University of Southern California Dissertations and Theses

Glucose metabolism of the kelp bass, Paralabrax clathratus Girard

(USC Thesis Other)

Glucose metabolism of the kelp bass, Paralabrax clathratus Girard

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content GLUCO I •TABOLISM OF THJ K~LP BA PARALABRAX CLATHRATUS GIRARD by Karen Laverne Bever A Di sertation Presented to the FACULTY OF TH GRADUAT~ CHOOL UNIV RSITY OF SOUTH R CALIFORNIA In artial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biological Sciences) 1arch 1975 , UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90007 This dissertation, written by Karen Laverne B ver under the direction of h .. ~-~- Dissertation Com- 1nittee, and approved by all its rne1nbers, has been presented to and accepted by The Graduate School, in partial /ulfillment of requirenzents of the degree of DOCTOR OF PHILOSOPHY Dean DI SERTA1 IO CO 1MITTEE -------~~ ------ Chairman Dr« .. DICATION To my paren sand my family who have . 1ven me a deep apprecia ion for the value of an education • • 11 ACKNOWL~DGMENTS I would like to thank Drs. Arnold Dunn , Peter M. Shugarman, M. Michael Appleman, Basil Nafpaktitis , and Arnold Brodie for their encouragement, understanding, an guidance throughout my graduate experience . A very special thank you is due to Dr. Russell Zimmer, Director of the Santa Catalina Marine Science Center, for without his su port, this roject could not have been undertaken. To Maymie, I can only say hat ut for your invaluable assistance as trapmaker, technician, and socia~ director, the past six years would certainly have been considerably less enjoyable. This work was supported by a National Science Foun dation Traineeship and Biomedical Sciences Support Grants from the United States Public Health Service. • • • 11.l. TABLE OF CONTENTS ACKNOWLEDGMENTS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Page iii LIST OF FIGUR • S .••. , ••.•• , •••...•••. , ..•• , , ..••. , . vi LIST OF TABLES . . • . . . . . • • . . . . . . • . .............. ... . . . Vll Chap er I. LIT RATURE REVIJW ..............•......•..... 1 Introduction , etabolic Pate e ilation of Plasma Glucose Regulation o Glycogen ~etabolism RePUlation of Fat etabolism Gluconeogenesis from Amino Acids Temperature Effects on enzymes of Hetero- therms Approach to the Problem I I . r/JETH ODS . • . . . . . • . . . • . . . . • • . • . . . . . . • . . . . . . . . . . 11 Care and ~aintenance of the Animals Surgical rocedures Experimental Procedures for Intact In vitro Assay of Muscle Glycogen phorylase Animals hos- III. RESULTS . . . . . • . • . • . . • . • . • . . . . . • . . .•..•...... 25 General Life His ory of the Kelp Bass Plasma Glucose Levels Twenty Four Hour Fluctuations Seasonal Fluctuations Glucose Loading xperiments Epinephrine Effects . lV Chapter Glucose Turnover Rate Introduction Isotopic Glucose Steady tate Conditions Non-steady tate Conditions Gluconeogenesis from Alanine Introduction Isotopic Alanine and Glucose Alanine Disappearance Alanine Appearance in lasma Glucose In vitro Assay of r~uscle Glycogen Phos phorylase Page IV. DI CU3 ION ..•••••••.•••••••••••••••••.•..•• 90 v. VI. Regulation of lasma Glucose Glucogenic ~ubstrates .ruscle Glycogen Phosphorylase sur . ...L\RY •••••••••••••••••••••••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 115 117 V LI, T OF FI U Figure Page 1. natomical Location of the Implanted Cannula in the Ventral Aorta of the Kelp Bass . . . . . . . . . . . , . . . . . . . . . . . . . . . . . , . . . . . , , . . 1 3 2. T~enty Four lour Variation in Plasma Glucose Concentrat· on . . . • • • • • • • • • . . . . . . . . . 30 3. easonal Variation in Plasma Glucose Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4. Glucose Loadin xperi ents ... • • • • • • • • • • • • 36 5. inephrine 8ffects upon Plasma Glucose Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 • • 10. 11. 12. 13. 14. e ~etabolic •ate of 3 of Glucose-6- 1 , -6- 1 1.~C 14 and C Labels . . . . . . . . . . . . . . . . ' . . . Steady tate Conditions • • • • • • • • • • • • • • • • • • • • t on-steady ~tate Conditions • • • • • • • • • • • • • • • • Ton-steady tate Conditions • • • • • • • • • • • • • • • • 1lon-steady "tate Conditions • • • • • • • • • • • • • • • • on-steady 14 c-4lanine 14 C-Alanine State Conditions I e e I t I t I t e • e t I t I Disappea ance from the lasma .. Appearance in Plasma Glucose .... 14 C-Alanine Appearance in Plasma Glucose • • • 15. The ~ffect of Temperature upon Partially Purified Sreletal ffuscle Phosphorylase Activit. ram Three Species of Fish ...•.... 16. Arrhenius Diagrams for Fish Muscle Glycogen Phosphorylase ............................. . 45 48 61 63 66 68 71 76 78 83 87 . Vl LI. T OF TABLES Table Page 1. Avera e Plasma Glucose Concentration in r.1g% Based on the Initial Sample . . . . . . . . . . . . . 27 2. Average lasma G uco...,e Gone~ tration in r g/a Based on the T'1eant::' of All Serial Samples in an · ..... xperiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 3. Liver and Muscle Glycogen Content in Fed and Fasted Kelp Bass expres ed as g Glycogen Glucose Per 100 M Tissue ·r et )J eight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 4. ~stimation of Glucose Parameters during Steady tate Conditions with a Single Injection of Tracer Quantities of Isotopes .. 52 5. Parameters of Glucose metabolism in Fed and Fasted Kelp Bass under Steady tate Con- ditions • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 6. A Comparison of Parameters of Glucose Wetabolism in 1ammals and Kelp Bass ......... 58 7. Incorporation of Alanine- 14 c into Plasma Glucose . • • • • • • • • • • • • . • • • • • • • • • • • . . . . . . . . . . • . 74 8. ~inetic Parameters of Kelp Bass Phosphor- ylase .. •••···••• .. •••·•••••••••············· 81 9. The 1 ffect of Te perature upon Total Phos phorylase Activity of Two Midwater Fishes as Compared with a hallow Water Fish .. . .... 85 vii CHAT 'RI LIT ~R '1 1 UR ' R VI J, Introduction Bon fishes are amon the most successful of organisms in term of the numbers of teleost species as well as their invasion of a wide variety of habitats. Their success lies in part, in their abili r to a just their metaboli m to et var in environm ntal conditions. woof the most ·m ortant o thee environmentally re ated factors to be di cussed below are chan~in~ tempe atures and otential starvation due to decreased food av~ilability. Fish have adapted successfully to a diversity of ther- mal habitats. ome ex~st at temperatures of -1 to -2 C, while others tolerate temperatures near 40 C. Shallow water marine fish may experience slowly changing seasonal thermal variations. On the other hand, some mesopelagic species may under o vertical migrations daily which expose them to as much as a 10 to 15 C chan e in ambient tempera ture. The metabolic and biochemical reor anizations which accompany such thermal adaptations have been under investi~ation for many ears. Fish are also able to adjust their metabolism in order to withstand lon period~ of food deprivation, periods 'which in mammalian terms constitute severe starvation The 1 most notable examples are almon and eels which go for extensive lengths of times without feeding during seasonal mi rations (69). Also, it has been proposed that open ocean mesopela ic species which mi rate to the surface to feed during evening hours may subsist at depth with minimal food for some time during reproductive eriods. Further more, the nature o the available food sources and the etabolic re uirement of deeper mesopelagic and bathy e lagic fishes has lon been a problem confrontin~ oceanic biolo ists. These ish live well below the productive near surface waters and must cope with a l"mited food supply ( 71) . uestions which need to be answered in understanding both adaptation to changing thermal environment and to with standing lon term fas-tin in fish are: 1) vVha t are the primary stora e forms of metabolic energy? 2) How can mobilization of these stora~e products be regulated? J) ow do the previously mentioned environmental factors pro mote metabolic reor anizations or alter preexisting patterns of ener v metabolism? B. Metabolic Rate Classic metabolic studies measuring oxygen consumption have indicated that fish (35, 36, 90, 92, 115) and other heterothermic animals (92) have lower metabolic rates than mammals of comparable size (92). In conjunction with the above observations, many authors have reported that the 2 blood glucose concentrations in a wide variety of fishes are enerally lower than mammals (7, 32, 65, 8) as would be expected with lowered metabolic demands. It is not sur prising therefore, to find carbohydrate metabolism as measured by the use of radioactive isoto es is markedly lower than in mammalian systems ( 15, L~7, 68) . Brown ( 15) demonstrated that following an intraperitoneal injection of glucose-1- 14 c or glucose-6- 14 c, the maximal specific acti vity of the released 14 co 2 occurred between 10 and 12 hours with significant release still occurring at 48 hours. Sim ilar findings were reported by Hochachka (47) and Liu, et al., usin 14 C-glucose and 14 c acetate in intact oldfish (15), Although utilizin isotope tracers is an excellent method for observing dynamic processes of metabolism of intact or anisms, such studies in fish are sparse, limited in art by the difficulties in sampling while maintaining a reasonably unstressed experimental animal. C. Regulation of Plasma Glucose Concentrations There is ample evidence that similar endocrine control mechanisms operate in regulating carbohydrate metabolism in both fish and mammals. The usual metabolic responses to rapidly acting hormones such as insulin (7, 31, 32, 65, 77, 78, 88, 89, 111, 113), glucagon (7, 31, 63, 88, 113), and epinephrine (7, 62, 79, 86), however, require hours to days to occur in intact fish, rather than minutes as in mammals (J3, 38). To avoid spurious results which mi ht arise as 3 a resu t of s ecies · ecificit of the proteinaceous hor mones, purified codfish in ulin has been used and the results are similar to those obtained with mammalian insu lin admi1istered to fish (77, 8) . The effect of purified fish ~1ucagon on plasma lucose has not been re orted. Recent data obtained by Tnorpe and Ince (113), however, indicated that lucagon may exert a hy er lycemic effect within JO minutes after intravascular injection of the 1or mone ·none to thee veek starv d ike, , ile codfish in u- in initiated an extended h po lvcemia ,hie began nine our afte o one a ini t at·on. T action o these to hormone ma e 1 be euarate te porally in fi lthouRh the nutritional state o t e ani al may certainlr determine the res onsiveness of metabolic . oce ses in teleo~t fisheo to glucose re ulatin hormones, t e role of insulin in controllin 6 lasma glucose levels seem to be of less importance tan glucagan over short tire periods (0-30 minutes for gluca on, J-24 hours for insulin) in fi~h. Furthermore, following the administration of a glucose load to both marine and fresh water fish, plasma lucose concen trations ma remain elevated for as lon as 48 hours indi catin~ a delayed response in endo enous ins11lin release and/or activity (7, , J2, 41, 8). The fact that plasma lucose responds to hormonal stimulation shows that endo crine mechanisms are functioning , even if delayed respon siveness indicates that the physiolo ical role of these 4 hormone is exerted over long time periods as compared to mammals. s a consequence of this delayed response, a dis- tinct temporal separation in hormone release and hormone action may be suggested. In conjunction ·vi th the delayed and loose hormonal con * trol of plasma glucose, many authors have reported that the glucose concentrations in a broad variety of marine and frech water fish is enerally lower than in mammals with wide variations in levels commonly observed in different s ecies as wel as in differen individuals o the same spec·es ( 2, 8) . D. Regulation of Glycogen Metabolism The glycogen content of fish liver renains table over lon periods of fasting (64, 72, 83, 106) indicatin that glycogen does not appear to be u~ilized as a primary reserve of metabolic fuel and may even be stored durin fasting unlike mammalian liver yco en (88). Other evid- ence does exist, however, that liver lyco en is depleted to a varyin extent during starvation and may be dependent upon .pecies or environmenta conditions (55, 64, 69). In addition, prolon ed exercise may stimu ate liver 1. co en oly is ( 55). Prior nutritional histor.r ma, affect o-lyco en utilization as well. I rats are fed a hiah protein, car bohydrate free diet and then fasted, mobilization of liver glyco en is depressed (JO). This certainly must be {ept in 5 min e inte r ting re u ·sf o di fer nt teleo t spe- cies v ich miFht hav wide v va in djetarv habits . cogef!_ ·sh u. c e rrlyco n is . eadily obilized by exer cise (2L~ , l~6, 86, 109), lthouo-h the effect of starvation upon mu cl level ·snot clear (52, 55 , 64). Th. enzyme controllin lyco enolysis, lycogen phos horyla e, ha been · olated from chondrichthyean (2 , 20) and teleost ( 21) muse e and its activity measured by everal inve t· a or (21, 57, 106) at o h methodolo ical differenc sin . . . . . e tu 1es ma e com l on dif lCU t. Ou data ln l- . C te evelc- of lvco n D 0 hor .rla e ctivitv in bo h el ba r t m cle ( 8, 94 Ji on et • ( ) , the nzy t·c l e C .c d do not se m to ed, her Ca ++ y be involved . V r m ln ol . Thi . is not unlike . . Sl C'. in view .. ca cium relea e in mu. cle contraction. egulation of Fat ~etabolism _ _._ _____ _...;..;_;;,..;;...;..~~-:.....;;;.;...~:..;;; ) . d t . rnl com on t 0 he e ho monally ct · - . ti ulatin P' yco of the role of Fish accumulate large amounts of lipid in liver and uscle (24, 46 , 69) , however, with the exce tion of the . extensive amounts 0 lipid utilized durin sq} on mi ra- b e tion~ (10), the role of lipid metabolism durino- tarvat ·on and temperature adantat · on remain. to be clarified. ech- - . anism o lipol,si. and lipogenesi have beens udied (10, 2 , 1 / r'J ) • t . . , , an . , wi h starv ti on , a r1 e n p asma fr 6 fatty acids (FFA) has been observed (11, 64, 72,110). In addition, ,vhile 14 c-acetate incorporation into lipids increases during cold adaptation (51, 60), total content decreases (60). Hormonal control of lipid metabolism in fish is even less well established than that of blood lucosc levels Insulin promotes decreased plasma FFA and phospholipids, althou~h the time required to observe the insulin effect may var rom 30 minute to 12 hours(??), while diabeto - en·c a ents. ch a allo an and streptozotizin do inc ea.e la a leve in oldfi ( 78) • . ne r1ne, a mam- m lian i o ytic hormone, ma increase FA (62, 66) or lower FA levels in fi h plasma (79). 1inick and Chavin su )gest that epinephrine effects may be insulin mediated followin • epinephrine induced hyper lycemia (79). Cate cholamines decrea e 14 c-acetate incorporation into liver lipid (67), yet have no effect upon activation of the long chain lipase of red muscle, a fatty acid metabolizing tis sue (10, 12). In mammalian adipose tissue, epinephrine is a otent stimulator of lipol si~. F. Gluconeo enesis from Amino Acids Proteins may act as potentially important ener y stor a e reserves in fish , ultimately contributin to the pro duction of plasma lucose (95). That fish utilize muscle protein and/or decrease protein synthesis during starvation has been suggested by many investigators (34, 56 , 69 , 107) 7 Furthermore, protein catabolism, subsequent transamination, and conversion to glucose or glycogen appears to be func tional and under hormonal control (17, J4, 107, 108) as in mammals. Larson and Lewander have reported increased liver glutamic-oxaloacetic acid transaminase (GOT) with starva tion (64). Storer d~d not observe such starvation induced cane in liver lutamic-pyruvic acid transaminase (GPT) of oldfi , lthou~h the enzymatic activity wa re pensive to cortiso injection (107). In trout, liver GPT activity is also increased b cortisol and cortisone, 1hile GOT activity does not change (J4). vi ence in mamm lian sys tems indicate that while glucocorticoids may stimulate transaminase activity, their primary effect is to enhance production of substrate from e trahepatic reserves under physiolo ical conditions (27), This mi ht exnlain the sometimes confusin and contradictory nature of the effect of lucocorticoids and starvation upon gluconeogenesis in both fish and mammals. For a comprehensive review of the literature concerning the im ortance of luconeogenesis from alanine in the ener y econom of fish and protein turnover in extrahepatic tissues,seo section IV, B. J. Temperature ffects on Dnzymes of Heterotherms Thermal adaptation has been shovm to alter metabolic rates in a compensatory fashion so as to offset a strict dependence of these rates upon temperature (16, 39 , 40, 90 , 8 93). Alteration of metabolic rates may involve a tempera ture promoted shift in the fraction of carbon flow throu h lycolysis, the pe11tose shunt, and the Krebs cycle (18, 47, 4, 51, 102). Although these physiolo ical readjustments have been inresti~ated at all levels, current emphasis is placed upon the kinetics and regulation of key enzymes in the above pathways. In many heterothermic animals, enzyme substrate affinity is highest at the environmental tempera ture (53, 98, 103). As the acclimation temperature i~ altered, so also is t e maximal enzyme-substrate affinity as measured by K. The evidence suggests either the pres- m ence of inducible thermall adapted isoenzymes (50, 74, 80, 81) or a temperature dependent alteration in the kinetics of a given enzyme (4, 99, 100). Hoskins and Aleksiuk have found thermal adjustments in the 1 1ichaelis constant (Km) in the liver su ernatant malate dehydro enase of a garter snake which experiences daily temperature extremes. Iso enzymes of the enzyme are present which promote enhanced velocities at low temperatures, however~ these isoenzymes are not inducible by long term adaptation (54). Independ ence of enzyme-substrate affinity from adaptation tempera ture has been observed in rainbow trout liver fructose diphosphatase (FDPase) by Behrisch and 1Iochachka (5), although they made no attempt to isolate specific thermally induced isoenzymes. A second effect of tern erature mi, ht involve the 9 affinity of enzymes or modulators. This icturc is some- 1hat unclear ·n that specie~ difference do occur, po~sibly a a re ult o environ ent needs. FDPase i olated from trout liver ha a tern er tur ~ensit"ve AP inhibition (5), whi et at of salmon and lun fish is temperature inde endent (J, 6). H. A roach to the roblem Thin dif'oertation research is directe.d toHards a tudy oft e turnov r of ~luco. e in kelp ba~ sin i otopic dilution techni ue ith the ultimate oal of understandin the impo tance of ~ uco e and gluco enic proce ~e . in car- nivorouc f" he . model is pro o.ed for the e lation of this metabo ic uel b hormonal and environmental factor int e int ct, a in fish, Paralabrax clathratu . Thece stu ies include the at·ve contribution to lucose s thesi from Coric clin o lactat, om 1 co enolysis, and . rom a anin ,luconeo ene is as e fected b lon:, and 1ort te~m starvation. Finally, a preliminary investi a tion into the effect of temperature on the kinetics of muscle glyco en phosphorylase is made in those enzymes iso lated from P. clathratu , a ~hallow water marine inhabit ant, and two midwater fishes of the ;an Pedro Basin: Trinhoterus mexicanus, and Leuroglossus stilbius. 10 CHAPTER II METH DS A. Care and Maintenance of xperimental Animals Kelp bass, Paralabrax clathra us, were obtained by rapping in Big Fisherman's Cove, Catalina Island. The trap, baited with squid, was set in depths less than 10 meters to inimize tress cased by swim bladder expansion in he ca tured fish a the tra wa raised. Bas wei hing 200 to 400 ram re selec ed and placed in 50 allon holding tanks with fre hr nning sea water at ambient ocean temperature. Squid was rovided daily as readily accepted food source. Re um tion of feeding after capture was used to indicate adaptation o the new environmen of the holding tanks Food was then withheld fro experimental animals whe fasting con itions were necessary. B. ~urgical Procedures • Anaesthesia Fish are anaesthetize with tricaine methane sulfo nate (1n 222, Aldrich) in sea water (1 /10 1) until they make no escape response when touched. At this point, res piratory movements still occur. Afte anaesthetization, the fish are transferred to the operating table, placed ventral side up, and sea water containing anesthesia is 11 perfused across the ills with a tube placed in the fish's mouth. This procedure insures an adequate supply of oxygen for the organism. 2. Cannulation Subsequent cannulation procedures were modified from those of mith and Bell (97). The operculum i. lifted back and anchored, exposin~ the ills. The beating heart can be seen lyin ventral to the gills and beneath the membrane linin, the ill chamber. An inci ion i made in the overlying membrane and an muscle tissue is retracted carefully, exposin the ventri cle and , e hulbous arteriosus ( fi re 1). Te cannula i polyethylene tubing ( 50), 70 cm in len th, attached to the shank of a 22 au ,e hypodermic needle bent at an angle of approximately 120 de rees (fig ure 1). The bevel opens into the oblique angle formed by the needle. The cannula is then filled with Courtland saline (119) containin 100 units of sodium heparine per ml. The saline is prepared without lucose. The needle is inserted into the ventral aorta anterior to the bulbous arteriosus and posterior to the first ill artery branch (figure 1). The ne dle can be carefully threaded back towards the bulbous arteriosus. As soon as the cannula is in lace, blood ulses back into the tubing. This blood can be pushed back into the aorta with fresh heparinized saline. The cannula is then anchored firmly to the skin 12 Figure 1. Anatomical Location of the Implante Cannula in the Ventral Aorta of the Kelp Bass 13 .... ~ gi 11 arter ANTERIOR cannula __ ..... v<2ntra I aorta bulbous - arteriosus DORSAL _ __..,.~ __ au r i c I~ ~ POSTERIOR ventricle __ ___,,,. ____ _ VENTRAL with ilk surgical sutures, makin sure the tubin exits at a location causing minimal interference with opercular movements. The cannula is washed free of blood and the free end is heat sealed. J. Recovery Fresh sea water withou anaesthetic is then cir culated across the gills of th anaesthetized fish until weak opercular movements b • in. The fish are then perma- nently placed in indivi ual experimental tanks also sup- plied with fresh running sea water. The entire nrocedure ~ should t keno loner than 25 minutes includin ecovery from anaesthesia. To in ur the abs ence o sur ical trauma, cannu lated animals are not used until the third or fourth day after surgery. The cannula is flushed daily to prevent bloo from clotting in the needle and tubing. During the time between surgery and experiment, the fish do not eat. If given a least a week of recovery, the animals would resume feeding. Unfortunately, however, the cannulas only remain functional for about five to six days. It should also be mentioned that the entire surgic2l procedure is best suited for fish with a wide opercular openin for max imum exposure of the ventral aorta. c. jxperimental Procedures for Intact Animals 1. Sampling protocol Fish are fasted for a prescribed period of time 15 prior to and including surgery and recovery as they do not feed after cannulation. Fish are classified as fed if they have received food up to two weeks prior to the experiment; fasted, if they have not fed for over two weeks. According to my observations, approximately two weeks are required for passage of food through the digestive tract of these fi h. Isotopically labelle 3 14 lucose-6- H, -6- C or ucose-6- 3 {, alanine-u- 14 c is injected via the indwelling cannula at time zero. The experimental animal is free swimming in the aquarium at this time and for the duration of the experiment. After isotope injection, the cannula is flushed several times by drawing blood up the full extent of the tubing and washing back with heparinized saline. This prevents contamination of the early blood samples by the infused isotopic compounds. Subsequent to the injec tio of isotope, serial blood samples of approximately 0.15 ml are drawn at appropriate intervals. To minimize stress from blood loss, the total blood drawn from any animal during a given experiment does not exceed ten percent of the total blood volume of the animal. The fish is sacrificed by slowly injecting one ml of Diabutal (sodium pentabarbital) via the cannula. Unfor tunately, most fish convulse during this procedure. No other method was found to be any more satisfactor y. 16 2. Glycogen assay The fish are quickly weighed and muscle and liver samples are taken to determine glycogen content as well as the incorporation of radioactive label into glycogen. The entire liver is removed while a portion of muscle dorsal to the lateral line between the first and last spines is dis sected out. Glycogen is extracted after the method of Good, et al. (J?) with boiling 30% KOH, precipitated in 95% ethanol, and repeatedl.v reprecipi ated from 95% ethanol. The lycogen is then hydrolyzed in boiling 1 N H 2 so 4 , neu tralized, an assayed for glucose. An aliquot (1.5 ml) is also placed in 12 ml of Nu's scintillation fluid composed of 5.0 g PPO, 0.2 g POPOP, 100 g napthalene, in 1 liter of dioxane (120) and counted in a Nuclear Chicago Unilux II liquid scintillation counter. Glycogen content is expressed as mg glucose/100 mg tissue; specific activity as corrected CPM/mg glucose. An alternative method for assay ing glycogen is the Krisman assay using an oyster glycogen standard and the reprecipitated glycogen unknown (61). An approximate ~orrelation between the two methods was made by hydrolyzing oyster glycogen standards and assaying for glucose content. After tissue samples are taken, the sex of the animal is determined. J. Plasma glucose assay Plasma is eparated from whole blood after 17 centrifugation for one minute in a Beckman 152 Microfuge. A protein free filtrate is prepared according to the method of omogyi (104) with a zinc-barium precipitation of 0.05 ml plasma. The final volume is 1.55 ml of which 0.5-1.0 ml i assayed utilizing a modified glucose oxidase (Glucostat, orthington Biochemicals) (73). 4. Isolation of Elasma glucose (25) In order to dete mine the 14 c, 3H of pla ma lucose, a plasma sam le of a 0 1 ml . spotted a continue u line on lS a s ecific activity oximately 0.05- hatman #1 paper. On e·ther side of the plasm sample, a small drop of lu ~ose is spotted to allow location oft e pl sma lucose after chromate raphic separation. The chromatography is carried out overnight with a descen ing solven system of n-propanol, ethyl acetate, and water (7:1:2). The"refer ence" chromatograms are sprayed with 0.1 ml aniline/25 ml 1.66% uthalic acid in n-propanol and developed by heating. Glucose can be then eluted from the chromatogram with dis tilled wa er using either descendin chromatography or a Reeve An el sleeve in a centrifuge tube. The eluant i diluted to an appropriate volume. An aliquot is assayed for ~ucose concentration and another is counted in Wu's cintillation fluid as previously described in section 2. uecific activity of both 14 c and 3 H lucose can be deter- mined and plotted on a semilog plot versus time. ubse- quent calculations of fractional turnover rates, gluco e 18 pool, and glucose space will be discussed in the Results section. 5, Tandem column separation of glucose, lactate, and alanine For separation of plasma glucose, lactate, and alanine, a triple column set up was employed according to methods communicated to us by Dr. Joseph Katz. Three sets of pasteur pi ette columns were prepared and placed one below the other in the following order. First, at the top, was a bank of columns each containing Dowex 50 x 4 (100-200 mesh), hyd1ogen form. These columns will retain amino acids. Below these were a set of AG 1 x 8 (100-200 mesh) acetate form columns for binding lactate and pyruvate. Finally, Dowex 1 x 8 (100-200 mesh) was prepared in the bora e form for retainin glucose. a. Preparation of resins 1. Dowex 50 x 8 (100-200 mesh) H form is cycled four times with 1~ NaOH, distilled water, lN HCl, finishing with the BCl. The resin is washed in distilled water until a neutral pH is reached. The pasteur pipettes are plugged with glass wool, then filled to the constric tion with resin. Finally, a plastic funnel is cut off 0.5 cm below the funnel and placed on each column. A tight fit between the column and the cut off stem of the funnel is required. 2. AG 1 x 8 (100-200 mesh) acetate form is 19 preyared by cycling the resin four times with lN NaOH, dis tilled water, and lN acetic acid. The resin is washed in water until a pH greater than 4.8 is obtained. Columns are prepared as described above. 3. Dowex 1 x 8 (100-200 mesh) chloride form is washed three times with lN NaOH, 3 times with distilled water, and 10 times with saturated boric acid. This resin can be stored in 4% boric acid or rinsed with distilled water. Columns are packed as described above. b. Preparation of sample An ali uot of plasma (0.05 ml) is precipitated with 1.0 ml of 61 perchloric acid and 1.0 ml distilled water. After centrifugin, the supernatant is titrated with JO% KOH to approximatel,r pH 6. If the titration is performed at O C, the neutralized supernatant can be sepa rated from the 1 c10 3 precipitate without centrifu ation. c. Separation of sample constituents The tandem columns are assembled as described previously and washed thoroughly with distilled water. After all columns have drained completely, 1.0 ml of sample is applied followed by 2,5 ml, 3.0 ml, and 2.0 ml of , water. Each complete fraction is collected separately after it has passed through all three columns. A 1.0 ml aliquot of the 3.0 ml fraction is counted in Wu's cintil lation fluid to determine the c0unts per minute (CPM) of JH in plasma water 20 At this point, the triple columns are sepa rated and each set is treated as described below in order to determine the corrected CP~ of 14 c, 3 H glucose, alanine, or lactate per ml plasma. In our laboratory, the triple column separation led to recoveries of 86 to 102% when 14 14 . known quantities of ·sotopic H 2 o, C-glucose, C-alanine, and 14 c-lactate were used. d. ·eparatio!}_procedur~for the individual ion exchangg__g_olurnns raction Volume Eluant lanine column Dowex 50 x 8 Hydrogen form 6.o ml 1,5 ml J.O ml* 1,5 ml O.llVI Phosphate buffer, pH 8.0 Lactate column AG 1 x 8 Acetate form Glucose column Dowex 1 x Borate form 1.0 ml 3.0 ml* 1,0 ml .0 ml J.O ml* 3,0 ml l.ON Formic acid 0.5N Acetic acid *One ml aliquots are counted to determine 3 H, 14 c content of eluted compounds per ml of plasma. D. In Vitro~ssay of Muscle Glycogen Phosphorylase 1. Animals used Phosphorylase was partially purified from three species of fish: Paralabrax clathratus, a shallow water fish with a seasonal temperature variation of 10 to 21 C, Leuroglossus stilbius, a rather diffusely distributed mid water fish (300-500 m depth distribution) experiencing a 21 thermal range of 5 to 10 C, and Triphoterus mexicanus, a vertically migrating mvctophid undergoing daily thermal chan es of approximately 5 to 15 or 20 C, dependin upon seasonal chan~es in surface water temperatures (500m - surface depth distribution). The shallow water fish, Paralabrax clathratus, was obtained wile SCUBA diving in less than ten meters by spearin the animals. Muscle tissue was filleted and frozen at -20 C. The midwater fishes, T. mexicanus and L. stilbius, were taken in the San a Catalina Basin off the outhern California coast, u ilizin an open mouth, 10 foot diame er Isaacs Kidd midwater trawl. Trawling depth was between 200 and 500 meters, although sampling of course occurred from depth to the surface. All trips were made on the University of Southern California ma ine sciences ves sel, R/V Velero, durin June hrough September, 1973. The entire animal was frozen immediately in liquid nitrogen with final separation of muscle from head, fins, and gut being performed just prior to muscle homogeniza tion. In effect, the "muscle" preparation contained skin and bone as well, due to the small size of the midwater fishes (approximately one to two grams). 2. Partial purification ang_assay procedures Initially, muscle tissue, frozen in liquid nitro gen, was powdered in a Spex freezer mill at the maximum speed for one minute, then homogenized in a buffer composed 22 of the following (1 g tissue/5 ml buffer): 0.04M sodium malate pH 6.5, 0.04M mercaptoethanol, 0.005M EDTA, and 0.02M sodium fluoride. Homogenization was carried out for one minute at top speed using the micro attachment to a Sorvall Omnimixer. Subsequently, we determin d that simple homogenization was equally efficient at releasing phos- horylase without the freezer mill pretreatment. The remainder of the phosphorylase isolations were repared by directly homo enizin thawed muscle in the above buffer for 2 to 3 inutes usin the Omnimixer. The micro attachment was employed whenever appropriate. The homogenate is centrifuged at J,OOOxg for 15 minutes in a ~orvall RC-2B . The supernatant is brought to 42% saturation at 20C with saturated ammonium sulfate (S S) and left stirring overnight at 4 C. The precipitate is collected by centrifugation at 16,000xg for 20 minutes, then dissolved in a diluting buffer containing O.OlM Tris, p 7.5, O.OOlM DTA, 0.04J mercaptoethanol in the following proportions: 1 ml diluting buffer for every 4 ml homoge nate precipitated. The A precipitation was necessary due to the low enzyme activity in the straight homogenate of the midwater species. For uniformity, kelp bass muscle phosphorylase preparations were treated in the same manner. Active and total phosphorylase levels were measured after the methods of Hedrick and Fischer (44). "Active" enzyme i assayed without AMP in the reaction mixture; 23 "total," in the presence of 2 mtl AMP. Activity is measured in the direction of glyco en synthesis with velo city expressed as umoles lucose-1-phosphate hydrolyzed/ min/g tissue. Preliminary purification of kelp bass muscle phos phorylase b was carried out according to the methods of Cohen, et al. for dogfish skeletal muscle phosphorylase b ( 20) • 24 CHAPTER III RE ULT A. GeneraL_!4_fe.J:!istor.i of the Kelp Bass In order to interp~et data concerning metabolism in cold blooded vertebrates such as the kelp bass (Paralabrax clathratus Girard), some knowledge of the organism's habits and life history is necessary. Environmental factor may have rofound effects upon metabolic pathways and their egulation in heterothermic or anisms as discussed previously. Kel bass are found in or near kelp beds along the Pacific coast from ~onterrey Bay, California to f/Ia dalena Bay, ~exico (122). All animals used in this study were captured at the Isthmus area of Santa Catalina Island in waters shallov,er than ten meters, al though many individuals can be found considerably deeper. In the capture location, seasonal temperatures may vary from 10 to 21 C, with the highest temperatur~ attained during summer months at 1.5 meters. Daily t mperatures in the capture area fluctuate only 1 to 2 degrees when measured with a continuously recording Ryan model D thermograph. This species of fish is moderately active, particularly individual3 in the 150 to 350 g weight range used in this study. They are usually found in the open, swimming among 25 the kelp stipes. tudies by the California Department of Fish and Game indicate that kelp bass generally do not migrate far from their local area (122). Larger individ uals appear more quiescent, although they move rapidly when disturbed (per onal observation). Kel bas are carnivorous, feedin on other fish, crustaceans, and mollusk. The majors ecies component of the diet may vary considerably as does the frequency of feeding. For example, durin he late winter and early prin of some ears, lar e numbers of quid (Loligo opalescens) come inshore to spawn , providing an abundant food source . Other times of the year, food is sparse. If considered in mammalian terms, "long term starvation" may be a normal condition in these fishes a a consequence of their opportunistic mode of feedin. exually mature individuals begin spawning as early as April with other members of the population continuing to spawn through late September (122). • Plasma Glucose 1 Glucose levels Unlike plasma ~1ucose concentration in mammals, this value varies considerablf from fish to fish and can fluctuate markedly with time in any individual. Table 1 shows plasma glucose values in fed and fasted kelp bass. "Fed" or "fasted" fish were classified as such based on an observed time of approximately two weeks for passage of 26 Table 1. verage Plasma Glucose Concentration in ~g % B9.sed on the Initial ..:>ample Condition ~o. of Fish Range Fed (1 - 14 day) 18 17,8 - 100.2 Fasted (15 - 42 days) 7 4.o - 44.4 ·----·----------- ➔(. tan ard error of the mean ♦ 11ean+S~M '( "i. 35.0 + 4.8 19.6 + 5.4 27 Table 2. Average Plasma Glucose Concentration in Mg% Based on the Means of All Serial Samples in an Experiment Condition No. of Fish Range if Mean+SEM ------------------ Fed (1 - 14 days) 17 16.4 - 105.2 48.3 + 5.7 Fasted (15 - 42 days) 7 15.6 - 44.2 25.9 + 4.J ____ , _____ , __ * Standard error of the mean 28 food through the digestive tract. Fish sacrificed prior to two weeks had material remainin in their intestine. Plasma lucose concentrations were determined from the initial samples drawn during isotope dilution experiments. Although variability within both fed and fasted fish is high, the glucose levels of both groups are significantly different (t test, P 0.02 level). In addition, the high est plasma glucose concentration attained in fed fish (100.2 mg%) was considerably greater than that found in fasted animals (44.4 m o) These data imply that glucose levels are maintained at hi her concentrations while food is present in the di estive tract, and plasma concentration dro s si ificantly with extended fastin. The variation amon individual kelp bass within bot nutritional oup- ins is too great to make any other conclusions as to the relationship between len th of fast and lucose concentrations. In the course of an experiment, plasma glucose levels may vary considerably. hen the mean plasma glucose concentration is calculated from all samples in a given experiment and all the experimental means compiled, there ; is still a significant difference between fed and fasted kelp bass, although the average concentrations have increased (see table 2). 2. Twenty four hour fluctuations Figure 2 illustrates the variation in plasma 29 Figure 2. Twenty Four Hour Variation in lasma Glucose Concentration The ata from four individual kelp bass has been graphed with zero ime re resentin 1300 hour (1:00 P.M.). JO \..A,) ~ 140 w V) 0120 u :::, t5100 <{ 2 80 V) <( 0: 60 ~ 0 l9 40 2 20 0 2 4 6 8 10 12 14 16 18 20 22 24 HOURS ~luco~e occurrin in four fed individuals over a 24 hour period. To minimize a stress induced glucose response to blood loss, the total amount of blood sampled per fish did not exceed ten percent of the estimated total blood volume. The maximal change in blood sugar is from 23 m % to 158 mg% in animal K38; the minimum is 54 m o to 92 m o in K31. These fluctuations in glucose concentrations indicate that this hysiological parameter is not re lated to the same extent as in mammals where little variation occurs. There is not sufficient information to determine the dependence of 1 cose levels u on he time of day as ould be he case if iurnal rhythms e e involved. Figure 2 is, however, raphed with zero hours re resentin 1300 ho rs. J. Seasonal fluctuation That seasonal changes seem to be without effect on plasma glucose levels is suggested in figure J where the initial plasma glucose concentrations are grouped by mon h and by nutritional condition. The highest values occur in January (92 mg%) and May (100 m %), however, these repre sent only ten ercent of the total fed experimental ani mals. Data is sparse between e tember and illarch due to consistent poor success in trappin fish during these months. his was due in part to winter storms, smashed traps, and a eneral reluctance on the part of bass to enter the traps at this time of year. The data presented in fi re 2 was collected during the reported onset of 32 Figure J. easonal Variation in Plasma Glucose Concentra ion. Upper line represents mean fe glucose levels; the lower line, mean fasted glucose levels. • , plasma gl~cose values of fed in ividuals; O , plasma glucose values of fasted individuals. 33 r---r-----,r----r----,----,---r--....-------- 0 0 0 ~ • 0 (X) • 0 <.D • • 0 • •• 0 Ill e 0 • • 0 N 0 • z 0 I 7 ~ z 0 72 <( LL 34 reproductive maturation and spawning in 150 to 350 g bass (122). Post-mortem examination of all experimental ani mals, however, confirmed a full range of reproductive stages: immature, mature, and spawned out. ~o conclusions can be dravm with respect to the effect of time of capture, ex, or de ree of e ual maturity on plasma lucose concentr tions. 4. Glucose loading experiments In or er to inve ti ate the h.~r::oio o ical regula tion of fish blood su ar levels, lucose loads were admini stered via the indwell"n aortic cannula. The resu ts of loa ing with 0.2 glucose perk fi hand 0.1 g ucose per kg fish are shovm in fi re 4. These loads are e uiva lent to 2.0 and 1.4 times the estimated glucose pools of the resnective animals (section II, C. J) lasma glucose concentrations increase to approximately 5,8 times the preinjection con rol value in fish K24 and 4.4 times in 32 ,ith the shar est rate of decline occurring within three hours in both (fi re 3). In K24, lasma glucose values remain at the lev8l attained at J hours for the remainder of the samplin eriod (20 hours) while in Y32, plasma lu cose concentrations reach control values by 12 hours . If we assume that the drop in the blood su ar is brought about by enhanced insulin secretion , we would have to conclude that the rate of release and/or the action of insulin at in ulin sensitive tissues is considerably slower than it is 35 Figure 4. Glucose Loading 1 xperiments The glucose load was injected at zero time with the data being expressed as a ratio of plasma glucose concentration at time, t, divided by the mean lucose concentrat·on o~ thee preinjection plasma samples . 0 , animal K32 (0.1 g lucose7 kg body wei~ht); e , animal K24 (0.2 g lucose/ k body wei ht). 36 0 M I.() (\J 0 (\J (/) l[) ~~~~=='==Jo ~ M C\J r- ( IOJ l uo:, / I OlU Z>W ~ J 2>dX2)) 3SO:)n79 v'V'JSv'ld Q'.: :::, 0 I 37 in mammals, Such a slow response of these fish to endoge nous insulin would in part explain the apparent loose regu lation of plasma glucose levels in this organism, although these represent only 2 experiments. These results in kelp bass are in fact paralleled by studies reported by other investigators (see section I, C). Although both lucose loads increase plasma con centrations greater than four fold, the control values are different in both fish: K24, 88 mg% and KJ2, 17 mg"fa). In K24, the capacity to remove glucose from the plasma may be depressed due to partial saturation of the sites of uptake of glucose by an already high blood glucose level (approxi mately 2.5 times that of a normal, fed animal). On the other hand, the K32 blood glucose control is only one half of that of a normal fed fish. This may partially explain why K24 glucose values never decrease lower than 3 6 times the control while the K32 values return to 1.0. An unsuccessful attempt was made to measure insulin levels with the double antibody technique against mammalian insulin (i::,chwartz/IIIann assay kit) ; however, either insulin levels were below detection or the interspecies difference was too great to cause displacement of 125 r-insulin (human) from the insulin antibody complex. There is evidence that the latter may be the case, at least in goldfish (14). In order to eliminate the possibility that the initial fall in glucose during the first three hours of the 38 loadin ex eriments was due to slow distribution of the load through the extracellular space, 3 }-allose was admini stered. Allose is a non-utilizable su ar used for determi nation of extracel1.ular volumes. Unfortunately, the activity of the tritiated su ar was too low to be detected in plasma at the dose given. Thus, the precise nature of the three hour decline in blood sugar is still not clear. 5. Effects of epine~hrine As shown in figure 5, intravascularly administered epine hrine (0.004 mg/k fish) causes a transient rise in blood su ar which peaks 30 minute after injection. Post injection lucose concentrations are expressed as a frac- ion of the avera e of 3 re-e inephrine plasma samples (29.9 + .1 m o). Th data from a secon animal was not included because the control values increased rom 9.3 to 41.2 mg% over the preliminary sampling ~riod. Any e ineph- ine effects would be masked by the non-hormone induced changes in plasma gluco e. A sham injection of saline should be inclu e- in control animals to correct for poten tial stress induce changes in glucose due to experimental procedures. C. Glucose Turnover Rates 1. General introduction Isotope dilution rocedures allow determination of lucose turnover in intact animals (i.e. hepatic glucose production and extrahepatic lucose u take). In such 39 Fi re 5. ~pinephrine ~ffects upon Plasma Glucose Concentravion ~ inephrine (0 . 004 mg/ k body weight) was injected via the cannula at time zero. The data is ex res ed as in fi re 4 . 40 0 ~ (\J 0 <D (\J 0 ( fOJ+UO) / fOl-Ul>W!J2>dXZ>) 3S0:)n79 v'l,/\JS'v'ld 41 studies the follo in assumptions are made: a. Glucose production occurs mainly in the liver. In mammals, nondietary glucose comes primarily from hepatic glycogenolysis and gluconeogenesis. The kid ney cortex can also add to production via gluconeoge esis although this contribution is only a few percent of the total. 1 e assume the situation in fish is the same. b. ~xtrahepatic tissues are the primary utilizers 0 cose. Thi is particularl true in these fish where liver lvco en level ~ are quite stable even durin exten ed fa tin vith le s than 1% of both the administered 14 c and 3 A lucose dose incor orated into total liver gl co en in fed animalu (table J). C Free glucose is found only in the circulatory and e~tracellular spaces since the sugar is pho phorylated upon entering cells. In general, following injection of radioactive glucose into the circulation and the mixing of label in the glucose pool, unlabelled gl cose produced by the liver dilutes the administered radioactive compound thereby lowering the lucose specific activity. The assumption is made that the plasma is a raid reflection of events occur ring in the total glucose space (circulatory and extra cellular space). Under steady state conditions, where there is no change in blood lucose concentration, 42 able J. Liver and f , 1uscle Glycogen Content in Fed and Fasted Kelp .t:Sass xpressed as ~g of Glycogen Glucose Per 100 ~~ Tissue ~et Weie~t. . e l Fasted Fish umbe Animal .. (8) ( l.t, ) ean ~an.ge ean ... ange ii' Liver 0.047 + 0.012 0.012 - 0.l0L~ 0.161 + 0.05 0.090 - o.478 Liver o,oq1 + O.OA4 0.001 - 0.142 0.00 0.002 0.000 - 0.00 n .,,~ uscle 0.01? + o. o.oos - 0.010 0.028 + 0.00 0 . 014 - 0.040 *Liver glycogen synthesis measured as incorporation of 3 H glucose and expressed as follows: ~ uose in Liver Glycogen= (CPrJI 3 H/mg P.:lycoaen P-"11:!cose) (~~ glucose/100 m~ tissue) X 10 ( CP1'1 1 H dose/100 g body weight) +=" w production equals utilization and the decline in specific activity follows first order kinetics. If the production ra e of the lucose changes, the rate of dilution of the label is also altered, resulting in a departure from first order kinetics. athe atic 1 treatment of data will be discussed in section III, C. J. 2. Is~topic glucose For 1ucose turnover studies, tracer uantities of lucose-6- 14 c, lucose-6- 3 _ were used. The s ecific acti vity of the injected dose ,as a follows: 0.27 millicuries lucose-6-JI/0.22 m ml an 0.0019 millicuries lucose-6- 14c/o.0065 mg/ml with varyin amounts injected (0.13 to 0.30 ml/fish) depending u on the experiment and the size of th an·mal. Glucooe-6- 14 c is considered to be a reversible tracer (59) in that the 14 c in three carbon fragments derived from lucose can be reincorporated into ucose via lactate return (Cori cycle). Glucose-6- 3 H, however, is an irreversible tracer since the tri ium label is lost to body water at the dicarbox. lie acid shuttle and during the fumarase reaction (fi re 6) (28). One third of the iso tope is ost in the carboxylation of pyruvate to o -aloace tate . After reduction of oxaloacetate to malate, the remaining tritium is lost to body water in the extremely rapid fumarase directed equilibration between malate and fumarate. One half of the remaining tritium disappear for 44 Figure 6. The Ietabolic Fate of 3 H and 14 c Labels of Glucose-6-31, -6- 14 c • ~, hydrogen labe ed as 3 H: • c, carbon labeled as 14 c. 45 HC=O COH COH COOH COOH COOH HOC ~ ~ ~ ~COP03 ., ~ COH •~c H > C=O --HCOH • ...re H2 ,. CH2 r.COH g I ucose-6- 3 H, P E P _5_14c pyruvic acid I act i c acid ~ COSCoA I C H 2 ~ ac<2tyl CoA COOH VJ H C=O • CH COOH OAA COOH ~9,_c r.CH COOH malic acid 1/2*H COOH ~~ COOH tumaric acid •c lost as •c~viaTCA cycle 46 every passage thro gh the fumarase step (28). arly exper iments using pyruvate-2- 14 c have shown considerable equil- ( 45) • ibration between OAA, malate, and fumarate The difference in behavior of the 14 3 lucose- C, - H can be u ed to determine the de ree of recyclin durin altered metabolic states such as starvation. Chan es in the lucose-6- 3 H s ecific activity permit calculation of lucose pool size, glucose space, and rates of replacement of lucose under steady tate condition. J. y state conditio s In ei ht nimals studied u ing glucose-6-JH, 14 -6- C, blood lucose levels remaine relatively stable during the entire experiment and the decrease in plasma glucose specific activity followed first order kinetics. A representative experiment is shovm in figure 7 in which lucose-6- 14 c, -6- 3 1 was administered intravascularly to . clathratus. The mean blood glucose was 31,4 + 7,8 m o and the dilution rate of 14 c and 3 1 lucose wa similar indicating that the incor oration o plasma lactate into lucose i minimal. It should be emphasized at thi point that experi mental animals were not e ercised. ~ Ji th exercise, blood lactate concentrations rise substantially and then fall slowly, requiring as much as 12 hours to return to pre exercise levels (14). Cori cycling would be expected to increase significantly under these conditions, although 47 Figure 7. Steady tate Conditions Glucose - 6- 3 . , -6- 14 c was injected via the indvellin cann a in kelp bass. Pe iodic blood amples were taken and assayed for plasma luco~e pecific activity. • , m-'l' f plasma glucose ; 0 , 3 H specific activity ; X , 14 c specific activity . 48 w 60 l/) 8 ::::, _J 40 l') ~ 0 O> E .....-.. 30 (\J 'O -c-- X 20 ...._,, >- f- - > I- 10 u <( u LL - u 5 w (L l/) (") I .. 60 120 180 ~ -c--u MINUTES 49 this hypothesis was not tested in kelp bass. In order to calculate glucose replacement and the degree of recycling, the estimations of Katz, et al. were used (59) with the following mo ifications. Because the metabolism of these kel bass is so slow (t1 of 3 H lucose 2 is 98 ± 37 minutes), the sin le injection isotope experi ments mu t be carried out for considerably loner than four hours in order to anpro ch zeros ecific activity, a re uirement for raphical interpretation o this data. In some cases, a lon as 20 ho rs would be necessary to reach 1000 CPr /m lucose. Because extrapolation between four and 20 hours would introduce extreme error, the data was fit to an exponential equation of the form: using manual curve peeling and a regression analysis for fitting ex onential curves provided with the s atistics pacl i-- ~ 0 10 LL 8 ~ 6 CL V) ~ 4 L_ __ --1, ___ ___J__ ___ -1.-___ .1.-__ _., 60 120 180 240 MINUTES 62 i re 9. .. on-steady ,=>ta"te Condition Legend is as described in fi re • 63 ~ 0.2--------..-----,,-----. rn 0 0 ~ -- c E -- en E .....__, a:: w l/) 0-1 0 80 u => _j (.9 ~ 40 0 0) E >- r- 8 > I- 6 ~N u'o 4 - ~ l.J.... X _......._,,, hJ Q_ l/) (""') 2 I I ... ------ r ... ____ _ 60 120 180 MINUTES 64 (i.e. altered production and utilization), require hours, rather than minutes as in mammals. Figure 10 resembles the previous two experiments in that production rates are elevated, however, there is little apparent lucose utilization. In this case, increased plasma glucose concentration is strictly due to hepatic production. A plateau in blood lucose occurs as reduction decreases and utilization be ins to increase. he second a terna ive re ultin in a net synthesis of lucose i shovm in fi re 1 . An elevated rate of pro duction is coupled with a si) ifican drop in lucose uptake between 60 and 120 minutes resulting in a 350% increase in blood lucose levels. As before, production ultimately declines, leading to a stabilization of plasma glucose concentration at 100 mgfo. Again, these regulatory responses require hours to become evident. D. Gluconeo@nesis from lanine 1. General introduction Evidence from previously described experiments indicate that although lasma lucose concentration drops after two weeks of fastin in kelp bass, the rates of pro duction and utilization remain the same during steady state conditions. To investigate the importance of amino acids in this production of glucose in both fed and fasted fish, alanine labelled uniformly with 14 c was injected via the cannula along with glucose-6- 3 H. Both isotopes were 65 re 10. on-steady State Conditions Le~end is as descrioed i fi re • 66 ~ 0.2------r-----,------,------,-------, en 0 0 ~ -- C E -- en E w (./) 8 :) _J <.9 ~ 0 en E >- ._ - > ~ 0-1 80 40 10 ~(\J~ 'o 5 u ~ - X LL .._., ~ V, (V) I --- ,-------- ----- 60 120 180 240 MI NUT ES Figure 11. fen-steady tate Condition Le end is as described in fi re 8 . 68 ~ Q. 2 Ol 0 0 ~ -.... C ·E 0.1 -.... CJ) E ....._,,, ct: w (./) 0 u 80 ::, _J (.9 ~ ~ 40 E ~ 30 N 'O ~ X 20 .......... >- I- - > I- u 10 <( u 8 - LL u 6 w 0... (./) 4 M I ~- .... ,_-, I ' I I I a I I t l ---- ---~----,_ ,------------- ._..._ ------ -------' 60 120 180 240 MINUTES present in tracer uantities. The lucose-6- 3 H permit determination of the rates of production and uptake of lu cose, while the appearance o 14 c lab 1 in plasma lucose ive a measure of alanine incorporation into lucose as well as the rate of d·sappearance of alanine rom the lasm. 2. Isotonic alanine and glucose The s eci ic activities o he injected tracers were 0.05 mil icurie alanine-u- 14 c/o.029 m ala ine/ml saline and 0.01 millicuries lucose-6- 3 H/0.0ll m 1 cose/ ml saline. gain, the quantity injected varied wi h the animal. For ease of comparison, all specific activity ha~ been correcte for a ose of 10,000 CPM/100 boy wei ht. ince the majori y of these experiments were not at a steady state, calculation o rates of roduction an uptake ~ere car ied out accor in to he method of Dunn, et al (29) as in ection III, C. 4 a terse aratio of ~lucose and alanine by either aper or tandem column chromatography and subsequent determ·nation of radioactive content. J. Alanine disappearance The d·sappearance of alanine from the extracellular space of fish can be expressed as the percent of the 14 c alanine dose remaining in that estimated space (21 ml/100 g body weight, see section II, C. )). In figure 12, a pro nounced decrease in 14 c alanine is observed between 15 and 30 minutes in these fed kelp bas with an average loss o 70 Fi ure 12, 14 C-Alanine Disappearance from t.c Plasma Alanine-u- 1 l}C ·ra i jected via the ind ·telling cannula and eriodic blood samples ,,ere taken. r he labeled alanine was se arated rom other isotopic com~ounds with the t ndemly a n ion e'Cl n e co mns described inc apte II . The data roM tvo experiments is presented as the percent of the administered dose remainin in the plasma at any time, t. 71 0 co ~ U) ow N I- ~ :::> z - 2 0 ~ ___ .___ __ ___._-"-___._--'------'---'-----' 0 0 0 0 L{) ~ 'v'JAJS'vld NI 3SO0 3N I N'vlv'- =>v~ °lo 72 n d · . d l L~ C l . b "l O . t 070 of the a ministere a anine y I minu es. y 60 minutes, essentially all of the dose is gone (less than 3% remains). If unpublished e timations of the extracellular space of marine fish were used in these calculations (sec tion II, C. 3), even more profound changes in the loss of alanine would be apparent as the published values are some what below that estimated by isotopic glucose techniques. Plasma levels of alanine were not measured and as a conse quence, specific activity data is not available nor is data available on alanine disappearance in fasted fish. 4. Alanine appearance in plasma glucose Results shown in table 7 sug~est that alanine glu coneo enesis plays an im ortant role in glucose production in both fed and fasted kelp bass. As much as 2% (mean= 1.2%) of the administered label is incorporated into each m of plasma glucose in JO minutes in fed fish. Based upon the estimated size of the total glucose mass, 7.6% of the d . . t d 14 c . . d . 1 1 a minis ere is incorporate into the tota body u- cose. This is about twice the incorporation previously observed in rats having a much higher rate of lucose turnover(??). Fastin increases the already high rate of alanine incorporation. For comparative purposes, the data of table 7 has been expressed as percent of alanine dose per 100 g fish per mg pla ma glucose , rather than as percent of the dose a pearing per estimated total glucose mass . This 73 Table 7 . Incorporation Animal Condition --- 1 (11) Fed 2 ( 39) ( 3 ) 4 ( 9) 5 (1) 6 (12) 7 (20) Fasted 8 ( 3) 9 (19) Days Off ood 4 l} 4 6 9 13 20 25 33 of Alanine- 14 c in o lasma Glucose ei ht _iQrams) 171 305 79 59 294 202 ft ean 202 355 245 Mean+ EM 1 0 Dose Given per 100 g Fish/mg Plasma Glucose~ ___}0 min 60 min 1.6 0 4 o.6 0 4 0.9 0.7 o.6 1.0 2.0 0 8 1.2 + 0.3 0 7 + 0 . 1 - - 1.2 1 . 2 1.8 o . 6 ----------------- ·:t- ___ 212m_1_4 ____ c __ _ mg plasma lucose cpm 14 c dose 100 body wei ht X 100 74 avoids makin unwarranted extr polations o ~lucose mass from steady state ke p bass to the luco e mass of non steady ~tate animals. At O minutes a ter isotope injec- t · on , t mean incor o at·on ·nto glucose by aste i h i reater than tat y ed fish , owever, with on y two such fasted indivi ual , statistical comparisons are not valid. t 60 minutes, the increase in incorporation of 14 c ala nine carbon into 1 co e in fasted fish is siRnificant (p 0.05) These ma actually be underestimations of the true luconeoaenic capaci y of kelp ass, however, this will be ct· cu se later. In bot ed and faste ish, the specific activity 0 4 c- lucose new r vn hesize fro alan·ne decreases i a lie anne to that o the luco e- _3 14 are repre ent ive ex e i ent. Thi 0 P..Ures 13 and ua ests that the dmin· e ed alanine i inco orate ·nto lucose well before the i st lasm ample is taken. Both paper and tandem colu n chroma o ra hie sepa ations of 11 cos 14 comparable results, that is, he slopes of C and . lVe . turn- over are similar. Unfortunately, the column eluant inter feres with the chromagen of the Glucostat assay so speci ic activities of plasma glucose could not be dete mined with the tandem column separation . Also, lucose concentra tions in the eluant were below the level of detection of the eckman Glucose Analyzer . 75 Fi re 13. 14 C-Alanine Gluco. e pearance ·n Plasma • 1 L~ • • • A anine-U- C was 1nJ ct.d c1 ~ co e- - 3 II vi t e indwellin _J cannula. eriodic plasma samples were ta1cen and assayed for 3.r{ and il}C gluco e pecific activity . The legend i as desc ibed in i re 7. 76 w ~ 30 u :::) ..J 20 (.9 ~ 0 10 O> E ~ 20 N •o ,-- X ...._,, >- 10 }- - > I- ~ u 5 LL - u w a.. V) 60 120 180 MINUTES 77 igure 14. 14 c- lanine Appearance in lasma Glucose The legend is as de cribed in figure 13, 78 20 ~ (N 0 ~ X 10 .....__,, >- r- - > J- u <( 5 u LL - u w a.. (/) 2 60 120 180 240 Ml NUTES 79 .J • ho.§]2horyla e Althou h liver lyco en levels remain relatively .table diring fastin~ or many da , muscle lycogen may be more readily utjlized as uel for contraction (24, 52, 55, 64, 72, 86, 106). P elimina y experiments by Nakano and Tomlinson (86) in icate that skeletal muscle ~lyco en levels fall rapidly in re ponse to exercise, while similar exoeri ent in liver are inconclusive. To ur her clarify the role o lyco en in carbohydra e metabolism in muscle, muscle lyco en phos hor lase was artially pur· ie from . ee ecie o i • • a alabrax clathratus (kelp bass) a shallow water marine fi h with a seasona te. era ure r n e o 10-2 C, Leuro lossus stilbius, a di fusel dis- -----_;..__~ ributed i water ( 00-500m) ex erienc·n a therma mi ratin myctoph·d ish under oin aily thermal changes of approximately 5-15 or 20 C, depending upon. easonal chan es in surface water temperature . The rationale for the use of these three species was as follows. Kelp bass are moderately active fish found in waters shallower than 20 m. ~nvironmental temperature changes are slow and for the most part seasonal in nature. Triphoterus mexicanus under oes rapid temperature changes durin its iurnal vertical migrations; one would herefore expect adaptation to rapid thermal change. In addition, re nounce activity of the lyco~en hosphorylase ystem would 0 Table Kinetic P r--=tm phorylase r s of l p a s Total (a+b) ------------- r Temperature m V max lscle Phos- b y m (C) (ill_! G-1-) (units/g wet wt) (mM G-1-) 5 10 15 20 25 30 6 11 6 16 14 4 10 14 33 50 6 19 16 81 be ex ected if lyco enolysis is indeed a ma,jor. ource of ene y for movement. Finally, Leuroglossus stilbius would experience minimal environmental variation (5-10 C) and act as a control for the T. mexicanus enzyme. 1. aralabrax clathratus Fish were taken durin July when the water tempera ture was app oximately 15-20 C. E axial mu cle was reed o trace of lateral 1·ne red muscle and ar iall purified accord·n~ top evio ly de cribed methods in ection II, D . 2 • .. nzy e acti vi .,y a assayed accord in o the me hod o e ric ad Fische (44) ti izin hi~h leve 0 lucose- 1-phosp ate (75 q); units of activity erg a o muscle are expressed s umoles in o ~anic phosnhate release per minute per gram. Total enzyme activit, (a+b) i measured in the presence of 2mM AM , Nhile the amount of the active form of the (a) . A~ independent . enzyme lS Fi 15 and table 9 show the effect of . . re increasing tern erature on total phosp orylase activity ( t F dependent , a b forms) an the ercent of phosphor lase in the actlve om (A independent, a). Total activity increases mark- edl between O and JO C. Throu h the seasonal temperature ran~e (10-20 C) , activity increases from 6.4 + 2.4 to 19 . 9 + 4 . 9 units per ram of muse e tissue, wet weight . The ratio of a/a+b does not chan e above 5 C, su~ estin that temperature does not stimulate either enzymatic or non enzymatic conversion of the b to a form at environmental 82 Figure 15, The ~ffect of Temperature upon Partially rified Skeletal ffuscle Phos horylase Activity from Three Species of Fish Total phosphorylase activity was measured in the presence of 2 fl A1P; phosphorylase a in the absence of A M P. Units are defined as umole of inorganic phosphate a pearing per minute per gram of muscle tissue. The upper panel represents the percent of the total enzyme activity which is A /IP indenenden t ( .A ) • The 1 ower panel shovs the effect of the assay temperature upon total phosphorylase activity in P. clathratus ( X ), L. stilbius ( 0 T, and T. mexicanus T • ) . 83 .o 40 0 -.._ 0 ~ 0 + 0 20 ~ 30 > J u <{ w l/) <{ _J >- 5 I Q_ l/) 0 I Q_ 20 10 0 10 20 30 ASSAY TEMPERATURE (°C) 84 Table 9. The ~ffect of Temperature upon Total Phospho rylase Activity of Two Midwater Fishes as Compared with a ~hallow Water Fish Temperature L. stilbius T. . P. clathratus mexicanus (C) (units7P-}* (uni ts/g' · (units7g}* 0 0.08 + 0.05 0. OL~ + 0.04 o.oo + o.oo - - - 5 O. 37 + O. 19 0.21 + 0 .12 2.33 + 0.90 - - - 10 0 • 7 + O .19 0.51 + 0 .12 6.40 + 1.80 - - - 15 ----- ----- 13. 30 + 0.00 - 20 1. 91 + 0.52 1.66 + o.4o 19.90 + 2.40 - - - 30 3.98 + 0.76 3.61 + 1.06 34.30 + 2.70 - - - -- - *Velocity+ S .1 5 temperatures. There is no Aa independent activity at or below 5 C. Throughout the ummer temperature ran e (15- 20 C), total activity ran es between 13.3 ! 4.9 units/ wet wei ht. o.o and 19.9 An Arrhenius plo of total muscle phosphory ase act·v·ty (f·gure 16) is nonli ea, su estin the ossib"l- ·ty of tempe ature ·nauc conformational chan~e in he enz e. Ab eak in the po occur at h lower imit o the sumer ada tation te perattre for kel bass (15 C) as 1ell as t 5 C. imilar results are .een for the active form (a) of mu cle phosp orylase. The broken lo may also indicate chan es in the energy of activation ( a) of he enzyme, althou h this is mo e enerally accepted o repre sent conformational chan e (75). The Michaelis-Mente constant (K) and the maximal m velocity (Vmax) rere d termined from L"neweaver Burke lot at four different assa tern eratures (table 8). Chan es in a say temperature, ho ever, had little e feet pon the Km' unlike that re orted for many re la ory enzymes of carbo hydrate metabolism (section I, G). Purification of hos phor la e b by the method of Cohen, et al. (20) did not substantially alter the Km for lucose-l-P0 4 (table 8). 2. Leuroglossus stilbius hosphorylase isolated from these midwater fish showed far less total activity than the kelp bass enzyme (figure 15, table 9) at all temperatures above O C. t 86 igure 16. Arrhenius Dia~rams for Fi h .uscle Gl co~en Phosphorylase The le end is as in figure 15. 87 r---. .0 + 0 '--" > J- - > J- ~ w (/) <( _J >- 0:: 0 I CL (/) 0 I CL _J <( ~ 0 J- 5.0 1-0 0.5 0 -1 0 -05 33 19.4 34 50-4 35 36 37 88 assay temperatures corresponding to environmental tempera tures (5-10 C) total activity (a+b) ranges from o.41 ± 0.3 to o.8 _ 0.3 units/g wet weight. In comparison, the bass enzyme activity measu ed at 5 to 10 C is 2.3 + 1.9 to 6.4 + 2.4 units/~ and at its summer temperature range (14- 20 C), 1 .3 + 0.0 to 19,9 + 4.9 units/~. In neither L. stil bius nor T. mexicanus could any A 1,1P independent phos- ~orylase be detected. The Arrhenius plot of L. stilbius phosphorylase activity is linear from 5 to 30 C (fi ure 17), unlike that of the kelp bass enzyme Nhich breaks at both 5 and 15 C. The properties of the L. stilbius phosphorylase are indi- cative of a more thermally stable enzyme, however, the low total activity of this enzyme possibly precludes it from playing an important role in the energy metabolism of this . organism J. Triphoterus mexicanus As can be seen from figure 15, there is little dif ference between muscle phosphorylase activity of T. mexi canus and L. stilbius. A~rhenius plots are similar, although the T. mexicanus enzyme appears to have two breaks; one at 5 C and one at 20 C. As mentioned before, T. mexicanus phosphorylase a activity could not be detected. 89 C APT~R IV I . CU .. IO Regulation of Plasma Glucose In certain interestin and ·m ortant ways, the metaboli m of kelp bas ·s uite unl"ke that of mammals Low plasma lucose concentrations, depressed Plucose tun over rates an an increase emphas·s on protein metaboli m in the ener y eco omy of kelp bass may be due in part to the ada t tion 0 hi col bloode 0 ani m to . env1.ro - mental tern er ur v ria ions and t e c uisition of a . • • D e he dif erences, C rn1vorou mo e 0 n ri 1.on. 0 e ish be ite ~ell • mod stem fore • ma u e a UCl a- tin hormon 1 rol m chan . . sly investi ated con sm p ev 0 o ly ,i th difficu ty in . her ebrate l ver • 1. lasma glucose levels major metabolic difference between fis and higher mammals is in the low concentration of plasma glu- case and its variability. hile rats maint in a constant fasting blood su ar of approximately 90 m o, this species of fish has a normal, fed mean level of 35 m %, al thou ha load induced blood glucose of 510 m o can be tolerated in this species ithout obv·ous effects on behavior (section III, B 4) Large variation may be seen ·n the 18 animals sampled (17. -100.2 m ,o) as well as within given 90 individuals (fi re 1). These low plasma lucose concen trations in kelp bass may partially result from the con sumption of a hi h protein-low carbohydrate diet since as much as 70% of the calorie intake of carnivorous fish is resent as protein (91). In this study, kelp bass were con idered a "fed" when the had food resent in their di estive tract and had eaten one to fourteen days rior to the ex eriment. P esumabl. , maximum bloo su ar concentra- tjon occur ate eedin. In the laborator. ra, lucose conce tra ion in the venous sv te ma . , xceed 180 fee in ( ?), however, their iet contains a la e propo - tion of digestable carbohydrate. This is in cont ast to the 35 m % found in fed kelp bass, fish which ely on a hi h rotein, low carbohydrate source of food. The normally low levels of lucose may iv an indication that circula in lucose may e less im orta t a an ener y source in kelp bass than in mammals. Cer tainly lucose ti nover measured isotopically is consider abl de ressed when compared to rats of e uivalent size (table 6). The replacement rate of lucose in both fed and fasted fish durin~ stead state conditions is 0.039 m 1 cose/min/100 bodJ wei ht, or 1/20 that found in rats. General oxy en consumption studies by other investi ators, however, indicate that total aerobic respiration is decreased as a consequence of the heterothermic condition of fish (i.e. their vulnerability to changing ambient 91 temperature) com ared .o the reeu ated, homeothe mic nature o mammals (sec ion I, A). The de ressed res i ation ound in cold blooded r·sh can be quite extreme. For exampl , if one measured oxygen consumption rates of two related spe cies of fish inhabitin quite ifferent environments such as the tlantic cod, Gadus morhua, and the deep ocean benthic macrourid, Coryphaenoides acrolepis, one finds hat respiration in the shallow water fish i two orders of ma itude hi~her than hat o the macrour·d (96). Com ara ble temperature were used in this case (3.0-3.5 C). . e av·oral modification de to he ecrea e oo avai a- bilit at 1230 meters certainly could modifv oxy en con umption in the macrourid as coul limited ada tation o hi ambie t essures. n e p·ra ion in the benthic . 1S was carried out at depth with an automated samplin device contajnin an oxy en electrode (96). Certainly such fac tors as activity, oxy en tensions, and ial variations could also modify oxygen consumption, but the fact remains that metabolism in intact heterotherms, whether measured by oxy en consumption or by the turnover of isotopic lucose, is much slower than in mammals. 2. Potential hormonal controls Glucose concentration in kelp bass is low and vari able, however, rem atory mechanisms do o erate to estab lish a certain degree of homeostasis with res ec to bloo luco e. he fluctuation in lucose levels observe over 92 a 24 hour eriod (fi re 2) can be viewed as lon term re latory respon es re uirin hours to occur rather than conds or minutes as in mammals. Thi is urther sup- ported b luco~e loadin ex eriments (fi re J) where at least 12 hou s may be re uired to di po e of the load, alt ou h the in"tial all in luco e occurs within hree hours. Thus, there a e to be an insul"n-like response in ke bass 1hich is emp r mentally unlike that in mam- mals .. hether his is due to a delayed release o ·nsulin, a decrea e sen itivit to in ulin at ·nsulin re pon ive tiusue , a slower respon 8 by tare tissues, or low levels o cir~1latin insulin i not clear. in an ibodies a ain and Fo deter ine that fa t·ng level o insulin in old i h ( 6 u /ml) are twice that in man, while tho~e o fat n to d ·sh (Onsanu s ., 19 u / 1) are approximately e ual o hoe of man. There appears to be no cross reactivity of fish ins lin in standard human anti-insulin assay~ (14). Gluco e (40 m o) and leucine in the presence of glucose stimulated in vitro insulin release from islet tissue after 60 minutes as did feedin prior to the experi ment in intact fish. Thus, it appears that plasma insulin levels are similar to those in mammals and are indeed res onsive to lucose stimulation, althou h the insulin release (60 minutes) and insulin mediated depression of lasma lucose concentrations (J-24 hours) require lon 93 time eriod in both te eosts and chondrich-thyeans (47, 88, 1) and recove ies are slow. The suggestion has been made that in fish, insulin ma.r be more immediately involved in controllin free fatty acid levels (FFA) since insulin depresses FFA levels before it induces hypo lycemia (??). The FFA effect predeeds the lucose effect b at least two and one hal hours in old fi h (77). Thi data is con i tent with a pro ooed model of cont ol of lucose via FFA oxidation and e abo ite eedback echani m thic i al o known s e 1 co e-FFA C. C e ( 7 , 112) C s n 1 vel of FFA in the a ma increa.e lucose uptake b the vat· lycolvsis. he o osed ch indirect y ac i ·s di cussed below, howeve the control is via pyruv te deh do enase and ho phofructokina e. ikew·se, elevated F concentra- tions depress luco e u take. uperimposed upon this potential non-hormonals control would be hormonal "fine tunin" and the capacity to handle excessive lucose loads. ith the onset of starvation in mammals, lasma lucose evel be in to all and FF concentration~ be in to rise. In order to conserve lucose fo tissues such as the ner vous sy em, it is importan to promote a switch over in muscle metabolism from lvcolysis to fat v acid oxidation. T ere are some difficulties in te tin this mo el in vivo, however hep r·ne induced elevate lasma FF in humans results in a decreased ability to handle an oral 1 cose 94 load (87, 112). In vivro experiments suggest that muscle fatty acid oxidation depresses glycolysis via a proposed acetyl CoA and citrate feedback inhibition of pyruvate dehydrogenase and phosphofructokinase respectively at phys iological levels of free fatty acids (87, 95). Gluconeog enesis in rat liver and kidney cortex has been found to be stimulated by millimolar quantities of fatty acids, but the only concentrations which are effective are unphysio logically high (95). Newsholme and Start su gest that the lucose-FFA cycle is most apparent at low glucose concen rations with the direct effect of insulin on lucose uptake by the periphery occurring only in the resence of excessive amounts of glucose (87). Fish, therefore, could be potentially u eful in investigations of this type of control because 1) insulin action affects plasma FFA con centrations long before the development of a hypoglycemic condition and 2) plasma ~lucose is normally low in fed fish and is further decreased by starvation. ot unexpectedly, other investigators implicate insulin in the control of protein metabolism. Insulin stimulates muscle protein synthesis in Fundulus heter oclitus as measured by 14 C-leucine incorporation (56) while Tashima and Cahill found increased in vitro incorporation of glycine-2- 14 c into toadfish muscle protein in the pres ence of insulin as well as increased immunoreactive circu lating insulin in vivo after a force fed high protein meal 95 (111). The latter occurrence would be ex ected to be pro nounced in carnivoroua fishes. In addition, elevated amino acid concentration remotes insulin release from isolated toadfish islet tissue (89). Insi ht into the nature of regulatory controls in fi h can be ained b stud in t ose kelp bass whic are not in a stead tate (i.e chan~ing blood lucose concen- tration). Int ese fish, inc . ase. in lasma lucose a e bro1 h about b either i crea ed ~ lucose p eduction and/or ec ea e gl1cose u ta e. A can be ee in fi211res ro 1, eleva ed glucose conce trat·ons esult in a rad al shutdo\ in it reduction ,ith limited concurrent ncreases in lucose uptake, dependin upon the ex eriment. The ultimate resu tis a plateau in blood glucose durin the course of the experiment. In some cases, t ere is a decline ubse uent to the plateau. These metabolic read justments require one hour or more, although in only one non-stead. state experiment di the replacement rate ever reac that value determined for the "steady state" animals. It hould be em hasized that these experiments were only carried out for a maximum of five hours. If one assumes a ro o ed la t·me fo lucose stimulated in ulin outnut in . kel bas~ usin the lucose loading ex eriments, a ro nounced increase in uptake due to insulin might be missed. In the lucose loading experiments, plasma glucose levels only return to normal after 12 hours or more. Thus , if the 96 experiment lasts for no more than five hours, it is not likely that an insulin effect upon lucose uptake will be seen. Nith the exception of fi§':ure 11, the most dramatic chanues in ~lucose turnover in these fish occurred in lu cose reduction ather than in uptake. This su~~e~t~ that the nredominant control mechanismc function in turnin lu cose production on or off rather than relyin upon the con certed action of re latory hormone upon both uptake and production. One cannot eliminate, the possibility that this is an example of the separation of hormonal events, namely, that insulin acts initiall to decrease lucose production from the liver, then subsequently increase lu co e uptake by extrahepatic tissues. The separation of these two events was su ested by Dunn, et al. in intact does, althou h not on the extended time scale which would be found in fi h (105). Certainl , one might account for the re orted lag between insulin administration and the onset of hypo lycemia in fish (section I, C) by the above explanation. If insulin acted to shutdown glucose produc tion without initially affectin utilization, one would not necessarily observe any decrease in plasma glucose levels. It would not be until glucose utilization began to increase that plasma glucose concentrations would then fall , partic ularly in li ht of the low rates of glucose uptake during the early art of the non-steady state experiments in 97 relation to thee evated rates of reduction. second notentia mech ism for regulat·n luco.e outnu involve glue ~on, hormone w ic is ener Jl cce te to be a potent hyp~r~lycemic . ent l mamma .. Deere in~ concentrations o ,l11ca on coul re 11 t in lo 1- ered rat of eduction of l1cose, althou h without he concommi ant action of insulin ·.n increaeoin uco e uptake b the e i hery, it i a·rricul to see how one could invoke hi as a control of glucose levels. lthou thi investi ator made no attempt to meas re o i elate kel bass luca on, p b ish d st dies indicate that 1 cagon i found in the eleost pancreas and de ree of suecies c· ici JY (31, 6J, llJ). ha a T e ol o this hormon . l till no clea, e haps a,:-, a esul o the r viou ly mentione s eci icity and/or mode o in c ion o the hormone in the reported ex eriments. Intr vase ar ini ctio of bovine luca~on at 2 m_/k e.ults i er lycemia at 0.5 1ours w ·ch persi ts for nine hour in the pike, ~sox lucius (113), while in qualus acanthias, between six and 18 hours are necessary before elevated plasma glucose can be detected (88). Jo effect upon liver l,yco enolysi could be discerned in • acanthias (88). In the lamprey, Lampetra fluviatilis, intra eritoneal y injected result in elevated plasma uca on (1 mg/k) did not luco e, however, in yellow ... els, s·m· ar doss reduced a ianifjcant hyper 1 cemia in three 98 hours and at 2 mg/k, the effect persisted for at least six hours. Glucagon did not induce the expected hyperlipemia in the eels (63). Parallel experiments in pike and in do - ish shQ.rks with codfish insulin shoqed maximal hypo l.1- cemia at ix hours and 24 hour respectively after intravenou. admin" trat"on Co fi h insul·n hould have reduced roblems ·nvolved in usin insulin isolated from a very di erent , 113). Theim ication rom ese e lt . l that te oral y, insulin and lvco en are sear te , with he actions o uca on exertin . l S ef ect more rapidly u on lasma lucose than insulin if 0.5 to six hours can be considered rapid! The persistence of hy er lycemia induced by gluca on makes it less likely that luca on alone cold be es onsible o hor onal re lation of lucose in kelp bas although thee oses of bovine luca on ma very well be in excess o that normally pre sent in ish. Blood lucose in cats will respond to a ittle ar- o. u luca on er kilogram of bod wei ht (33). Ot er evidence or cont o of lucose in kelp bass can be found by compa in fed an fasted animals . Plasma lucose concentrations are maintained at 35 me-% for two 1eeks, then fall to 19. m 1 o from 4 to 42 days a te the last feedjn. Te minimal lucose mass appears to be sli htly lar er in fed fish, however, the minimal mean transit times of lucose are not si ificantly different. Likewise, glucose replacement rates are similar , althou h 99 the small number of fasted, stead, state animals make com arisons difficult in li ht of the individual variations jn almost any measurable parameter. The above data su ge,;,t that althou h lasma lucose level drop with prolon ed fastin, roduction and u take o lucose can be maintained at com arable steady state rate in fed and fasted el bas . B. Glucogenic Sub trate 1. Glycogen Assum·n then, that blood lucose concentration ·n kelp ba s re ated albeit so~ , what could be the source of lucose roduction during both fed and fasted state? It appears that in kelp bass, liver glyco en levels are maintained and may bes ored durin a 42 day fast (table J). Little meanin ful information on muscle lvco en could be obtained since the method of sacrificin V the animal caused m t:"cle spasm, presumably resultin . ln prom t 1. cogenol s · s (table J) . In addi ion onl. 0,091% o t e admi istered b1cose-6-3 dose was incorporated into iver ~lycogen in the fed nimals. he indication is that liver l1co en in these fish is rather inert. Literature re orts of liver lyco en levels sug ests that glyco en mobilization under conditions of fastin i quite variable . Thus , species specific differences may exist (64 , 72 , 83 , 88 , 106). Furthermore , liver lycogen content was not altered by lucaJon injections of 2 m /k in do f i sh , 100 although t_is dose does promote p olon ed hyper~lycemia from six to 1 hours ( ). 2. Lactate co e Coric clina a pears to lay a limited role in glu ed ction in el b s as only 6.9% recycling of lu- cose carbon could be measured in fed fish unlike that in post-aboor tive rat~ where 23% of the lucose-6- 14 c is recycled via lactate. A~ mentioned p eviously, Co i eyeing would be ex ected to play a more important roe in r·sh after exerc·se ~hen blood actate levels are elevated (section II, C. 3). sonly to steady state fasted ex er iments were carried out with both lucose-6- 3 H and lucose- 6-1l}C, nothin can be said about the relative ·ncrease in uco e reduction J. Alanine om actate du in fa tin . 1 X eriment were und rta en to estimate the contri bution o alanine J.uconeo;!!enesis to lucose prod1ction b . h f 14 c 1 . b . 1 easuring tea earance o a anine car on in p. asma glucose. 'I.'racer quantities of gluco e-6- 3 H allowed the monitoring of parameters of glucose metabolism as discussed previously. In both fed and fasted kelp bass, the specific activity of the 14 c lucose newly synthesized from ~lanine and lucose-6- 3 H decrease with similar slopes over the identical time periods (fi res 13 and 14). This observa- tion sunports the existence of a rapidly operatin .... lu- coneogenj_c mec anism in kelp bass since the 14 c abe . is 101 incorporated well before the first blood sample (30 minutes). If earlier samples were drawn, increasing 14 c .f. t· ·t ld b t d d ucose- speci 1c ac 1v1 y wou e expec e ue production of labelled ~lucose from alanine-u- 14 c. to the The important role of alanine in kelp bass metabolism can be inferred also from the disappearance rate of alanine from the lasma (fi re 12). By 30 minutes, only an avera e of 13 0 of the administered alanine dose remain~ in the circu- lation. y 60 minutes, essentially all isotopic alanine has isa peared. For comparative pur oses, the followin est·mation of the luconeo enic capacity of kelp bass was ade: % alanine dose/100 /m lucose=CP 1 14 c ;CPM 14 c alanine m glucose 100 body wei ht By 30 minutes, a proximately 1.2% of the dose was incor porated into each milli ram of plasma lucose in fed fish in contrast with 0.2% maximal incorporation at 10 minutes in post-absorptive rats (27). 'ince no earlier samples were taken in fish, it is difficult to estimate either the maximal conversion of alanine to lucose or the time at which this occurs. A com arisen with rats is instructive . . Jhile the glucose turnover in rats is 25 times that of the fed and fasted kelp bass (table 6), the time of maximal appearance o alanine carbon in ~lucose cannot be any more different than 20 minutes assumin~ 30 minutes for fish and 10 minutes for rats. Furthermore , fed fish incorporate 102 1.2% of the dose as compared with 0.2% in rats. To deter mine the degree of difference between the latter two num bers, comparative alanine pool sizes must be determined. The reasons for this will be explained below. As reported b Dunn, et al. (27) plasma alanine concentrations in the rat are 0.159 umole /ml/100 body wei t. In catfish fasted 4 hours, plasma leve s are estimated to be much hi her, O. O umoles alanine/ml/100 body wei~ht (117). It is possible tat not only are t e differences in alanine luconeo enesi between rats and fish si ificant, but ue to the potentially lar e alan·ne pool size in ish, these differences may be even more profound. Fasting increases alanine conversion si ificantly in kelp bass (table 7, 60 minutes) even though the glucose production rates do not change. This suggests that a sizeable increase in the amount of plasma lucose ori ina tin from alanine takes place under conditions of prolon ed fasting. No data is available on possible nutritionally altered alanine pool sizes in kelp bass. After ei ht months of starvation in Cyprinius carpio, plasma alanine levels drop 501o (69), however, ei ht months and 42 days are hardly comparable time intervals. Thus it appears as if luconeogenesis is of prime importance in maintainin blood glucose levels both in fed and fasted kelp bass. The data has been expressed in the above manner(% dose per 100 g incorporated/mg plasma glucose) in order to 103 minimize the number of assumptions which would necessarily be required to estimate the percent of the dose incorpo rated into the total lucose mass. The majority of the alanine experiments were carried out in fish which were not at a teady tate with respect o lucose (i.e. production e ceeded u ake). hi net ynthe ·so ~ ucose as at lea t wo cone uence : 1. The luco e a. la r t n t e ti ate ro ld be exnected to b or teady tate condi ·on, . 1nce no time did the ate o o uc ion o gluco e in ete mined for the alanine e periments rop be ow tha teady state. In only one fish (table 7, . anima number ) did glucose output equa uptaKe. Alo, u take of glucose never exceeded production. Increased production results in a net synthesis of luco e with an increase in the glu cose mas, ma ing extra ol tion of the total 14 c dose incor orated ·nto this mas rom speci ic activity data an underestimate based on the steady .tate measurements. 2 . net vnthesis o lucose would tend to increa e the a ready hi h an·mal to animal variat·on in the amo nt o alanine converted to asma lucose. ~his is because all data sug est that alanine is a major luco enic substrate, compared with lactate or glyco en. An added complication occurrin ·n these non-steady state animals is due to altered alanine pool sizes related to nutritional conditions. Without plasma alanine 104 concentrations and alanine specific activity data, the extent of dilution of the 14 c alanine dose cannot be deter mined. This would certainly affect the apparent conversion of alanine carbon to that of glucose as measured isotopic ally since a arge cold alanine pool would extensively dilute the injected dose of alanine and less 14 c label would ap ear in lucose, not necessarily because of a ow ~ uconeo~enic ate, but beca1 ve oft e lows ecific acti vity of the alanine pool. All of the above factors could cau e variable results, however, the tendency would be to un erestimate the magnitu of gluconeogenic capability of these kelp bass. Thus the tabulated values in table 7 would be too small. In the one alanine experiment carried out under steady state conditions, estimation of alanine carbon appearance in lucose resulted in appar nt elevated levels of gluconeo enesis compared with the other two fasted fish (table 7, animal number ). Tis tends to support the statemen~ made above. It is not unexpected that luconeo~enesis should be a major mechanism for production of glucose in carnivorous fishes in view of the low carbohydrate-high protein content of their natural diet, with approximately 70% of the ingested calories present as protein and the apparent absence of significant mobilization of liver glycogen durin starvation (section I, C. l; IV, B. 1) (91). Levels 105 of alanine aminotransferases in the tissues of native chan nel catfish are considerably hi her than those reported in do~s, particularly in the liver, t and spleen. Further- more, catfish cultured on a diet high in protein have si - nificantly increased alanine aminotransferase activity in liver, kidney, and brain (116). Neither aspartate amino transferase nor glutamate dehydrogenaoe showed such pro nounced alteration in activity with the modified diet. That fish liver mitochondria are capable of directly utilizing alanine as a potential Krebs cycle intermediate was determined by Gubmann and Tappel by meas- 14 uring the release of CO 2 from both the carboxyl and car- bonyl carbons of alanine (L}2). The rate of release of the carboxyl carbon exceeds 50 times that from the carbonyl carbon. he difference is due to the delayed removal of this carbonyl carbon of pyruvate until the second turn of the cycle (42). Using intact animals, Nagai and Ikeda found a decreased release of 14 co 2 from uniformly labelled alanine in carp fed a low carbohydrate diet (77% protein) versus a diet high in carbohydrate (19% protein) (84, 85). In the low carbohydrate diet, alanine oxidation was nearly identical to th~t of uniformly labelled 14 c acetate, indi cating direct conversion of alanine to pyruvate with subse quent decarboxylation via pyruvate decarboxylation system and the Krebs cycle; however, as stated before, the frac tion of alanine oxidized by carp receivin the low 106 carbohydrate diet was one half that used by fish fed on the high carbohydrate diet. These investi ators attempted to · t· f 14 c · t 1 1 measure the incorpora ion o in op asma g ucose. Unfortunately, the variability of the data was so hi h, no conclusions could be drawn with regard to the effect of diet on luconeo ~enesis from alanine. The results reported . this dissertation demon- in str te ra id and ~ubstantial incorporation of 14c alanine carbon into lasma lucose of P. clathratus which exceed that rate of conversion found in fasted rats of comparable size (27), This conversion of alanine to lucose has, fur thermore, been shown to increase with conditions of pro longed fasting (table?, animal number 9). Thus, luco neogenesis from alanine seems to be rapid, hormonally sensitive, and of potential importance in the ener~etics of fish feeding rimarily on a hi h protein food source. Support for this hypothesis can be found in com- arative studies in both birds and rats. ~isenstein, et al. have demonstrated a three fold increase in ~luconeo en esis from 14 c alanine in perfused livers isolated from rats fed a hi h protein-carbohydrate free diet as compared to Purina controls. They believe this increase is due to a dietary stimulation of plasma glucagon production (JO). imilarly, M igliorini, et al. found significantly higher incorporation of 14 c alanine carbon into media glucose in liver slices taken from the black vulture, Coragyps 107 at atus, as compared to the activity of slices obtained from the common domestic chicken (76). The black vulture is granivorous, and as such, consumes a diet hi h in carbo hydrate (76). In addition, elevated levels of PP carboxy kinase and glucose-6-phosphatase were observed in livers of the vulture as compared to the chicken livers. If luconeogenesis from amino acids is an important provider of metabolic fuel, a rapid turnover of proteins in extrahepatic tissues would have to occur with concommitant release of the free amino acids into the circulation. There are few observations available on the effects of starvation on protein turnover, alt ough temperature in uced alterations in rote·n synthesis have been studied. Jackim and LaRoche determined that decreased 14 c leucine uptake into muscle proteins resulted from fasting Fundulus heteroclitus for three, five and seven days compared to fed controls. Furthermore, resumption of feeding increased 14 C-leucine uptake to that of fed fish within one day (56). These investi ators were unable to duplicate these results durin late summer, implying a pronounced effect of season upon protein turnover. Their results are supported by a considerable bod of literature based upon increased prote olysis, decreased proteinaceous nitrogen, and increased muscle water content with prolonged starvation in both marine and fresh water fishes (69). luscle protein synthesis, as measured by 108 14 c-leucine uptake, exhibited marked compensatory effects in that 5 C acclimated goldfish demonstrated higher net protein synthesis at 5 C, 15 C, and 25 C than fish accli mated to 25 C and measure at the latter three temperatures. There was, however, little difference in net synthesis of rotejn in livers of warm and cold ada ted fish (43). aschemeyer also indicated increased iver synthesis with cold adaptation (43) s do Dean and erlin (23). All of the above tu ies are com 1·cated by the possibi ity that a ·ncor orate radio-active label i releaced from protein durin de radation, it can be rapidly reutilized b protein synthetic machinery. In an attempt to correct this deficiency, ~omero and Doyle utilized a double labelling technique where the 14 c-leucine wa injected at time zero followed by a subsequent injection of 3 H-leucine at a suf ficient time interval to allow peak incorporation into pro tein of the previous 14 c label (101). Tritium to 14 c ratios can then be observed as a relative index of degrada tion with time. There appears to be no thermal accelera tion of de radation in the muscle of warm adauted Gillich- thyes mirabilis (26 C) as measured by Somero and Doyle. In fact, cold adapted fish (5 C) a pear to be breaking down muscle protein more rapidly than warm adapte formc (101). These findings are considerably different from those of Das and rosser who estimated increased protein degradation in the muscle of warm adapted fish (22), however, 109 methodological differences make direct comparisons diffi cult between these two sets of experiments. If temperature induces alterations in the mobilization of protein stores, release of free amino acids from extrahepatic tissues such as muscle would certainly be expected. C. Muscle Glycogen Phosphorylase his preliminary ·nvesti ation of partially urified kel bas mu cle glyco en phosp oryla e u est tat its re not n ike those o enzymes· olated from othe col blooded o . anisms: lobster (1), fro (75), do :,- fish shark (20), and tout (121). · At tern eratures encount ered by_. clathratus during the summer, total enzyme activity (a+b) is substantial (13,J to 19.9 units/g muccle mear-ured in the direction of glyco en synthesi at 15 to 20 C) and, at JO C, is nearly equal to that found in rat a trocnemius muscle (94). Arrhenius plots indicate con ormational tability to temperature above 13 C for both a and total activit.r ith an energ of activation ( 7 ) of a a proximately 2 kca (fi re 14). This val1e is close to those re orted or phosphorylase b of fro ~ muscle (15 kcal) and lobster ho horylase a (15.9 kcal) ( , 75). Below the environmental tern eratures of kelp bass, markedly. . .J increases a ot unexpectedly , the maximal velocity of the reaction falls with decrea in temperature. Gordon determined maxi mal rates of oxygen utilization of white muscle minces for 110 thirteen species of fish taken from Southern California offshore waters. An avera e oxygen consum tion of those fish, at 25 C, excludin those rapid, continuous swimmers (two carran ids and one Pcombrid), is 320 ml o 2 /min/k (39). This would be equivalent to 0.04 umoles glucose consumed er ram per minute. To produce equ·valent energy anae ob·cally 1ould re uire a proximatelv 0.7 umoles of uco~e er ram ner minute. If 50% of the total phospho - ylase activity was in the active form (a) operating at one half of the estimated ma imal velocity, then there would still be sufficient phosphorylase activity at 10 C to account for the metabolic needs of muscle in these kelp bass. s there is no visible red muscle mass, the major repulsive force and therefore t e major expenditure of ener y ould occur in the anaerobic, white muscle mass. nfort nately, t e amount o available lyco en in the dorsal white muscle of ke p bass cannot be accurate_y esti mated from either the in vitro or in vivo experiments due to the robable exhaustion of these stores when the fishes were sacrificed. As can be seen in table 3, measured ly co en levels are extremely low and variable. ~nzyme-substrate affinity for ,lucose-1-phosphate (G-1-P) remains relatively temperature independent in kelp bass. The estimated Km for G-1-P in the partially purified reparation is lower than that reported for shark (24mM) or lobster (54m~) phosphorylase b under similar assay 111 conditions, ho,ever, it is comnarable to that found in trout (15m ). It should be remembered that the kinetics were determined on a mixture of a and b orms of the enzyme. althou h elim·nary results with purified phos- phor.rla0e b from el bass re ulted in a s·milar value or the Km for G-1-P (table ). It is possible that the Km for lyco en, the enzyme's affinity for modulators uch as AMP, or even other nzyme~ in the cascade might be sensi tive to temperature. This would ltimately confer a tem perature sensitivity upon lycogenolytic recesses. In summary, muscle lycogen phospho ylase in ke p bass certainl has the otential for contributin . Sl if·cantl tote ener etic needs of mu cle in thi moderately active ish. The ca alytic capa i itie of ho horyla e indicate that th enz e i relatively ·nde endent o tern eratu e over env·ronment ran es althou h conformational chan es in the enz e of summer fish is indicated below 15 C by the Arrhenius dia rams. The activities of total phosphorylase in the two mid water fishes are an order of ma itude lower than that of . clathratus at all temperatures measured, although from the Arrhenius plots, the phosphorylase of these midwater fishes appears to be more thermally stable over a wider ange of temperatures (5-30 C) with a minimal~ of 19.4 a kcal for T. mexicanus and 16.3 kcal for L. stilbius. Pre- .umably , neither Leuroglossus nor Triphoterus encounter 112 tem eratures lower than 6 C (C. Rainwater, personal commu nication). Althou h the migratory patterns of L. stilbius are not clear, T. mexicanus mi ates from 550 meters to near surface waters (5-15, 20 C). Current information on L. stilbius shows it restricted to 550 to 450 meters depth in anta Catalina Basin (5-10 C). The suggestion is that L. stilbius may indeed be a vertical migrator in the shal lower an Pedro Basin where the experimental animals were taken (C. Rainvater, personal communication). This could explain the similarities in the enzymatic activities of the hosphorylases from these fish and as well as t e similari ties in their behavior at di ferent temperatures. In spite of the low activities for lyco enol sis in these two midwater fi hes, suf icient energy could be pro duced from lyco en reakdown to account for respiratory rates at daytime depth, particularly if one considers that both T. mexicanu and L. stilbius are thought to be inac tive during the day (19). Childress and 1 ygaard (19) esti mate the rate of oxygen consumption by these fish to be approximately 0.18 ul o 2 /hr/mg wet weight. This corre sponds to complete oxidation of 0.022 umoles of glucose/ ' min/g wet weight which is well within the capability of glycogen phosphorylase, even at 5 C. Of course pressure mi ht affect catalysis, however, -. affinity of the enzymes of mi ratin midwater fishes seem to be independent of ressure unlike those of benthic species (49, 2). 11 113 of the estimations of lyco en breakdovm are just that - estimations. vithout more detailed study, one can say only that phosphorylase activity of the muscle of these two mid water fishes providey a maximal catalytic rate sufficient to account for measured respiratory rates assumin~ complete oxidation of hexose to CO 2 and water. 114 CHA TER V SUMMARY Tracer quantities of both glucose-6- 3 H and glucose-6- 14c were used in cannulated, intact kelp bass (Paralabrax cla hratus Girard) to measure luco e replacement rates, lucose mass, and recyclin of carbon from lactate in free wimming, unanesthetized fish. Alanine U- 4 c in conjunc tion with lucose-6- 3 allowed simultaneous measurements of gl coneogenesis rom a ino acids and lucose turnover rates. Under eady sta e conditions, the rate o glucose replacement an the minimal mean glucose mass are less, the inimal mean transit time of glucose is increased, and the minimal glucose mass is one third that found in rats of comparable ize. All of these values are variable, however, only the glucose mass per 100 g boy weight appears to change substanti lly with fastin up o 42 ays. Data from non- tea y state experiments indicate that regu latory res onses to elevated plasma lucose levels occur with a shutdown in production followed by an increase in glucose uptake. These regulatory responses require hours to days to occur and may be temporally separated events. ubstantial incorporation of alanine carbon into plasma glucose occurs JO minutes after isotope injection in these fish. Fasting significantly elevates this incor- 115 poration, suggestin that alanine gluconeogenesis may be a major source of glucose for energy metabolism in these carnivorous fishes in light of a low rate of Cori cycling and the apparent stability of liver glycogen durin both fed and fastin states. 116 BIBLIOGRA HY 1. Assaf, S .. and D. Graves. 1969. Structural and catalytic ro erties of lobster muscle lyco en hoC" phor lase. J. iol. Chem. 244:5544-5555. 2 . A '""af, , . A. and . . Yuni c • 1971 . A com ara ti ve stud of er~ stallized ·1~y and lemon shar muscle 3. 1 co en phosphory a es. Int. J. iochem. 2: 04-60 . ehrisch, {. 1969. Tern erature and the re enz e activity in poi ilotherm. Fructose p, atase ro m·gratin salmon. Biochem. J. 69 . lation of iphos- 115:6 7- 4. Behrisch , 1972. 1~olecular mechanisms of adapta- tion to lov tempe ature in marine poikilotherms ome regulatory properties of dehydro enase from two retie pecies. Har . Biol. 1.}:267-275. 5. ehrisch, . and .. oc achka. 1969. Temperature nd the re lation of enzyme activity in poikilo therm'"", roperties of rainbow trout fructose diphos phatase. Biochem. J. 111:287-295. 6. Behrisch, H. and ) .. ~ ochachta. 1969. and the re lation of enzyme activity in therms. Properties of lun fish fructose ta e. Biochem. J. 112:601-607. Temperature poikilo diphospha- 7. Bentle. , P. J. and B. K. Foll et . 1965. ffects of hormones on the carbohydrate metabolism oft e lam pre ·, Lampetra fluviatilis. J. ~ docrinol. ]1:127- 137. 9. 10. ever, K. and A. Dunn. 1974. Un ublished observations. Bever, K. and A. Dunn. 1974, Carbohydrate etabolism in intact fi.h: studies with 3H, 4c lucose and 14c alanine. Fed. Proc. Jl:1560. ilinski, ~. 1969. Lipid cataboli~m in fish muscle in Pish in Research, 0. ~. I' euhaus and J. w • alver eds. cademic ress, rew York. gs. 135-155, 117 11. ilineo i, -'. and L. J. Gardn r. 196 . ~ffect o star- 2 • 13. vation on free fatty acid leve in blood p asma and m1scular tissues o rai bov trout (Salmo gairdneri). J. i~h. e • d. Can. 25:1555- 5 0. ilinc i, •. nd Y.C. Lu. 19 9. to,ard onrr-c ain tri ~c rids in o rainbow trout ( almo eairdne i). C n. 26:1 57-1866. Lipol tic activity ateral line mu cle J. Fi~h. es. Bd. Bi 1 in. k i , i' • and R . 1 • • • Jonas . enz e A and carnitine o atty rainbow trout mitochondria. J. 2 7 : 8 5 7 - 8 6Li, • 1970. ◄ ffects of co acid oxidation by 1 i, h. es. Bd. Can. 14. Black , '•, A.C. Robertson, and R.R. arke~. 1961. 15. 16. ome a~pects of carbohydrate metabolism in f·sh in Comparative hysiolog" of Ca bohydrate ,1etabolism in Heterot er,ic Animal . Univ. o Ja hin 0 ton -eattle. P s. 89- 24. Bro\·m , , . D. 1960. Gluco~ e metabolis C 11. Com. P eo·o1 . ..2,2: 1- 5. . in ca J. u lock, T. 1 ✓55, Com en.ation for tempe ature i em taboli.m and act·v·t of o· ·1othe m. iol. ev. JQ: 1-342. t e , 196 . si in Iorth A Gen. Co docrin. ormonal control of ican ee ( n illa 10: 5-91. - 8 . Cald,el , .. 1969. Ther al co pensation o re pi ratory e Z"'r,es in tissue of the oldfish (Carassiu auratu L .. Comp. Biochem. Physiol. Jl:79-93, 19. Childresc, J. and L y aard. 1973. The chem·cal composition of idNater fiohes aL a function of depth of occurrence off 1 outhern Ca ifornia. Deep Sea Research. 20:1093-1109, 20 . Cohen, . , '.l'. uewer, and ➔ • H. Fi sher. 1971, hos- p orylase from dogfish muscle. urification and a comparison of its physical properties to those of rabbit muscle phosphor lase. iochemi t y. 10:2683- - ? 69L~. 21 . Cra tree , B. and 1➔ .A. ewsholme . 1972 . The activi ties of hosphorylase, hexokinase , phosphofructocinaoe lactate dehydro enase , and lycerol-1-phos ate dehy dro~en e in muse e from v rtebrates and inverte- br tes . Biochem . J. 126 :lJ-9-58 , 118 22. as , . and C.L. rosser. 1967. Jiochemical chan es in tissues of oldfish acclimated to high and lo, tem peratures: I. rotein ynthesis. Comp Biochem. hysiol. 21:449-467. 23. Dean, J.JJI. and J.D. Berlin. 969. Alterations in 24. hepatocyte function of thermally acclimated rainbow trout ( almo _ai dneri). Comp. Biochem . Physiol. 29: 07-312. Dean, stu y b. e 299, J .. and C.J. Goodni ht. 1964. comparative of ca bo drate m tabolj in ish affected erature nd e erci . Phy 10 . Zoo . 17:280- e oco , . 1959. h siol. ]Z:175, ✓ romatop,: Can. J. Biochem. 2 6 . Dunn, . , K. Bever, . , en owe t and J. Katz . 197 5. Thee. t·mat·on o glucose ecyclin~ ·n vivo u in 3 and 14c lr.bel ed lucose. Fe . roe. In res. 27. unn, . , H . Cheno ,eth and J. . emington. 1971. The relationship of adrenal glucocorticoids to transami nase activity and gluconeogene i~ in the intact rat. Biochem. Biophys. eta. 237:192-202. 28. Dunn, A. , r . Chenoweth and L. . chaeffer. 1967. stimation of glucose turnover and the Cori cycle using lucose-6-t-14C. Biochemistry. 6:6-11. 29. unn, .. , B. Friedmann, . ,aass , G.A. eichard and . : einhouse. 1957. 7 ffects of insulin on blood glu cose entry and removal rates in nor al dos. J . iol. Chem. fil:225-237- 30. 1 .. isen. tein, . , I. track and . teiner. 197L}. Glucagon stimulation of he atic gluconeo enesis in rats fed a hi~h-protein, carbohydrate free diet. etabolism. 2J:15-2J. 31. pple, A. 1971, The endocrine pancreas in Fish Phys- iology, Vol. II , .. Hoar and D.G. Randall, eds. cademic ress, Iew York. Pgs. 275-319. 32. Falkmer , S . 1961 . •'xperimental diabetes in fish. eta ndocrin . uppl. JZ:3-122. 33 . oa, P . 1972. The secretion of glucagon in Handbook of Physiology , ection 7, Vol. 1, American hysiolo - ical 'ociety, ashington, D.C. s. 261-277. 119 J4. Freeman, l .C. and D.R. Idler. 1973. ffects of corticosteroids on liver transaminases in two salmo noids, the rainbow trout ( almo airdneri) and the brook trout ( alvelinus fontinalis). Gen. Comp. ~ndocrin . 20:69-75, J5 . 1 ry , F. . . 1971. he eff ct of environmental fac- tors on the physiolo y of fish in Fish hy iology, Vol. VI, ed . # •• 1 oar and D. J. andall, ew York , cad mic Pres . P s . 1-9 . 3 . r, H' . ~.J. and J .. Hart . 191~ . Relation of tem perat re too~ en consumption in the oldfi~h. io. u 1. 94:66-77 . 37 · Good , C., H. Kramer and M . omoayi. mination of ~lycogen. J. Biol . Chem. 933 . The deter- 100:485-591, J8. oo man, L .. and A. Gioman, es . 1965, The a- cological Ba ic- of Therapeutic , f.acfiillan Co. Yor. 39. Gordon , ,1...:) , 1972. Comparative studieu on the metab oliom of shallo 1 ater and deep sea marine fishes. I. Nhite mu~cle metabolism in shallow water fishes. ~lar . Biol. 1J:222-2J7, 40. Gordon , J •• 1972, Com arative studies on the metab oli m of shallow water an deep sea marine fishes. II. Red muscle metaboli~m in shallow water fishes. Mar . iol. 1.5.:246-250. 41. Grant , ' .C., F.J. Hendler and . 1. anks. 1969. tudies on blood su ar regulation ·n the little skate, a.ia erinacea. hysiol. Zoo .· 42: 231-247. 42. Gumbmann , 1. and A. Ta pel. 1962. yruvate and ala nine metabolism in carp liver mitochondria. Arc . Bioc em. io hysics. 98:502-575, 43. Haschemever , A. 1 ,V 1968 Compensation of liver pro tein Sfl]thesis in temperature-acclimated toadfish, O sanus _tav . io. Bull. 135: 130-140. 44. edriclc, J. L. and J. I . 1 isher. 1965. On the role of pyridoxal 5' phosphate in phosphorylase. I. Absence of classical vitamin B6-dependent enzymatic activities in muscle g~Jcogen phosphorylase. Biochem. 4:1337- 13L} J • 120 45. Hiatt, LI., M. Goldstein, J . Larean and B.L. orecker. 1958. The pathway of hexose synthesis from pyruva te in muscle. tT. Biol Chem. 231: 303- 307. 46 .. ochachka, . J. 1961. The effect of physical train ing on oxygen debt and glycogen reserves in trout. Can. J. Zool. ]2:767-776. 47. ochachka, . r.J. olism in fish. 1941. 1961. Can. J. Glucose and acetate metab iochem. Physiol J-2:1937- 48. ochachka, P.d. 1968. ction of temperature on branch points in o-luco e and acetate metabolism. Com. Biochem. Phy iol. £2:107- 1 . 49. "!ochachka, . N. 1971. ~ zyme mechani ms on tempera ture and pressure ada tation of offshore benthic or.er ni ms: The asic Problem. A. 7.ool. 11:425- 437. 50 . Ho cha ch a, . ~. a1 d B . Clayton- ochachka. 197 3. luco~e-6-pho phate dehydrogena~e and thermal accli- mation in the ullet fish. t ar. Biol. ,251-259. 51, Hochachka, .. and T •. Haye . 1962. The effect of tern erature accliration on pathwayc- of g-luco e metab oli in the trout. Can. J. Zool. 40:261-270, 52. .lochachka, . J. and A. C. inclair. 1962 Glyco en stores int out tir-sue before and after stream plant in . J . 1 i h . Res . d . Can . 12 : 12 7 -13 6 . 53. 54. oc ac ka, P. J. and Gt! Somero. tion of enzymes to tern erature. hy. io . 27:659-66 . 1968. The ada ta Com. Biochem. Ou in , q • tern erature from a cold arietali .. . . and . leksiuk. 1973. ~ feet of on t e kinetics of malate deh dro enase climate reptile, T1amnop i si inalis Co . iochem. Ph. siol. !±..2]:34 - 53. 55, Inu·, Y. and Y. Oshima. 1 66. 1 ffect of tarvation on etaboli and ch mical compo~ition of eels. ull. a . oc. ci. Fis . _E:492-501. 56. Jackim, J • and G. LaRoche. 1973, in Fundulus heteroclitus mu cle. hysiol . 44A :851-866. Protein synthesis Comp. Biochem. 121 57. Janssens, P.A. 1965. hosphorylase and glucose-6- phos hataoe in the african lun fish. Com. Biochem. Physiol. 16:317-319. 58. Katz, J., A. Dunn, n. Chenoweth ands. olden. 1974. etermination o srnthesis, rec cling, body mass of lucose in rats and rabbi ts ·in vivo w· th 3 1 {-and 14C- l u cos e . i o chem . , 11+ 2 : 1 71-18 3 . 59. '"atz, ,J., r. Rostomi and . Dunn. 1974 . .c,volution of luco~e turnover, body mas~ and recyclin with rever, ible nd ·rreversible tracers. Bioche . J. 11+2: 161-170. 60. Knipprath, ·J.G. and J.F. I~ead. 1968. rrhe effect of environmental temperature on the fatty acid composi tion and on the in vivo incorporation of l-14c ace tate in oldfish (Caraosius auratus L.). Linids. J: 121-128. 61. Krisman, C. 1962. , 1 ethod for colo i 1etric e tima tion of glyco en with iodine. nal. Biochem. 4:17- 23. 62. Lars, on, . and norenine 20: 55- 67. 197 3. Tl1etabolic effects of epinenh ine rine in the eel. Gen. Cornn. ndocrin. 63. Larsson, A. and K. Lewander. 1972. ~ffects of luca on administration to yello eel . Com. Biocher . .c hy. io . 4JA: 831- 36. 64. Lar~son, . and K. Le¥ander. 1973. eT ects of starvation in the eel, An ioc1em. Physiol. 44A:J67-374. ,, etabolic illa, h. Comp. 65. Leibson, L. and ,'. 1 1. lisetskaya. 1968. :!.ffect of insulin on blood su ar level and ~lycopen content in organs of some cyclostome and fish. Gen. Comp. ~ndocrin. 11:381-392. 66. Leibson, L.G., ~- "· Plisetskaya, and T.L. Jazina. 1968. Concentration of non-esterified fatty acids in blood plasma of cyclostomata and fishes and its changes under the influence of adrenaline and insulin. Zh. ,vol. Bi ochim. Fi ziol. L~: 121-127. ( In Russian, cited by Chem. Abstr . .§.2:1041). 122 67. Lip hav, .. , . P tent and P. Foa. 972. •ffect. of ninephrine and none inephrine on t e epatic ipids oft e nurse shark, Jin~lymo toma cirratum. or . tab. e . L~:34-3 . Liu, D. 1 I . , " . reu er nd C. ~ a g. at ways for gluco e in the c·chlid bimaculatum. Co . Bioc1 . hy ·01. _970 . Catabolic ieoh, Cichla. oma .l§:173- 1. 69 . Love, L. 1 • 1970. The C_h_e_i_c..;_a;,_,l __ _;.__ ......... '---o __ f_F_i_· _sh~e_s, cademic r "S, 1 ew York . 70. 71. 72. 73. 74. Lutz, P.I. 1972. ody compartmentalization n . ion di tribution in the te eo. t ( erca fluviatili ). Comp. · och m. P y('.'<io . 41" :18 - 9J . . 1ar hall , . 1960 . wimbla der ea i he. ·n relation to thei s o Di co e Re . ]1_:1- 22. J. . and D. • and f edin on e ic n e (An e. iol~.-=,..;.~~ o m . or oo 29.'327- tructure of eep tematics and biol- ffects o te - etabolism in ta Le, u u ) . 5. •va uation o a determinat"on. er--ter, . , D . Io The "nfluenc of ior of 1-alanine: 1e s 1 eletal u. c fo~ ili('.'< L.). Co cheocu and . 1 ic le cu. 9 3. d t ion te perature on the beh v- 2-oxo ~lutarate aminotran. erase of e of the ond loach ( i~gurnus p. Biochem . P ys·o1. 45B:923-9Jl. 7 5 . 1e t z er , B . :\ . , L . G 1 as e and ~ . .1Ie lmr e i c . 19 6 8 . urification and propertie of frog skeletal mu cle pho phorylase. iochemistry. Z:2021-20J6. 76 . . igliorini, H., C. Linder, J. Moura and J. Veiga. 1973. Gluconeogen sis in a carnivorou~ bird (black vulture). Am. J. hysiol fil:1389-1392, 77. 1inick, r . C. and rJ . Chavin. 1972. 1 ffects of verte brate insulins upon serum FFA and phospholipid levels in the oldfish. Comp. Biochem. hysiol. 41A:791- QL}. 123 78. Iiinick , l• .C. and . Chavin. 1972. •ffects of alloxan, streptozotocin, or -mannoheptulose upon seru free fatty acid and serum lucose levels in oldfish. Comp 13iochem. hysiol. L~2B: 367- 376. 79. llinick, r.1 . C. and v. Chavin. 197 3. ffects of ca te- cholamines u on serum FFA levels in normal and dia betic oldfi h. Com. iochem. Phy iol. 44A:1003- l008. O. 100n , T. and P . TJ . 1 ..rochachla . 1971, ffect of ther- a acclimation on multip e for s of the liver solu- ble NA lined isocitrate dehydro enase in the amilv almonid e. Comp. Biochem. Ph. siol. 40 :207- 213. 81 . Ioon , T. and P.., . Jochac l<:a. 1971. Temperature and enzyme activity in poilcilotherms . Isoci trate ehy dro enase in rainbo 'v trout liver. Biochem . J. 123: 695-705 , 82 . ,loon, T., T. dustafa and .. Iochachl<:a. 1971. The adaptation of enzymes to press re. I . com- nari on of muscle FK fro urface and mi ''later fishes wit the omolo ous enzyme from an off'""hore benthic species. m. Zool. 11:491-503. 83. ragai , I. and . Ikeda. 1971. •ffect of starvation on blood lucose level~ and hepatopancreatic gl cogen and lipid content in car. Bull . Ja. Soc. Sci. Fi'""h. l'Z.:404-410. 4. agai, 1. and . . Iked . 1972. Carbohydrate metabo i in fir--h III. ~ffect 4 of dietar composition on etabo ism of ~lucose-u-1 C and 1 ta ate-U- 1 ~c in carp. ull. Jap. oc. • 1 ci . Fish. J.§.(2) :137-143. 85. a ai, ,, . and . Ikeda. 197 3. Carbohydrate metabo li in fish IV. ~ffect of dietar com o ition on metabolism of acetate-14c and L-alanine-u-14c in car. Bull . Jap. oc. ci. p·ch. J.2(6) :633-643. 86. Nakano , n. and N. Tomlinson . 1967. Catecholamine and carbohydrate concentrations in rainbow trout in relation to physical disturbance. J . Fish . es . Bd. Can. .?4 : 1701-1715 . Newsholme , etabolism. . and C . "it art . Wiley and ons, 1973. _.§_gUlation in I e v York . 124 • • 90. 91. 92. Patent, G. 1970. Com ari on of some hormonal effects on carbohydrate etabolism in an elasmobranch and a holocep alan. Gen. Comp. ~ndocrin. 14:215- 242. atent, G. and P.O. •oa. 1971. Radioimmunoassay of insu in in fis es, e~periment in vivo and in vitro. Gen. Comp. ~ndocrin. 16:41-46.- ei. s, C .. and J. Field. 1950. ~e.Jniratory metab olis of excioe tis ue~ of Yarm and cold ada ted fish. Bio. Bull . ·213-224. hillips, . 1969. Iutrition, dige tion, and energy utilization in _ish Phy iologtt, Vol. I., ca~emic res, I e York. gs. 391-}J2. o e , C.L. and F .. Brovn . 1961. imal , . vaunders Co. , s. 15 Com arative hiladel hia. 93. Roberts, J.L. 1966. ystemic versus cellular accli mation to temperature b poikilotherms. ~elgolander iss. eer~unter. 14:45 -465. 94 . cha e ff er , L . D . r . C en o 1e th and . Dunn . 19 6 9 . drenal corticostero·d involrement in the control of pho p1orylase in mu cle. ·o. Chim. Bio hy~. Acta. 92: 304- 30 . 95. c tton, .C. ad L . Utter. 196 Th re lation of ~lycol. sis and uconeo ,cnes·s in animal tis e . Ann. ev. B · ochem. ]7_: 21r - 302 . 9 . mith, K.L. and I .R. He.·sler. 1974, espiration of benthopel ,ic f 0 shes: In s·tu measure ents at 1230 mete . 184:72-3, 97. mi t , L. and G. \. Bell. 1964. technique for pro lon ed blood samplin~ in free- wimm·n salmon. J. Fi . ' e s • B d • Can . £_ : 7 -- 71 7 . 98. omero, . r. 1969. 'nzymatic mec anisms of tern era ture compe sation: immediate and evolutionary ef ·ect of temperature on enz mes of a uatic oikilo t 1erms. m. ·aturalist. 103: 517- 530 99, . r, 1969. Pyruvate ina e variant. oft e la a in~ crab. videnc for a temp rature depend ent interconv r. ion between ti..ro forms av·n di. tinct d ada tiv in,tic p o er ies. iochem. 111~:2 7- 24. 125 10 . o ero, G .. 1973. ermal odllat·on of pr v te met boliun in the fi~h Gillichthy. mirabi i~: The ro e of lactate dehyd o enase. . Com . Bio chem. P , ,:, i o 1 . I J .i, B : 2 0 5 - 2 0 9 . 10 . . omero, G, l'l. and D. Doyle. 197 3. Tern era ture and rates of otein de rad tion in t1e fish G · , irabilis. Cop. iocl e. Phy iol. 46 :4 1 O 2 • 1 om er o , G . T • , • G i e e and D • hJ o hl ch 9 6 8 • Cold a a tation of the Ant retie fish, Trematomvs be nacc Comp. Bioche . Phy iol. 2b:22J-2JJ. 10 J. Somero, G. 1 • and P. · J. rochachka. 1968. The effect 1 o te ature on ct 1 tic and re ato function~ te kinase th rainbo, tout an the i. , re be ioch m. - . L~ • . 945, De . b ood 0 1, erm n 10n 0 .u 1. • 1 0:6 -73- 0 t ele, B. Pl-io Dunn, T tszuler, I . , , • •• I. at1 eb an . C I de"Jodo. 196.5. In ibition b insulin of hep tic lucose production . t al 1n nor 0 • A • J. 1 siol. 208:J01-J06 06. tip on, J.H. 1965. Comparative a pect of the control of ~lyco en utilization in vertebrate liver. Conp. ioc e . lh iol . .l.5.:187-197- 107. torer, J.H. 1967. tarvation nd the effect of 108. co ti ol in the oldfis (Carassius auratus L.). Conp. iochem. Physiol. 20:939- 48.-- ~ 1allo v, R. L. and • . !i:emmin . 1970 Th e feet of o .. aloacetate, ACTL, and cortisol on the liver lyco ~en lev s of Tilapia o samb·ca. Co . Bice em. P s·o1. }2:9J-98, Ta r, I .L. . 969. blooded vertebrates etaboli , in ~ish J . · J. al ver , ed .. Contr t bet een fish and varm in enz e yste of intermediary · • 0 • Ieuha nd . ss, w York. 10. ·ma, L . and G.T. Cahi 1. 19 5. Fat met bolism · s in l_andbook of Physiolo, r, Section 5: Adi ose Tis e, erican P ~iolorricql ociet , ~ shington , .c. p u l 369-371. 126 111. 112. 13. Tas1ima, L .. and . Cahill, Jr. insulin in the toadfi h, Qpsanu •ndocrin. 11:262-271, 1968. 1 ~ffects of tau. Gen. Comp. '1 1 epper an, J. 1968. Hetaboli c and ◄ndocrine h,rsi olo r, er Boo 1 dic-1 Publi hers, Inc., Chicago. Thor e, . and P ,L. Ince. 1974, a tic hormo 1es, c techo a ine. , and blood m tabol·te n the rorth n ~ndocrin. £}:29-14. f ects o pancre lucose loadin on Gen. Comp. 4. Thor on, T. 1 61. Tl e par itionin of )ody rat r in Oste · e : phyla netic nd ecolo · cal impJ.ica- iono u tic verteb at . io . u 1. 20:2 2 51-1, • lernbe , . . 954. e iratory etabolism o tis e. of a in teleosto ·n relation to activit d body ize. Biol. u 1. 106:360-370. 1 6 . il on, . P . 1973. Jitrorren metaboli min c ann 1 catfish, Ictaluru unctatus I. Tissue distr:bution of a~p rtat~ and alanine aminot an fera e and 1 ta mic del dro ena e. Comp. iochem. h. iol. 46 :617- 621-J-. 1 7 . Jil on, . ~ and .. oe. 1974. lism in channe catfish, ctaluru elative nool sizes of free amino com ound in variou ti~sues oft iochen. ysiol. 48B :545-556. itrogen metabo B ) 1.cta tu III. · cid and related e catfish. Com. 1 . r i 1 i am on , J . . , · J • C eung , "L Co es and B . er c zig . 1967. Gl. colytic control mechanism~ IV. inetic of 1. col tic intermediate cha1. es durin electrical dischar~e and recovery in the main or an of •lectro phoru C" ele ctricus. J. i o . Chem. 2L1-2: 5112-5118 . 119. 20. T·Jolf , _,,. , te eo 0 t .... . 19 J. hy io o ical saline for freoh water ro~r. Fi h. Cultur. ~:135-140. Ju , 1964. imultaneous stud · es of pho transpo t and ,lycolysis by a sam le J.iqui tion countin procedure vith 32, Cl4, and poundd . nal. Biochem. 2 :207-214. phate scintilla HJ com- 121. ammamoto , I • 1968 . F'ish muscle lycogen phosphory la .. :>e . Can. J . Bi oche . 46 : 42 J-Li-32 . 127 122. oung, arle. 1963. The kel bass (_aralabra clathratus) and its fishery, 1947-1958. Fish ulle tin 122 Resources A ency of California, et. o 1 i hand Gae. 128 KAREN LA VERNE BEV R 1946 Born in Long B ach, California 1964 Graduated from range High cho I, rang , ali fornia 1968 B .. , Biological Scien e , Univer ity of outhern Cali fornia, Los Angeles, California 1968 72 National S ience oundati n Traineeship, Univer ity of Southern a1ifornia 1968, Tea hing A si t n t, University of Southern California 1972-74 1968-75 Graduate tudent, Uni ersity of outhern California 1969-74 Biomedical Sciences upport r nts, Univer ity of outhern California

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The effectiveness of criminal justice sanction strategies in the deterrence of drug offenders

PDF

Perceptual accuracy in issues related to marriage, parenthood and religion among engaged couples planning a Catholic Church wedding

PDF

Empathy as a function of guided daydreams in counselor education

PDF

The pulpit and platform career and the rhetorical theory of Bishop Matthew Simpson

PDF

A study of the effects of foster home placement on a selected number of "rejected" children

PDF

Action of DDT on two clones of a marine green alga Pyramimonas

PDF

Transnational society vs. the state-centric system : a preliminary analysis of the multinational corporation as an agent of systemic change of instability.

PDF

An innovative play environment for handicapped children

PDF

The treatment of juvenile delinquency in Germany

PDF

Investigations of the oxidative metabolism of phagocytizing polymorphonuclear leukocytes

PDF

The contribution of Mohammedanism to Philippine culture

PDF

Pastoralism in the novels of Mary Shelley

PDF

Clothing as a factor in the social status rating of men

PDF

The influence of Pietro Aretino on English literature of the Renaissance

PDF

Population genetics and recruitment of the kelp bass, Paralabrax clathratus

PDF

An investigation of oxygen-pulse time course as a measure of cardiorespiratory adaptation to exercise

PDF

Claus Spreckels of California

PDF

Exploring the genomic landscape of giant kelp: biotechnological implications and sustainable development

PDF

A study of the lipid composition of Streptococcus faecalis and Streptococcus faecium

PDF

Effects of glucose modulation on adipocytes maturation and metabolism

Asset Metadata

Creator Bever, Karen LaVerne (author)

Core Title Glucose metabolism of the kelp bass, Paralabrax clathratus Girard

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Biological Sciences

Degree Conferral Date 1975-03

Publication Date 03/10/1975

Defense Date 03/10/1975

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag glucose metabolism,kelp bass,OAI-PMH Harvest

Format theses (aat)

Language English

Contributor Digitized in 2022 (provenance)

Advisor Dunn, Arnold (committee chair), Appleman, M. Michael (committee member), Brodie, Arnold F. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC112723828

Unique identifier UC112723828

Identifier Ph.D. B.Sc '75 B571 (call number),etd-BeverKaren-1974.pdf (filename)

Legacy Identifier etd-BeverKaren-1974

Document Type Dissertation

Format theses (aat)

Rights Bever, Karen LaVerne

Internet Media Type application/pdf

Type texts

Source 20230201-usctheses-microfilm-box6b (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu