GLYCEROL SYNTHESIS IN THE RAINBOW SMELT OSMERUS MORDAX

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1 The Jornal of Eperimental iolo 198, (1995) Printed in Great ritain The ompany of iologists Limited 1995 JE GLYEROL SYNTHESIS IN THE RINOW SMELT OSMERUS MORDX JMES. RYMOND Department of iological Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154, US ccepted 6 September 1995 Rainbow smelt, Osmers morda, maintain high glycerol levels in winter to avoid freezing. fter intramsclar injection of 14 -labeled glcose, [ 14 ]glycerol was fond in the blood, liver and mscle, indicating that glycogen is a sorce of glycerol. Levels of both the active and inactive forms of glycogen phosphorylase were higher in mscle in winter than in atmn, althogh the fraction in the active form did not change significantly. More of the phosphorylase was in the active form in the liver than in the mscle. Short-term starvation reslted in a significant decrease in the level of glycogen soon after the stomachs were emptied, presmably to replace glycerol lost to the water. However, tisse glycerol levels remained relatively Smmary high, despite a near depletion of glycogen reserves. Triglyceride levels increased slightly dring starvation, indicating that triglycerides were not involved in glycerol synthesis. fter intramsclar injection of 14 -labeled pyrvate, [ 14 ]glycerol was fond in the blood, liver and mscle, indicating a second rote, presmably from mscle protein, to glycerol synthesis. phosphoenolpyrvate carboykinase activity was slightly higher in winter, possibly to assist in the conversion of pyrvate to glycerol. Key words: glycerol synthesis, Osmers morda, glcose, pyrvate, glycogen, glycogen phosphorylase, phosphoenolpyrvate carboykinase. Introdction The rainbow smelt, Osmers morda, is nsal among freeze-resistant fishes in that it prodces a large amont of glycerol in winter that acts as a colligative antifreeze (Raymond, 1992). Unlike some fro and insects that also prodce glycerol in winter (Storey and Storey, 1988), the smelt live in an aqeos environment and are ths sbject to glycerol losses throgh their epithelia. These losses can eceed 1 % of body glycerol stores per day (Raymond, 1993) and appear to doble the daily ener reqirement (Raymond, 1995). In insects and fro, glycerol is synthesized from glycogen. Glycogen also appears to be a sorce of glycerol in the smelt, as acclimation and acclimatization stdies of rainbow smelt and the related srf smelt Hypomess pretioss in Japan (J.. Raymond, H. Hattori and K. Tsmra, in preparation) have shown an inverse relationship between glycogen levels and glycerol prodction. However, these stdies also revealed some pzzling aspects of glycerol prodction in the smelt. Glycogen levels appeared to be nsally depressed after short-term starvation and glycerol levels remained relatively high, despite the low glycogen levels. The present stdy was ndertaken to investigate these phenomena as well as to obtain more direct evidence that glycogen was a sorce of glycerol. Materials and methods Winter-acclimatized rainbow smelt Osmers morda Mitchell were caght by fishermen with nder-ice traps in the Kochibogac River and Miramichi River estaries in New rnswick, anada, between late Janary and late Febrary of 1993 and Srface water temperatre dring these periods was approimately.4 to.7. dditional fish were caght in late November 1993, before freeze-p, from a similar trap (water temperatre approimately 3 ). For a glycogen depletion stdy, approimately 4 fish (average body mass 4±21 g, mean ± S.D.) were retrieved from a trap on 19 Febrary, approimately 16 h after it was set, and maintained in two filtered, aerated 47 l ice chests withot feeding for several days at a nearby motel. The water temperatre was kept near 1 by moving the ice chests in and ot of doors. The water was replaced on the second day. The fish were calm and appeared to be in good health. Samples of fish (N=5) were taken from the ice chests for tisse samples and stomach analyses at intervals of h. Stomach fllness was estimated visally, with 1 % corresponding to a fll stomach. Serm samples were taken as described previosly (Raymond, 1992). The livers and a sample of mscle from the rear ventral flank were taken, wrapped in alminm foil and qickly frozen between blocks of dry ice. In the laboratory, glycerol levels were measred sing a diagnostic kit (Sigma no. 337) (Raymond, 1992). Glycogen levels were measred according to the method of Keppler and Decker (1974) ecept that glcose was measred sing a Sigma glcose H-K kit. This had the effect of treating glcose 6-phosphate as glycogen, bt

2 257 J.. RYMOND as glcose 6-phosphate levels appear to be small compared with glcose levels (Storey and Storey, 1984), the error was assmed to be small. Triglycerides were measred with a diagnostic kit (Sigma no. 337) (based on measrement of glycerol before and after lipase digestion) sing the same tisse homogenate (in.5 mol l 1 HlO 4 ) sed for the glycogen analysis, dilted by a factor of 2 to redce glycerol levels. Triglyceride concentrations were based on an estimated moleclar mass of 88 Da. For the radioisotope stdies, fish were transported in an aerated ice chest to Mont llison University, Sackville, New rnswick. Water temperatre was maintained in the range to 1 by occasionally moving the ice chests otdoors. Water was replaced approimately daily by miing eqal parts of fresh sea water and well water and allowing it to cool to abot 1 otdoors. In each eperiment ([ 14 ]glcose and [ 14 ]pyrvate), two fish were injected intramsclarly with approimately.55 Mq of sbstrate and kept in a 1 l gently aerated bcket that was placed in one of the ice chests for temperatre control. The fish were killed 24 h later and tisse samples were taken as described above. Liqid etracts of the tisses were filtered with ltrafiltration tbes (Millipore, 1 M r ct-off pore size) and the filtrate was applied to a carbohydrate HPL colmn (Phenomene, RM), as described previosly (Raymond, 1992). The elate was monitored with a refractive inde detector. Fractions were collected so that, as far as possible, they corresponded to the refractive inde peaks. Samples of the HPL fractions (2 l) were then conted in a liqid scintillation conter. Glycogen phosphorylase activity was measred according to the method of Storey and Storey (1984) with slight modifications. Tisse samples were homogenized in homogenizing bffer (5 mmol l 1 imidazole, 1 mmol l 1 NaF, 5 mmol l 1 EDT, 5 mmol l 1 EGT, 15 mmol l 1 -mercaptoethanol, adjsted to ph 7.5) at a concentration of 5 lmg 1 tisse. The homogenate was qickly centrifged at 16 g for 3 s. Phosphorylase a activity was measred by monitoring the increase in absorbance at 34 nm of a mitre containing 95 l of reaction mitre (5 mmol l 1 phosphate bffer, ph 7.3, 2 mg ml 1 glycogen, 15 mmol l 1 MgSO 4,.25 mmol l 1 EDT,.5 mmol l 1 ND,.1 mmol l 1 glcose 1,6-diphosphate,.5 mmol l 1 dithiothreitol and 1 nit each of phosphoglcomtase and glcose-6-phosphate dehydrogenase), 5 l of phosphate bffer and 1 l of spernatant. (Unit enzyme activities are a defined by Sigma.) The phosphate bffer was replaced with 5 l of 2 mmol l 1 MP in order to measre total phosphorylase activity. Phosphoenolpyrvate carboykinase (PEPK) activity was measred sing the method of Sebert and Hth (1965), in which oaloacetate was sed as the sbstrate, with a few modifications. tisses that had been stored at 7 for months were homogenized in 5 volmes of ice-cold 5 mmol l 1 imidazole Tris, ph 7.5, with 1 mmol l 1 EDT and 15 mmol l 1 2-mercaptoethanol. pproimately 15 l of homogenate was incbated for approimately 2 min with gentle shaking in a water bath at 15. The amont of phosphoenolpyrvate (PEP) generated was converted to lactate in a reaction mitre containing 3 mmol l 1 NDH, 3 mmol l 1 DP, 15 mmol l 1 MgSO 4 and 5 mmol l 1 Tris, ph 8.. The reaction was started by adding 2 l of a soltion containing approimately 3 nits each of pyrvate kinase and lactate dehydrogenase. The reslting oidation of NDH was monitored at 34 nm. Initial eperiments confirmed that the amont of PEP prodced was proportional to both the incbation time and the incbation temperatre. Reslts [ 14 ]glcose metabolism Twenty-for hors after the intramsclar injection of two fish with [ 14 ]glcose, the 14 label appeared in glycerol in the blood, liver and mscle of both fish. Eample 14 activity (log conts min 1 ) e Retention time (min) Serm Mscle Fig. 1. Metabolism of intramsclarly injected [ 14 ]glcose in winter-acclimatized rainbow smelt. Lower crves, HPL chromatograms of serm and spernatant from homogenized tisses sing a carbohydrate colmn with refractive inde detection. Upper crves, conts per minte of HPL fractions. () Serm; () liver; () mscle. e, electrolytes;, glcose;, glycerol;, rea;, nknown.

3 Glycerol synthesis in smelt 2571 Glycogen phosphorylase activity ( mol NDH min 1 g 1 ) tmn Winter tmn Winter Mscle Fig. 2. Glycogen phosphorylase activities in liver and mscle of atmn- and winter-acclimatized rainbow smelt. Filled bars, total phosphorylase; stippled bars, phosphorylase a; open bars, phosphorylase b. Sample size for each bar is five fish. Error bars show 1 S.D. chromatograms are shown in Fig. 1. n nknown 14 species appeared in the liver and mscle samples with an eltion time of approimately 35 min. This peak, which was otside the region of mono- and disaccharides, cold not be attribted to phosphoenolpyrvate, oaloacetic acid, citrate, acetyl coenzyme, acetate or bicarbonate. Glycogen phosphorylase activity Total phosphorylase activity in mscle tisse was significantly higher in mid winter than before freeze-p in the atmn (t-test, P<.1) (Fig. 2). The activities of phosphorylase a (the active form) and phosphorylase b were also higher in winter (P<.5 for each). Sample sizes were not large enogh to determine whether similar differences were present in the liver. Total phosphorylase activities were not significantly different in liver and mscle tisses. However, a greater percentage of the phosphorylase was in the active form in the liver than in the mscle both in the atmn (77 verss 28 %) and in the winter (75 verss 35 %) (P<.1 for each). Short-term starvation The stomachs of fresh, winter-caght rainbow smelt were relatively fll, indicating that they were feeding actively in winter. Mysid shrimp, amphipods and polychaetes were the main food items. Stomachs were almost empty within abot 2 days after captre (Fig. 3). glycogen (Fig. 3), liver glcose (Fig. 3) and serm glycerol (Fig. 3E) levels all decreased and serm triglyceride (Fig. 3D) level increased in the period after the stomachs emptied. In the final three sampling periods, glycogen, glcose and glycerol levels were significantly lower (P<.1) and triglyceride levels were higher (P<.5) than in the first for sampling periods. Females in the sample (11 of 35), which were preparing for spawning, tended to have emptier stomachs and lower levels of liver glycogen and serm triglyceride than the males and, ths, were largely responsible for the early variability in these parameters. The lower levels of liver glycogen in the females were partially compensated by the fact that the livers were significantly larger in the females than in the males (3.2±.4 verss 1.8±.4 % of body mass, P<.1). This is a common phenomenon among spawning fishes (e.g. Henderson et al. 1984). Serm glycerol levels were correlated with liver glycogen levels (r=.39, N=35, P<.2), althogh when the latter dropped sharply, serm glycerol levels decreased by a relatively small amont. No correlation was fond between serm glycerol and liver triglyceride levels. glycerol and triglyceride levels (data not shown) closely resembled those in the serm, althogh the absolte glycerol vales were slightly lower in the liver becase of its lower water content. Mscle glycogen levels (.6±.4 mg g 1 at 2 h, Glycerol (mmol kg 1 ) Triglyceride (mg g 1 ) Glcose (mg g 1 ) Glycogen (mg g 1 ) Stomach fllness (%) Time after captre (h) Fig. 3. hanges in stomach fllness and metabolite levels in winteracclimatized rainbow smelt dring short-term starvation. () Stomach fllness; () liver glycogen; () liver glcose; (D) serm triglyceride; (E) serm glycerol. Sample size for each point is five fish. Error bars show 1 S.D. D E

4 2572 J.. RYMOND 14 activity (log conts min 1 ) e, p p p 1 p Serm Mscle Retention time (min) Fig. 4. Metabolism of intramsclarly injected [ 14 ]pyrvate in winter-acclimatized rainbow smelt. Data and symbols are as described in Fig. 1. p, pyrvate. 1.2±.7 mg g 1 at 5 h and.4±.3 mg g 1 at 124 h) were very low compared with those in the liver and did not show a significant trend dring the 5 day starvation period. Mscle glycerol levels, determined only at 5 and 124 h, were similar to those in the serm. Mscle triglyceride levels were too low to measre accrately. [ 14 ]pyrvate metabolism Twenty-for hors after intramsclar injection of two fish with [ 14 ]pyrvate, the 14 label appeared in glycerol in the blood, liver and mscle of both fish. Eample chromatograms are shown in Fig. 4. [ 14 ]pyrvate was present in the blood and liver, bt not in the mscle, where it appeared to have been converted to glcose. The 14 label was also fond in the mscle in an nidentified peak at 35 min. PEPK activity samples were taken from fish collected in early November (before freeze-p, water temperatre approimately 3 ) and late Janary (water temperatre.7 ). PEPK activity of liver tisse was slightly bt significantly higher in the winter (1.64±.47 mol PEP g 1 min 1, N=7) than in the atmn (1.21±.13 mol g 1 min 1, N=8) (P<.5). Discssion Winter-acclimatized rainbow smelt mst maintain high glycerol levels (approimately 2 4 mmol kg 1 ) for freezing avoidance (Raymond, 1992), despite significant glycerol losses (approimately 3 9 mg h 1 1 g 1 ) into the water throgh the gills and skin (and probably feces) (Raymond, 1993). The estimated ener loss associated with the glycerol loss, 2.1 J 1 g 1 day 1, is comparable to pblished vales for the metabolic rates of polar fishes (Raymond, 1995) and ths wold reqire an approimate dobling of the minimm food reqirement. The observed active feeding of the smelt in winter appears to be a conseqence of this. Starvation and cold acclimation stdies have previosly shown that the glycerol is synthesized rather than obtained throgh the diet (J.. Raymond, H. Hattori and K. Tsmra, in preparation). That stdy, by showing that there was a high demand on glycogen in winter and that glycogen levels were more depressed in fish acclimated to 1 than in fish acclimated to +1, strongly pointed to glycogen as a sorce of glycerol. In the present stdy, the conversion of [ 14 ]glcose to glycerol confirms that glycogen is a sorce of glycerol in rainbow smelt. This is frther spported by (1) the increase in glycogen phosphorylase activity in mscle, and perhaps liver, in winter and (2) the rapid depletion of liver glycogen reserves dring short-term starvation. The latter reslt appears to be nsal among fishes, as other stdies have shown that liver glycogen levels do not normally change significantly dring short-term starvation (Dave et al. 1975; Ottolenghi et al. 1981; lack and Love, 1986). The rapid consmption of glycogen in the smelt strongly sggests that it was being sed to replace glycerol that had been lost to the environment. Glycogen depletion rates are presmably lower in other fishes becase they do not eperience a significant carbohydrate effl. The ability of the smelt to maintain relatively high glycerol levels despite a near depletion of glycogen after both 5 days of starvation (this stdy) and 2 weeks of starvation (J.. Raymond, H. Hattori and K. Tsmra, in preparation) sggested that another glycerol synthetic pathway was present. This wold be a departre from other glycerol-prodcing animals in which glycogen appears to be the only sorce of glycerol (Storey and Storey, 1988). The pattern of relatively constant or possibly increasing triglyceride levels in starved smelt (Fig. 3D) was similar to that observed in a longer acclimation stdy (J.. Raymond, H. Hattori and K. Tsmra, in preparation) and indicates that triglycerides are not involved in glycerol synthesis, in agreement with previos stdies (Storey and Storey, 1988). This reslt is not srprising in view of the small contribtion of glycerol to the moleclar mass of triglycerides and the inability of animals to convert fatty acids into glconeogenic precrsors (Mathews and van Holde, 199). nother potential sorce of glycerol is protein. Dring

5 Glycerol synthesis in smelt 2573 periods of starvation, the catabolism of mscle protein yields several glconeogenic precrsors, inclding pyrvate (Mathews and van Holde, 199). This rote for glycerol prodction is sggested (bt not proven) by the appearance of [ 14 ]glycerol in all tisses eamined shortly after [ 14 ]pyrvate injection and by the increase in the activity of PEPK in the liver in winter. PEPK is a key glconeogenic enzyme that mediates the conversion of pyrvate to phosphoenolpyrvate. These reslts clearly demonstrate that there is a second rote to glycerol prodction in the smelt. Frther stdies sing labeled amino acids are planned to more clearly define this novel synthetic pathway. I thank K. allendar, J. Daigle and K. Sweezey for generosly providing the fish sed in this stdy, Dr W. Driedzic, Mont llison University, for providing facilities for holding the fishes sed in the 14 stdies, and Drs K. and J. Storey for helpfl advice on measring glycogen phosphorylase activity. This stdy was spported by NSF grants DPP and OPP References LK, D. ND LOVE, R. M. (1986). The seqential mobilisation and restoration of ener reserves in tisses of tlantic cod dring starvation and refeeding. J. comp. Physiol. 156, DVE, G., JOHNSSON-SJOEK, M.-L., LRSSON,., LEWNDER, K. ND LIDMN, U. (1975). Metabolic and hematological effects of starvation in the Eropean eel, ngilla angilla L. I. arbohydrate, lipid, protein and inorganic ion metabolism. omp. iochem. Physiol. 52, HENDERSON, R. J., SRGENT, J. R. ND HOPKINS,.. (1984). hanges in the content and fatty acid composition of lipid in an isolated poplation of the capelin Mallots villoss dring seal matration and spawning. Mar. iol. 78, KEPPLER, D. ND DEKER, K. (1974). Glycogen: Determination with amyloglcosidase. In Methods of Enzymatic nalysis (ed. H. U. ergmeyer), pp New York: cademic Press. MTHEWS,. K. ND VN HOLDE, K. E. (199). iochemistry. Redwood ity, : enjamin/mmin. 1129pp. OTTOLENGHI,., PUVINI,.. ND RIGHENTI, L. (1981). Glycogen in liver and other organs of catfish (Ictalrs melas): Seasonal changes and fasting effects. omp. iochem. Physiol. 68, RYMOND, J.. (1992). Glycerol is a colligative antifreeze in some northern fishes. J. ep. Zool. 262, RYMOND, J.. (1993). Glycerol and water balance in a nearisosmotic teleost, winter-acclimatized rainbow smelt. an. J. Zool. 71, RYMOND, J.. (1995). Responses of marine fishes to freezing temperatres: new look at colligative mechanisms. In Thermobiolo (ed. J. S. Willis), dvances in elllar and Moleclar iolo series, Greenwich, N: JI Press (in press). SEUERT, W. ND HUTH, W. (1965). On the mechanism of glconeogenesis and its reglation. II. The mechanism of glconeogenesis from pyrvate to fmarate. iochem. Z. 343, STOREY, K.. ND STOREY, J. M. (1984). iochemical adaption for freezing tolerance in the wood frog, Rana sylvatica. J. comp. Physiol. 155, STOREY, K.. ND STOREY, J. M. (1988). Freeze tolerance in animals. Physiol. Rev. 68,