned sculpin herts. Agin, species with high physiologicl levels of extrcellulr glucose hve greter cpcity to increse rtes of crdic lctte production under hypoxi. It should e noted tht lthough not significntly different when ssessed using two-wy ANOVA, involving only dt otined t physiologicl levels of extrcellulr glucose, the verge rte of lctte production y herts from short-horned sculpin ws 17-fold higher under hypoxi thn normoxi, so it my e tht this species hs some cpcity to increse lctte production, ut rtes would e less thn 1% of tht oserved in Atlntic cod or rinow trout. Enhnced rtes of lctte production occurred under hypoxi t oth 5 nd.5 mmol l 1 in Atlntic cod nd short-horned sculpin herts. Given tht glucose metolism ws not incresed under three of the four conditions, this finding suggests tht glycogen ws eing clled upon. Anlysis of dt for short-horned sculpin y one-wy ANOVA reveled significnt increse in lctte production under hypoxi, consistent with the suggestion ove tht herts from this species hve t lest limited cpcity for neroic glycolysis. The rtio of lctte production to glucose metolism provides further insight into the reltionship etween extrcellulr glucose metolism nd lctte production. At physiologicl levels of extrcellulr glucose, the lctte production:glucose metolism rtio for Atlntic cod, rinow trout, lumpfish nd short-horned sculpin ws 5.1, 7.5, 15 nd 3.4, respectively. A lctte production: glucose metolism rtio greter thn implies tht glycogen is eing moilized in ddition to extrcellulr glucose. The decrese in tissue glycogen in Atlntic cod nd rinow trout herts is consistent with this contention. It is likely tht lctte production y lumpfish nd short-horned sculpin herts is eing supported y glycogenolysis ut tht this is eing missed in the nlysis ecuse of the vriility in glycogen levels nd/or the low rte of lctte production reltive to the mount of glycogen. Conclusions It is generlly viewed tht in most fish species, hypoxi results in n increse in crdic neroic metolism involving oth glycogen nd glucose s metolic fuels to support lctte production (Gmperl nd Driedzic, 9). This scenrio is pulled together from rnge of studies ech contriuting prts of the story ut without one single experiment ctully mesuring rtes of glucose metolism under hypoxi. The current investigtion with isolted hert preprtions from Atlntic cod nd rinow trout supports the ccepted prdigm. Here it is shown tht in herts from these species receiving perfuste contining physiologicl level of extrcellulr glucose (5 mmol l 1 ), hypoxi leds to n increse in glucose metolism s ssessed y the rte of relese of 3 H O from [- 3 H]- glucose. As well, there ws concomitnt increse in the rte of lctte production nd glycogen utiliztion. Studies were lso conducted with lumpfish nd short-horned sculpin, which hve norml plsm glucose levels of less thn 1 mmol l 1. Herts from these species perfused with medium contining physiologicl levels of glucose, 1 mmol l 1 for lumpfish nd.5 mmol l 1 for shorthorned sculpin, hd rtes of glucose metolism similr to Atlntic cod nd rinow trout under normoxi ut filed to show ny increse in glucose metolism when sujected to hypoxi. Similrly, lthough the rte of lctte production ppers to e enhnced under hypoxi, rtes re still mny fold lower thn oserved in Atlntic cod nd rinow trout. These findings led to the conclusion tht crdic neroic metolism from plsm glucose is compromised in species with especilly low levels of plsm glucose. This is possily due to reduced glucose grdient from the extrcellulr to the intrcellulr spce. This conclusion is supported y the oservtion tht herts from Atlntic cod perfused with medium contining low concentrtion of glucose (.5 mmol l 1 ) did not show n incresed in glucose metolism. The position of limited rtes of glucose metolism is sed upon isolted herts performing similr levels of crdic work oth cross species nd under oth oxygen levels. As contrctility is the mjor determinnt of energy demnd, the next stge in the progression of this experimentl progrm should e to ssess rtes of glucose metolism t species-specific levels of pressure development nd frequency under normoxi nd hypoxic rdycrdi. The response of the isolted hert from short-horned sculpin mtches the whole-niml response. Short-horned sculpin sujected to hypoxi, t the limit of their tolernce, hd rtes of - deoxyglucose uptke nd lctte levels similr to those of control nimls. Aneroic metolism ws not ctivted; insted, hert rte nd crdic output were reduced, response tht would contriute to decrese in energy demnd (McCormck nd Driedzic, 7). The min messge of the present study is tht in some fish, low level of extrcellulr glucose compromises hert neroic energy production; therefore, hypoxic survivl of this tissue must depend on other fctors, such s decreses in contrctility, ion trnsport or protein turnover. In Europen se ss (Dicentrrchus lrx), whole-niml hypoxi tolernce is ssocited with the hypoxic performnce of ventricle strips (Joyce et l., 16). If hert hypoxi tolernce proves to e n importnt determinnt of whole-niml sensitivity to oxygen deprivtion, then the current findings of the consequences of low lood glucose on hert energy metolism ecome especilly importnt s mny mrine ecosystems re fcing oxygen reduction. Acknowledgements The uthors thnk Dr K. Gmperl for the use of instrumenttion to mesure oxygen consumption. We lso thnk the field services unit (Deprtment of Ocen Sciences) for the collection of short-horned sculpin, D. oyce (Deprtment of Ocen Sciences) for the supply of Atlntic cod nd lumpfish, nd Chris Dwe (Mrine Institute, Memoril University) for rinow trout. Competing interests The uthors declre no competing or finncil interests. Author contriutions Conceptuliztion: K.A.C., C.E.S., W.R.D.; Methodology: K.A.C., C.E.S., W.R.D.; Forml nlysis: K.A.C., C.E.S., W.R.D.; Investigtion: K.A.C., C.E.S.; Writing - originl drft: W.R.D.; Writing - review & editing: K.A.C., C.E.S., W.R.D.; Visuliztion: W.R.D.; Supervision: W.R.D.; Project dministrtion: W.R.D.; Funding cquisition: W.R.D. Funding This work ws supported y Nturl Sciences nd Engineering Reserch Council of Cnd Discovery Grnt nd the Reserch nd Development Corportion of Newfoundlnd nd Lrdor. W.R.D. holds the Cnd Reserch Chir (Tier 1) in Mrine ioscience. Journl of Experimentl iology 978

10 Journl of Experimentl iology (17), doi:1.14/je References Arthur, P. G., Keen, J. E., Hochchk, P. W. nd Frrell, A. P. (199). Metolic stte of the in situ perfused trout hert during severe hypoxi. Am. J. Physiol. 63, R798-R84. iley, J. R., Sephton, D. H. nd Driedzic, W. R. (199). Oxygen uptke y isolted perfused fish herts with differing myogloin concentrtions under hypoxic conditions. J. Mol. Cell. Crdiol., iley, J. R., rter, T. nd Driedzic, W. R. (). Mintennce of resting tension in the Americn eel (Anguill rostrt L.) hert is dependent upon exogenous fuel nd the srcoplsmic reticulum. J. Exp. Zool. 86, ecker, T. A., DellVlle,., Gesser, H. nd Rodnick, K. J. (13). Limited effects of exogenous glucose during severe hypoxi nd lck of hypoxi-stimulted glucose uptke in isolted rinow trout crdic muscle. J. Exp. iol. 16, Chvin, W. nd Young, J. E. (197). Fctors in the determintion of norml serum glucose levels of goldfish, Crssius urtus L. Comp. iochem. Physiol. 33, Clow, K. A., Rodnick, K. J., McCormck, T. J. nd Driedzic, W. R. (4). The regultion nd importnce of glucose uptke in the isolted Atlntic cod hert: rte-limiting steps nd effects of hypoxi. J. Exp. iol. 7, Clow, K. A., Short, C. E. nd Driedzic, W. R. (16). Extrcellulr glucose supports lctte production ut not eroic metolism in crdiomyocytes from oth normoglycemic Atlntic cod (Gdus morhu) nd low glycemic short-horned sculpin (Myoxocephlus scorpius). J. Exp. iol. 19, Driedzic, W. R. nd Gesser, H. (1994). Energy metolism nd contrctility in ectothermic verterte herts: hypoxi, cidosis, nd low temperture. Physiol. Rev. 74, Driedzic, W. R. nd Hrt, T. (1984). Reltionship etween exogenous fuel vilility nd performnce y teleost nd elsmornch herts. J. Comp. Physiol. 154, Driedzic, W. R., Scott, D. L. nd Frrell, A. P. (1983). Aeroic nd neroic contriutions to energy metolism in perfused isolted se rven (Hemitripterus mericnus) herts. Cn. J. Zool. 61, Driedzic, W. R., Clow, K. A. nd Short, C. E. (13). Glucose uptke nd metolism y red lood cells from fish with different extrcellulr glucose levels. J. Exp. iol. 16, Driedzic, W. R., Clow, K. A. nd Short, C. E. (14). Extrcellulr glucose cn fuel metolism in red lood cells from high glycemic Atlntic cod (Gdus morhu) ut not low glycemic short-horned sculpin (Myoxocephlus scorpius). J. Exp. iol. 17, Gmperl, A. K. nd Driedzic, W. R. (9). Crdiovsculr function nd crdic metolism during environmentl hypoxi. In Fish Physiology, Vol. 7 (ed. J.G. Richrds, A.P. Frrell nd C. J. runer), pp London: Elsevier. Gesser, H. (). Mechnicl performnce nd glycolytic requirement in trout ventriculr muscle. J. Exp. Zool. 93, Gillis, T. E., Regn, M. D., Cox, G. K., Hrter, T. S., runer, C. J., Richrds, J. G. nd Frrell, A. P. (15). Chrcterizing the metolic cpcity of the noxic hgfish hert. J. Exp. iol. 18, Hutton, J. J. (197). Rdiometric micromethod for quntittion of glucose utiliztion y the erythrocyte. Anl. iochem. 45, Joyce, W., Ozolin, K., Muduit, F., Ollivier, H., Clireux, G. nd Shiels, H. A. (16). Individul vrition in whole-niml hypoxi tolernce is ssocited with crdic hypoxi tolernce in mrine teleost. iol. Lett. 1, Keppler, D. nd Decker, D. (1974). Glycogen, determintion with myloglucosidse. In Methods of Enzymtic Anlysis, Vol. 3 (ed. H. U. ergmeyer), pp New York, NY: Acdemic Press. Lgue, S. L., Speers-Roesch,., Richrds, J. G. nd Frrell, A. P. (1). Exceptionl crdic noxi tolernce in tilpi (Oreochromis hyrid). J. Exp. iol. 15, McCormck, T. J. nd Driedzic, W. R. (7). The impct of hypoxi on in vivo glucose uptke in hypoglycemic fish, Myoxocephlus scorpius. Am. J. Physiol. 9, R133-R14. Neely, J. R., Denton, R. M., Englnd, P. J. nd Rndle, P. J. (197). The effects of incresed hert work on the tricroxylte cycle nd its interctions with glycolysis in the perfused rt hert. iochem. J. 18, Polkof, S., Pnsert, S., Soengs, J. L. nd Moon, T. W. (1). Glucose metolism in fish: review. J. Comp. Physiol. 18, Rodnick, K. J., iley, J. R., West, J. L., Rideout, A. nd Driedzic, W. R. (1997). Acute regultion of glucose uptke in crdic muscle of the Americn eel, Anguill rostrt. J. Exp. iol., West, T. G., Arthur, P. G., Surez, R. K., Doll, C. J. nd Hochchk, P. W. (1993). In vivo utiliztion of glucose y hert nd locomotory muscles of exercising rinow trout (Oncorhynchus mykiss). J. Exp. iol. 177, Journl of Experimentl iology 979