The Journl of Experimentl Biology 9, - Pulished y The Compny of Biologists doi:./je. Glycerol production in rinow smelt (Osmerus mordx) my e triggered y low temperture lone nd is ssocited with the ctivtion of glycerol--phosphte dehydrogense nd glycerol--phosphtse Willim R. Driedzic, *, Kthy A. Clow, Connie E. Short nd K. Vny Ewrt Ocen Sciences Centre, Memoril University of Newfoundlnd, St John s, Newfoundlnd, Cnd AC S7 nd NRC institute for Mrine Biosciences, Oxford Street, Hlifx, Nov Scoti, Cnd BH Z *Author for correspondence (e-mil: wdriedzic@mun.c) Accepted Jnury Rinow smelt (Osmerus mordx) ccumulte high levels of glycerol in winter tht serves s n ntifreeze. Fish were sujected to controlled decreses in wter temperture nd levels of plsm glycerol, liver metolites nd liver enzymes were determined in order to identify control mechnisms for the initition of glycerol synthesis. In two seprte experiments, decreses in temperture from C to C over period of dys resulted in increses in plsm glycerol from levels of less thn mmol l to pproximte men levels of (first experiment) nd mmol l (second experiment). In third experiment, decreses in temperture to C resulted in plsm glycerol levels pproching mmol l. The ccumultion of glycerol could e driven in either Decemer or Mrch, thus eliminting decresing photoperiod s necessry cue for glycerol ccumultion. Glycerol ccumultion in plsm ws ssocited with chnges in metolites in liver leding to increses in the mss ction rtio cross the rections Summry ctlyzed y glycerol--phosphte dehydrogense (GPDH) nd glycerol--phosphtse (GPse). The mximl, in vitro ctivity of GPDH, incresed twofold in ssocition with shrp increse in plsm glycerol level. The metolite levels nd enzyme ctivities provide complementry evidence tht GPDH is regultory site in the low temperture triggered synthesis of glycerol. Indirect evidence, sed on clculted rtes of in vivo glycerol production y liver, suggests tht GPse is potentil rte-limiting step. As well, trnsient increses in glycerldehyde--phosphte dehydrogense nd lnine minotrnsferse suggest tht these sites re components of suite of responses, in rinow smelt liver, induced y low temperture. Key words: rinow smelt, osmerus mordx, glycerol, glycerol-- phosphte dehydrogense, glycerol--phosphtse, low temperture, freeze resistnce. Introduction Rinow smelt (Osmerus mordx) is smll fish (up to cm in length) tht remins ctive nd forges for food in icy sewter during winter. Rinow smelt void freezing y producing n ntifreeze protein (Ewrt nd Fletcher, 99) nd y ccumulting high concentrtions of orgnic solutes, especilly glycerol (up to mmol l ), tht lower the freeze point colligtively (Rymond, 99; Rymond, 99). Plsm glycerol levels in rinow smelt re su-millimolr from spring to lte summer. Under conditions of nturl wter temperture nd sesonl photoperiod, glycerol egins to ccumulte in the plsm when temperture reches pproximtely C in Novemer. As the wter temperture decreses further, over the next weeks, to out C, there is shrp increse in plsm glycerol level to out mmol l. As winter progresses, glycerol increses to vlues in excess of mmol l (Treerg et l., ; Lewis et l., ). Temperture hs regultory role in glycerol ccumultion, s mintennce of fish t wrm ( C) tempertures over the winter suppress glycerol ccumultion (Lewis et l., ) nd premture exposure of fish to cold tempertures for weeks in the fll results in glycerol ccumultion (Rymond et l., 99). In this study, we ssess if the onset of glycerol ccumultion is due to temperture trigger lone or whether shortening photoperiod is required. Photoperiod ws considered to e potentil requirement since in nother freeze resistnt species, winter flounder, light nd not temperture, is the min environmentl fctor regulting ntifreeze production (Fletcher et l., 99). All studies, to dte, tht show glycerol ccumultion in rinow smelt hve involved decreses in temperture in ssocition with shorter dy lengths. Assessing the direct impct of temperture on the metolic response leding to freeze resistnce ws lso considered to e importnt since in rinow smelt the decrese in glycerol level in spring
Activtion of glycerol production in smelt 7 is not coupled to n increse in temperture per se (Treerg et l., ; Lewis et l., ). In the synthetic pthwy leding to glycerol production, dihydroxycetone phosphte (DHAP) is converted to glycerol -phosphte (GP) nd susequently to glycerol vi the rections ctlyzed y glycerol--phosphte dehydrogense (GPDH) nd glycerol--phosphtse (GPse), respectively. This contention is sed on higher mximl in vitro ctivities of GPDH nd GPse in rinow smelt liver compred to other species tht do not produce glycerol (Driedzic et l., 99; Treerg et l., ), correltion etween liver GPDH ctivity nd plsm glycerol level over sesonl time frme (Lewis et l., ), decreses in liver GPDH mrna ssocited with decreses in plsm glycerol level when coldcclimted fish re trnsferred to wrm wter (Ewrt et l., ), nd increses in liver GPDH mrna in ssocition with the initil sesonl increses in plsm glycerol level (Liescher et l., in press). We report here the reltionship etween cute decrese in temperture nd indices of metolic ctivtion t GPDH nd GPse. Rdioisotope nd stle isotope studies revel tht the cron sources of glycerol re glycogen/glucose nd free mino cids (Rymond, 99; Rymond nd Driedzic, 997; Wlter et l., ). It is likely tht liver is the exclusive or t lest the mjor site of glycerol synthesis. Isolted liver sections produce glycerol (Driedzic et l., 99), glycogen levels re much higher in liver thn other tissues of rinow smelt (Short nd Driedzic, unpulished), nd liver glycogen decreses in ssocition with the ccumultion of glycerol in plsm, liver, nd muscle during the fll-winter trnsition (Treerg et l., ). The importnce of mino cids s fuel for glycerol production in liver vi truncted gluconeogenesis, referred to s glyceroneogenesis (Hnson nd Reshef, ), is consistent with higher ctivities of enzymes ssocited with mino cid metolism in rinow smelt cptured in winter thn other non-glycerol producing species cptured t the sme time (Driedzic et l., 99). In ddition, on sesonl sis, there is correltion etween in vitro lnine minotrnsferse (AlT) nd phosphoenolpyruvte croxykinse (PEPCK) ctivities in liver with plsm glycerol levels (Lewis et l., ) nd similr pttern of PEPCK mrna levels in liver s plsm glycerol (Liescher et l., in press). Another enzyme tht is potentilly criticl to increses in glycerol production is glycerldehyde phosphte dehydrogense (GAPDH) s this enzyme is necessry component of gluconeogenesis. Although there is now n understnding of the metolic pthwys leding to glycerol production nd evidence tht the ctivities of some enzymes re ltered in concert with this production, detils of the control mechnisms re yet to e fully defined. In the current experiment, the impct of controlled nd cute decrese in wter temperture on glycerol production is determined t different times of the yer. Levels of key metolites nd ctivities of enzymes were mesured to ssess control points of glycerol production during the trnsition stte. The most importnt new findings re tht decrese in temperture lone is sufficient to ctivte glycerol production nd tht n increse in GPDH ctivity plys criticl role in the erly stges of the process. Mterils nd methods Animls Rinow smelt (Osmerus mordx Mitchill) were collected y seine netting from Long Hrour, Plcenti By, Newfoundlnd in lte Octoer nd, trnsported to the Ocen Sciences Centre, Memoril University of Newfoundlnd, nd trnsferred to l () or l tnks () with flow-through sewter. Fish were kept on nturl photoperiod with fluorescent lights set on n outdoor photocell, nd fed diet of chopped herring twice week. Stock fish were held t pproximtely C from the time of cpture to the initition of experiments. Whole niml temperture trnsition or step down experiments were performed in Mrch (collected in Octoer. ), Decemer (collected in Octoer ) nd Mrch (collected in Octoer ). For these experiments, smelt were trnsferred to l tnk set t C, two weeks prior to controlled decrese in temperture. Mss of smelt in Mrch, Decemer nd Mrch studies ws.±. g (N=),.±. g (N=), nd 9.7±. g (N=), respectively. There ws no significnt difference in initil condition fctor mongst the three groups which overll ws.7±. nd decresed etween nd % during the course of the experiments, dependent upon the length of time held t decresed temperture. Initil heptosomtic index (HSI) ws.±. (N=) nd.7±. (N=) for the Decemer nd Mrch experiments, respectively. This significnt difference etween groups my e due to the holding of the sme popultion of fish for longer period t elevted tempertures nd lortory feeding protocols. HSI did not chnge over the course of the temperture decrese chllenges in either experiment. HSI ws not recorded for the Mrch study. All fish remined in pprent good helth s evidenced y colour nd ctivity ptterns. Experimentl protocol Mrch The purpose of this initil experiment ws to determine if rpid temperture decrese, is sufficient to ctivte glycerol ccumultion. Furthermore, this study ws conducted when dy length ws incresing, such tht photoperiod should not e trigger. Wter temperture on dy ws C nd ws then decresed to,,, nd C on dys,,, nd, respectively. On dy of the experiment, fish were rndomly selected, weighed, mesured for length, nd lood ws drwn vi the cudl vessel. Fish were then killed with low to the hed, nd livers were removed, freeze clmped nd susequently stored t tempertures elow C. Blood ws centrifuged t 9 g immeditely fter smpling, plsm ws collected nd frozen in liquid nitrogen. Blood nd liver smples were tken t ech time point similr to dy.
W. R. Driedzic nd others Decemer The gols of this experiment were to repet the Mrch study t time when dy length ws decresing nd to determine chnges in levels of metolites ssocited with glycerol production. The experimentl protocol ws similr to tht of Mrch, except smpling occurred on dys,,,,, nd t wter tempertures of,,,,,. nd. C, respectively. Glycerol ssys were performed on oth the liver nd plsm. Liver ws lso nlyzed for the following: pyruvte, dihydroxycetone phosphte (DHAP), lctte, glycerol - phosphte (GP), glucose nd inorgnic phosphte (P i ). Mrch The ojectives of this study were to increse the time t C to ssess if the phse of glycerol ccumultion could e extended nd to determine ctivity levels of enzymes ssocited with glycerol production. Smpling occurred on dys,,,,, nd 9, t wter tempertures of,,,.,, nd C, respectively. Glycerol ws mesured in the plsm s descried ove. Liver ws ssyed for the following enzymes: glycerldehyde--phosphte dehydrogense (GAPDH, EC...), glycerol--phosphte dehydrogense (GPDH, EC...) nd lnine minotrnsferse (AlAT, E.C....). Biochemicl ssys Glycerol level in the plsm ws determined directly using colorimetric detection kit (F, Sigm-Aldrich, MO, USA). Smples were red t nm fter -min incution t room temperture. Liver ws homogenized in nine volumes of % perchloric cid, the homogente ws centrifuged t g nd the superntnt ws ssyed for glycerol s descried ove. For metolite nlysis, pieces of frozen liver were weighed nd homogenized in nine volumes of ice-cold % perchloric cid. Following homogeniztion, smples were centrifuged t g in n Eppendorf centrifuge for min t C nd neutrlized with mol l KOH. Pyruvte nd DHAP ssys were performed immeditely; lctte, GP, glucose nd P i ssys were performed on the frozen extrcts weeks lter. All ssys were performed on Beckmn DU spectrophotometer t nm with the exception of P i mesurements. Assy conditions were s follows. Pyruvte nd DHAP: l of liver extrct were dded to mmol l triethnolmine uffer (ph 7. t room temperture) nd. mmol l reduced nicotinmide-denine dinucleotide (NADH). Smples were red fter min efore dding either IU ml lctte dehydrogense (LDH) for the pyruvte ssy or IU ml of GPDH for the DHAP ssy. Asornces were red for nother min or until stle. Lctte nd GP: - l of liver extrct were dded to n ssy medium contining glycine uffer (Sigm, ) nd. mmol l oxidised nicotinmide-denine dinucleotide (NAD + ), ph 9. t room temperture. Smples were red fter min efore dding IU ml LDH for the lctte ssy or. IU ml GPDH for the GP ssy. Asornces were red for nother min or until stle. Glucose: ssy conditions were sed on procedure modified from the method of Bergmeyer (Bergmeyer, 97). Briefly, l of liver extrct were diluted : with the ssy medium ( mmol l imidzole, mmol l MgSO, mmol l ATP nd. mmol l NADP + ). l of glucose- -phosphte dehydrogense were dded to remove ny endogenous glucose -phosphte. Asornce ws red fter min, nd hexokinse ws then dded nd the sornce red fter min. P i : ssy conditions were sed on those of Rockstein nd Herron (Rockstein nd Herron, 9). A l smple of liver extrct ws dded to l.% mmonium molydte in N sulphuric cid nd l distilled wter. Colour ws initited y dding l of. mol l ferrous sulfte nd the sornce ws red t 7 nm fter min. For enzyme ctivity nlysis, pieces of frozen liver were weighed nd homogenized in nine volumes of ice-cold extrction uffer ( mmol l imidzole,. mmol l EGTA,. mmol l EDTA, mmol l mercptoethnol, mmol l sodium fluoride nd. mmol l phenylmethylsulfonyl fluoride, ph 7. t C) nd centrifuged t g for min t C. All three enzyme ssys were performed immeditely following homogeniztion nd centrifugtion. Control rection rtes were determined prior to the ddition of the sustrte. Assy conditions for GAPDH nd AlAT re descried y Treerg et l. (Treerg et l., ). GPDH ssys conditions included mmol l imidzole, ph 7. t C,. mmol l NADH nd mmol l DHAP tht initited the rection. Enzyme ctivities determined t nm were clculted sed on the millimolr extinction coefficient of.. All enzyme ctivities were determined t C to fcilitte nlysis t higher ctivity levels since we were primrily interested in reltive not solute ctivities during exposure of fish to decresed temperture. This ws considered cceptle with the ssumption tht enzyme ctivity is proportionl to enzyme content. Dt nlysis Mss ction rtio cross GPDH ws clculted s: [GP][pyruvte]/[DHAP][lctte]. This is sed on the premise tht the LDH rection is t equilirium nd, s such, the [pyruvte]/[lctte] rtio reflects the [NADH]/[NAD + ] rtio. The mss ction rtio cross GPse ws clculted s: [glycerol][p i ]/[GP]. Mens were compred with one-wy nlysis of vrince (ANOVA) for ll mesurements followed y Tukey s post-hoc test. A P vlue of <. ws considered to e sttisticlly significnt for ll studies. Results Plsm glycerol Rinow smelt mintined t C nd smpled in Mrch, Decemer nd Mrch, hd very low plsm glycerol levels of.,.7 nd. mmol l, respectively (Fig. ). When fish were chllenged with drop in wter temperture from to C over dys (Mrch nd
Activtion of glycerol production in smelt 9 [Glycerol] (mmol l ) A Mrch () B Decemer () C Mrch 7, () () () () () Temperture ( C) Dec. ; Fig. A,B), significnt increse in plsm glycerol occurred (P<.). A further drop from C to C in the Mr study, resulted in plsm glycerol level of mmol l. These experiments show tht temperture decrese lone is sufficient to drive n increse in glycerol production nd tht the glycerol increse is not photoperiod dependent. Fish chllenged with decrese in temperture in the Mrch experiment (Fig. C) showed similr pttern of glycerol increse (P<.). Rinow smelt kept t C, to dy 9, ccumulted extremely high levels of plsm glycerol ( mmol l ). Metolite nlysis (Decemer study) A significnt increse in liver glycerol prlleled tht mesured in the plsm (compre Fig. nd Fig. B). Liver glycerol levels incresed significntly (P<.) s compred to C vlues once tempertures reched C on dy. Liver,,, () () () () (),c () c () () () () c ( 9) Fig.. Plsm glycerol levels in rinow smelt sujected to controlled decrese in temperture during three seprte experiments: (A) Mrch ; (B) Decemer ; (C) Mrch. Dys following the temperture trnsition re shown in prentheses. Vlues re mens ± s.e.m. In Mrch nd Decemer experiments N= t ech temperture with the exception of Decemer,. C where N=. In the Mrch experiment N= for ll points except tempertures of. C where N= nd C where N=. Different letters indicte significnt differences etween two tempertures. glycerol level ws sustntilly lower thn plsm glycerol t this sme time point, 7 mol g in liver, compred to mol ml in plsm. Lower levels of glycerol lso occurred in liver thn in plsm t. C on dys following the trnsition. Glycerol synthesis requires the conversion of either glucose/glycogen or mino cids y DHAP nd GP. Significnt (P=.) chnges occurred in DHAP levels s function of time. There ws tendency for DHAP to increse during the initil decrese in wter temperture with mximum vlues occurring t C; this ws followed y significnt decrese to the lowest vlue t. C. Neither GP nor P i, one of the rekdown products of GP, showed significnt chnge s temperture decresed. Pyruvte, potentil sustrte for glyceroneogenesis showed tendency to chnge over temperture (P=.) with the men level eing lower t. C (.±. mol g ) thn t C (.±. mol g ). Lctte did not chnge significntly over the course of the experiment. The level of glucose in liver chnged with temperture (P=.) with the vlue t. C eing lower thn the highest mount noted t C. The mss ction rtio cross GPDH nd GPse were clculted from the vrious metolites mesured ove. The overll GPDH mss ction rtio (Fig. A) chnged significntly (P=.) s temperture decresed. Although the nlysis did not revel ny significnt difference etween specific points, the men vlue t. C (.±.) ws three times s high s the rtio t C (.7±.). The mss ction rtio cross GPse (Fig. B) incresed significntly (P=.) s the temperture decresed, peking t C. Enzyme ctivity levels (Mrch study) Activities of key enzymes involved in gluconeogenesis (GAPDH), the terminl steps to glycerol synthesis (GPDH) nd mino cid rekdown (AlAT), either chnged significntly or showed strong tendency to chnge s temperture ws decresed (P=., P=. nd P=., respectively) (Fig. ). Men ctivity levels of ll three enzymes were higher t C, compred to wrmer tempertures in ssocition with n increse in plsm glycerol (compre Fig. nd Fig. C). The elevtion in plsm glycerol ws ssocited with.-fold chnge in men liver GPDH from ± to 9± mol g min etween. C nd C. Averge GAPDH nd AlAT ctivities were.- nd.-fold higher, respectively during this trnsition. Discussion Plsm nd liver glycerol levels Plsm glycerol egins to increse s temperture flls elow C. This contention is sed on the grdul elevtion in men plsm level shown in ech of three independent experiments. This is followed y shrp increses in glycerol levels t tempertures of C to C. These findings re very similr to chnges oserved under nturl decreses in wter temperture nd photoperiod (Lewis et l., ). The level of
W. R. Driedzic nd others glycerol in liver followed similr pttern to plsm glycerol, with significnt elevtion reltive to the initil level, in ssocition with tempertures of C nd elow. A decrese in temperture lone is sufficient to result in n ccumultion of glycerol. A decrese in photoperiod is not requirement for glycerol production s the low temperture response my e induced in either Decemer (shortening dy length) or in Mrch (incresing dy length). It ws previously shown tht rinow smelt mintined t high temperture do not ccumulte glycerol when sujected to sesonl decreses in photoperiod (Lewis et l., ). Tken together these studies revel tht lowering of temperture is the key environmentl cue to glycerol ccumultion, wheres photoperiod is not fctor. Once elevted levels in plsm re chieved, the concentrtion of glycerol in intrcellulr wter must e sustntilly lower thn tht in the extrcellulr spce given levels of mol g liver vs mol ml in plsm (Decemer study). This finding is consistent with the rtio of plsm to liver glycerol in rinow smelt following sesonl chnges in temperture (Treerg et l., ). The true difference in glycerol etween the two comprtments wits the ccurte determintion of intrcellulr wter. Regrdless, the qulittive oservtion implies n ctive export of glycerol from liver cells to plsm ginst concentrtion grdient. The yest, Scchromyces cerevisie, ctively trnsports glycerol into the cell (Holst et l., ), whether similr ctive trnsport mechnisms exist in rinow smelt to move glycerol out of liver into the plsm is not known. An quglycerporin cdna from se rem, mrine teleost, hs een sequenced nd [Glycerol] [DHAP] [GP] [Pi] [Pyruvte] [Lctte] [Glucose]..... when expressed in Xenopus oocytes results in n increse in glycerol permeility (Sntos et l., ). A similr mrna trnscript hs een detected in rinow smelt liver (J. R. Hll nd W.R.D., unpulished) nd my ply role in glycerol permeility; however, n quglyceroporin, which could serve s glycerol conduit, in itself cnnot ccount for the lrge extr- to intrcellulr differences in glycerol levels. The mximl rte of glycerol synthesis in liver my e estimted from the increse in plsm glycerol. A considertion of the Dec. study etween dys nd serves s n exmple. Over this time period, plsm glycerol incresed from to mmol l. A typicl ody mss for.......,, 7, Temperture ( C) Fig.. Glycerol, dihydroxycetone phosphte (DHAP), glycerol -phosphte (GP), inorgnic phosphte (P i ), pyruvte, lctte nd glucose levels in liver of rinow smelt sujected to controlled decrese in temperture. Vlues re mens ± s.e.m., N= t ech point with the exception of. C where N=. Different letters indicte significnt differences etween vlues. Trnsition time nd plsm glycerol levels for this experiment re presented in Fig. B. rinow smelt is g, of which out g re one. Although glycerol content my e lower in liver thn in plsm, the content in most other soft tissues is similr to tht of plsm (Rymond, 99). Thus, the increse in glycerol in the totl fish etween dys nd would pproximte mol g (i.e. level t dy ) mol g (i.e. level t dy ) g, which equls 9 mol. A typicl liver mss, in g rinow smelt is.7 g. Accepting tht liver is the primry site of glycerol formtion, the rte of glycerol production over the dys would e 9 mol glycerol.7 g min which equls. mol glycerol g min. Given tht the loss of glycerol to wter is out % per dy (Rymond, 99),,,,,
Activtion of glycerol production in smelt our est estimte of the rte of glycerol production y liver is. mol glycerol g min t C. Similr clcultions for the Mrch experiment etween dys nd nd for Mrch etween dys nd yield rtes of. nd. mol g min, respectively. Sites of metolic regultion GPDH ctlyzes the conversion of DHAP plus NADH to GP plus NAD +. Independent ssessment of metolite levels nd enzyme ctivities suggests tht this site is criticl to ctivtion of glycerol production during cute low temperture chllenge. The initil stges of glycerol production re ssocited with trnsient increse in DHAP followed y decrese in this metolite. The mss ction rtio of this rection incresed t. C, reltive to higher tempertures. The mss ction rtio is clculted on the premise tht the rection ctlyzed y LDH remins in equilirium nd thus the pyruvte/lctte rtio reflects the NAD + /NADH rtio. This ssumption my not e correct during the trnsition period to high rtes of glycerol production nd my ccount for the clculted chnge in mss ction rtio occurring only fter [GP] [Pyr] [DHAP] [Lc] [Glycerol] [Pi] [GP] A B 7 Fig.. Mss ction rtios in liver of rinow smelt sujected to controlled decrese in temperture: (A) cross glycerol--phosphte dehydrogense; (B) cross glycerol -phosphtse. Vlues re mens ± s.e.m., N= t ech point with the exception of. C, where N=. Different letters indicte significnt differences etween vlues. Trnsition time nd plsm glycerol levels for this experiment re presented in Fig. B., Temperture ( C),, stedy stte levels of liver glycerol re chieved. As such, lthough fine detils of the temporl nlysis my e in question, ctivtion of GPDH sed on metolite nlysis, is reveled t high levels of glycerol ccumultion. In seprte experiment, there ws -fold chnge in, in vitro GPDH ctivity t C reltive to. C, in ssocition with increses in plsm glycerol level. Agin this implies tht this site is criticl in the process of high rtes of glycerol production t low temperture. The mximl in vitro ctivity of GPDH is ~7 mol g min t C. Assuming Q of this would equte to mol g min t C, vlue tht is well in excess of the clculted rte of glycerol production y liver. Even if the Q ws higher, for instnce, if the Q equlled, AlAT (μmol g min ) GPDH (μmol g min ) GAPDH (μmol g min ) 9 7 A B, C, 7, Temperture ( C) Fig.. Activities of enzymes in liver of rinow smelt sujected to controlled decrese in temperture: (A) glycerldehyde--phosphte dehydrogense (GAPDH); (B) glycerol--phosphte dehydrogense (GPDH); (C) lnine minotrnsferse (AlAT). Vlues re mens ± s.e.m., N= t ech point with the exception of. C, where N=, nd C, where N=. Different letters indicte significnt differences etween vlues. Trnsition time nd plsm glycerol levels for this experiment re presented in Fig. C.,,,
W. R. Driedzic nd others the mximl enzyme ctivity would still e more thn times the estimted rte of in vivo glycerol production. The high constitutive ctivity of totl GPDH, mesured in homogentes, needs to e reconciled with the fr lower estimted rtes of flux cross this rection. There re t lest genes encoding cytosolic GPDH in rinow smelt (K.V.E., R. S. Richrds nd W.R.D., unpulished). Crude enzyme ctivity mesurements report the ctivities of ll encoded GPDH isoforms, wheres it my e tht only one form is involved in the cold-induced mechnisms of glycerol production. Although there is now considerle dt tht points to the importnce of GPDH s n importnt regultory enzyme in glycerol production, there is mounting evidence tht other enzymes contriute to control of the process s well. The finl step in glycerol production requires the conversion of GP to glycerol plus P i ctlyzed y GPse. The importnce of GPse to glycerol production ws first implied y mximl in vitro enzyme ctivities in liver, which were significntly higher in rinow smelt thn two other species of non-glycerol producing species cptured in winter (Driedzic et l., 99). In the current experiment, GP nd P i levels in liver remin constnt s glycerol increses in ssocition with decrese in temperture. As such, the increse in the concentrtion of products, reltive to sustrte results in n increse in the mss ction rtio cross the rection ctlyzed y GPse fter the initition of the temperture trnsition. The mximl in vitro ctivity of GPse t C, previously reported (Driedzic et l., 99), ws.9 mol g min ; similr ctivity ws found y Rymond nd Hssel (Rymond nd Hssel, ). Assuming Q of, the rte of GPse ctivity would pproximte. mol g min t C; Q of would result in rte of. mol g min. Given these ssumptions, the clculted rtes of glycerol production y liver in vivo nd the mximl ctivity of GPse in vitro re closely mtched. Tken together, the chnge in mss ction rtio in ssocition with n increse in glycerol production nd the similrity in estimted rtes of glycerol production to mesurements of in vitro ctivity of GPse, suggests tht the rection ctlyze y GPse is rte limiting nd regultory. The three enzymes (GPDH, GAPDH, AlT) mesured in this study, ll follow the sme pttern, with n increse in verge ctivity etween dys nd, in ssocition with the upswing in plsm glycerol level. It is unlikely tht the increses in men ctivity of the three enzymes is spurious ut rther reflects chnge in metolic orgniztion. AlAT is required to chnnel cron from lnine into pyruvte. Increse in ctivity of this enzyme is consistent with correltion etween mximl in vitro ctivity in liver nd plsm glycerol level during sesonl chnge in temperture (Lewis et l., ) nd with nucler mgnetic resonnce findings tht show neighoring crons from injected lnine ppering in glycerol (Wlter et l., ). GAPDH is required for glyceroneogenesis from pyruvte nd mino cids. Activities of oth AlT nd GAPDH re well in excess of clculted rtes of glycerol production nd GPse. Although it is unlikely these sites re rte limiting, n increse in ctivity of these rections is component of the suite of low temperture responses of rinow smelt nd my contriute to metolic control. Generl conclusions In rinow smelt, decrese in wter temperture to less thn C, is sufficient to ctivte the metolic processes leding to glycerol ccumultion. This nticiptory response is similr to ctivtion of glycerol production in numer of insects (Storey nd Storey, 9) nd in the gry treefrogs, Hyl veriscolor nd H. chrysoscelis, where n increse in plsm nd orgn glycerol occurs in response to extended cold cclimtion t tempertures ove C (Lyne nd Jones, ; Irwin nd Lee, ). This sitution differs from the ccumultion of glucose in these frogs nd other frog species tht occurs only in response to freezing (Storey nd Storey, 99; Lyne nd Jones, ; Irwin nd Lee, ). Glycerol ccumultion is ssocited with ctivtion of GPDH. This contention is sed on metolite levels reported here, enzyme ctivities reported here nd elsewhere (Driedzic et l., 99; Lewis et l., ), nd chnges in liver GPDH mrna in the direction of chnges in plsm glycerol level (Liescher et l., in press); however, the high in vitro rtes of GPDH ctivity reltive to clculted rtes of glycerol production remins to e resolved. It my e tht in rinow smelt liver there is specific GPDH isoform involved in temperture-ctivted glycerol production. The rection ctlyzed y GPse lso ppers to e importnt in the metolic sequence leding to glycerol production. This position is sed on mss ction rtios of metolites t this rection nd the similrity etween clculted mximl rtes of glycerol production in vivo compred to the mximl ctivity of the enzyme in vitro. On the sis of levels of three cron intermedites nd glycerol, Churchill nd Storey (Churchill nd Storey, 99) proposed tht in the lrve of Epilem scudderin, GPse is suject to ctivtion nd then inhiition, during the trnsition to ccumultion of glycerol nd susequent mintennce of high glycerol content. How GPse ctivity is regulted in high glycerol producing nimls is yet to e ddressed. Increses in the ctivity of other enzymes, including AlT nd GAPDH re lso ssocited with the trnsition to glycerol ccumultion. This work ws supported y the Nturl Sciences nd Engineering Reserch Council of Cnd. W.R.D. holds the Cnd Reserch Chir in Mrine Bioscience. This is NRC puliction numer -. We thnk Drs Luis Afonso nd Sue Dougls (NRC IMB) for reviewing this mnuscript. References Bergmeyer, H. U., Gwehn, K. nd Grssl, M. 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