The rates of incorporation of [U-14C]proline and [U-14C]glucose
into CO2 and glycogen were assessed in Nautilus pompilius under both in vitro
and in vivo conditions. Both substrates exhibited tissue-specific rates of metabolism.
However, overall higher rates of incorporation into CO2 and glycogen were
observed with glucose, both with tissue slices and in the intact, catheterized
organism

Fields, JHA

Hochachka, P.W.

Pacific Science

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Pacific Science (1982), vol. 36, no. 3© 1983 by the University of Hawaii Press. All rights reservedGlucose and Proline Metabolism in Nautilus·J. H. A. FIELDS2 and P. W. HOCHACHKA3ABSTRACT: The rates of incorporation of [U-14C]proline and [U-14C]glucoseinto CO2 and glycogen were assessed in Nautilus pompilius under both in vitroand in vivo conditions. Both substrates exhibited tissue-specific rates of metabo-lism. However, overall higher rates of incorporation into CO2 and glycogen wereobserved with glucose, both with tissue slices and in the intact, catheterizedorganism.MATERIALS AND METHODSthat in Nautilus, glucose is far more vigorouslyoxidized than is proline in all tissues analyzed.Not surprisingly, glucose is also incorporatedinto tissue glycogen at much higher rates thanis proline.Experimental AnimalsAll experimental Nautilus were obtainedfrom local fishermen working out of Bindoyin Tanon Strait, Negros Orientale, Republicof the Philippines, in October 1979.In Vitro ExperimentsTissue slices were prepared as described byHochachka and Fields (this issue), and, ingeneral, all other manipulations were alsoessentially identical. An important difference,however, was in the incubation medium used.In the first study, Nautilus blood was useddirectly as an incubation medium. Nautilusblood contains a mixture of amino acids,glucose, and octopine (Hochachka, French,and Meredith 1978; Storey et al. 1979), pre-sumably in addition to other unidentifiedmetabolites. A total of 5 ml of blood wasadded to experimental flasks containing 2 /lCiof either [U-14C]proline or [U-1 4C]glucose.Although the blood levels of proline andglucose are similar (about 0.5-1.0 /lmole/ml;Hochachka et al. 1978, Storey et al. 1979), thetissue levels of proline can be assumed to bemuch higher, particularly in muscle. Since the337PROLINE IS AN ABUNDANT COMPONENT of thefree amino acid pool in cephalopod tissuesand is considered to contribute to aerobicmetabolism (Mommsen and Hochachka 1981,Storey and Storey 1978). At the same time,high activities ofglycolytic enzymes and amplesupplies ofglycogen (Ballantyne, Hochachka,and Mommsen 1981; Hochachka and Fields,this issue) also imply an important rolefor carbohydrate in cephalopod intermediarymetabolism. However, there is little informa-tion on the relative contributions of prolineand glucose to different metabolic processes.In preliminary experiments with squid andoctopus, Hochachka and Fields (this issueand unpublished data) found higher rates ofproline oxidation than of glucose oxidation.The data suggest higher proline oxidationrates in fast-swimming squid than in the moresluggish octopus. Since the whole-organismmetabolic rate ofNautilus is lower than that ofOctopus macropus (Johansen, personal com-munication), we were interested in comparingits relative rates of glucose and proline incor-poration into CO2 and glycogen. We found1 This research was conducted as part of the AlphaHelix Cephalopod Expedition to the Republic of thePhilippines, supported by National Science Foundationgrant PCM 77-16269 to J. Arnold. This study was alsosupported in part QY an operating grant from the NaturalSciences and Engineering Research Council ofCanada toP. W. Hochachka.2 University of Washington, Department of Zoology,Seattle, Washington 98195.3 University of British Columbia, Department ofZoology, Vancouver, British Columbia, Canada V6T2A9., , • , , ",. , '" ~~ ... , , , """ "~" ,..,.,.. , ,,,,>rn """'~ ".,.,338tissue levels were not ascertained in these ex-periments, the exact specific activities of pro-line and glucose were not known. Thus, therates of metabolism were only qualitativelycomparable.In a second set of experiments, the sameprocedure was used but the blood was firstvacuum dialyzed.In Vivo ExperimentsIn four separate Nautilus preparations, thevena cava was catheterized using proceduresdescribed elsewhere (Johansen, Redmond,and Bourne 1978). In two preparations, 10IlCi of [U-14C)glucose were taken up in 3 mlof seawater and injected into the blood, fol-lowed by a I-ml washout injection of sea-water. In the other two preparations, 10 IlCiof [U-14C)proline were injected in similarfashion. Small-volume (0.1-0.2 ml) bloodsamples were drawn at 5, 10, 30, 60, 90, and120 min following initiation of the experi-ment. The 14COZ was driven off with per-chloric acid, collected in KOH, and countedin Aquasol. Similarly, tissue glycogen was ex-tracted, purified, and counted, in both casesusing procedures described by Hochachkaand Fields (this issue). The 14COZ yields in theblood showed tremendous variation betweenindividuals. For example, one Nautilus retainedup to 3000 disintegrations per minute (DPM)per ml blood at about an hour after injectionof 14C-proline, while another showed between100 and 200 DPM for the duration of theexperiment, although in both cases the timefor 50"10 removal of radioactive metabolitesfrom blood was about 15 min. In the glucoseexperiments, the yields of 14COZ in the bloodwere not so variable but showed drastic dif-ferences in time course. Since, under theseconditions, unknown and variable amountsof 14COZ were also lost across the gillepithelium, this variability in the in vivoexperiment was not surprising. However, itdid preclude direct comparison of the in vivooxidation rates of proline and glucose usingthese procedures. At the completion of theseexperiments (2 hr after injecting labeled sub-strates), the animals were gradually chilledin ice-cold seawater for another 2 hr. ThePACIFIC SCIENCE, Volume 36, July 1982animals were quiescent through this manip-ulation and were in effect cold anesthetizedwhen sacrificed. The tissues were quicklyremoved, glycogen was extracted, and 14C_glucose incorporation was compared to14C-proline incorporation as described byHochachka and Fields (this issue).RESULTSProline and Glucose Oxidation RatesTwo interesting implications arise from as-sessing rates of COz release from proline andglucose. First, as indicated in Tables 1 and 2,rates of oxidation of glucose by all tissuesexamined greatly exceed rates of proline oxi-dation. Although large differences in intracel-lular pool sizes of proline and carbohydratemay contribute to the different rates of 14C_proline and 14C-glucose incorporation into14COZ, the overall pattern stands in strikingcontrast to that observed for Octopus mac-ropus kidney and gill (Hochachka and Field,this issue) and squid kidney, heart, and innermantle (Mommsen and Hochachka 1981;Mommsen et aI., this issue), where 14C-prolineoxidation rates exceed those of glucose.Second, despite the general predominanceofglucose metabolism in essentially all tissuesexamined, some clear-cut tissue specificitiesemerge. Heart oxidation rates of proline arehigher than in any other tissue (particularlywhen dialyzed blood is utilized as the incu-bation medium); at the same time, glucoseoxidation rates by the heart are relatively low,lower than for kidney, pericardial gland, orgill (Tables 1, 2). In other cephalopods ex-amined, a similar relatively high capacityfor proline metabolism has been observed(Mommsen et aI., this issue).Proline and Glucose Incorporation intoGlycogenAs in our parallel studies of glutamateand proline incorporation into glycogen inOctopus macropus, our experiments with Nau-tilus establish the capacity for this process inessentially all tissues studied (Tables 1, 2)." ... ~,,- ""'~'~"" ,.,. .... "-." ......... ,, "~,,-,,,"$.,~.,", ... ,," .......... ,-~- ...... - T'~' ,,,_,,. r-,. " ....".n"' ...Nautilus Metabolism-FIELDS AND HOCHACHKATABLE I[U- '4C]PROLINE AND [U-'4C]GLUCOSE INCORPORATION INTO CO, AND GLYCOGEN IN Nautilus TiSSUES339SUBSTRATE14C-Proline'4C-Glucose14C_GLYCOGEN14COZTISSUE (DPM/g tissue) (DPM/tlmole glucose) (DPM/g tissue)Kidney 225 57 2,390Pericardial gland 182 75 1,166Gill 669 480 2,543Systemic heart 946 72 2,868Retractor muscle 263 25 621LiverKidney 14,677 6,332 223,513Pericardial gland 10,456 5,696 73,484Gill 48,695 48,007 326,446Systemic heart 7,220 3,560 192,231Retractor muscle 2,503 483 12,037Liver 4,780 491 4,176NOTE: Undialyzed blood was used as Ihe incubation medium. Temperature. 2ZOC.TABLE 2[U- '4C]PROLINE AND [U-'4C]GLUCOSE INCORPORATION INTO CO, AND GLYCOGEN IN Nautilus TissuESSUBSTRATE14C-Proline'4C-Glucose14C-GLYCOGEN14COZTISSUE (DPM/g tissue) (DPM/tlmo1e glucose) (DPM/g tissue)Kidney 284 32.9 2,723Pericardial gland 204 70.3 3,481Gill 808 270.3 2,731Systemic heart 2,162 27.1 2,515Retractor muscle 272 29 2,413Liver 34.6 1,313Kidney 15,003 4,826 459,946Pericardial gland 1l,1l3 1,365 68,951Gill 29,376 17,920 206,081Systemic heart 8,259 3,182 320,129Retractor muscle 5,738 813 83,607Liver 2,979 1,463 48,584NOTE: Dialyzed blood was used as the incubation medium. Temperature, 22°C.However, the marked tissue specificity seen inOctopus macropus (Hochachka and Fields,this issue) is not so strikingly displayed. Also,the actual incorporation rates are low in com-parison with Octopus (Tables 1, 2), whetherthe incorporation is expressed in terms of 14Cincorporated per micromole glucosyl glycogenor in terms of 14C-glycogen formed per gramof tissue.In contrast, very high rates of incorpo-ration of 14C-glucose into glycogen typifiesmost of the tissues analyzed. Lowest ratesoccur in liver slices; highest rates, in kidney,gill, and systemic heart (Tables I, 2).In Vivo Proline and Glucose Incorporation intoGlycogenBecause of large percentage differences inthe amounts of glycogen present in differentindividuals, pooling of data from differentspecimens seemed to cover up more informa-tion than it summarized; therefore, the incor-poration rates are given for each Nautilus• , '" , ,'~, - - • ...~, ~.. " , • ~"" "-"< - , •340 PACIFIC SCIENCE, Volume 36, July 1982TABLE 3IN VIVO [U- '4C]PROLINE INCORPORATION INTO GLYCOGEN IN Two Nautilus FEMALES14C-PROLINE INCORPORATION INTOGLYCOGEN/4 hrGLYCOGEN LEVELTISSUE (Jlmole glucose/g) (DPM/Jlmole glucose) (DPM/g tissue)Nautilus # 1 (679 g total weight)Kidney 13.5 31.8 429.3Pericardial glandGill 5.9 27.9 164.1Systemic heart 23.0 22.3 512.9Retractor muscle 8.6 45.7 391.2Liver 3.0 17.0 50.7Nautilus # 2 (789 g total weight)Kidney 43.1 75.4 3,248Pericardia1 gland 32.6 20.4 665Gill 12.7 83 1,054Systemic heart 61.9 28 1,733Retractor muscle 89.1 45.7 476Liver 48.1 8.2 394NOTE: Tissue levels of glycogen varied extensively. so the data are not pooled.TABLE 414C-GLUCOSE INCORPORATION INTO GLYCOGEN IN Two Nautilus FEMALES14C-GLUCOSE INCORPORATION INTOGLYCOGEN/4 hrGLYCOGEN LEVELTISSUE (Jlmole glucose/g) (DPM/Jlmole glucose) (DPM/g tissue)Nautilus # 1 (678 g total weight)Kidney 26.3 881 23,176Pericardial gland 4.0 13,593 54,099Gill 1.7 120,241 199,600Systemic heartRetractor muscle 3.3 4,386 14,431Liver 1.4 4,523 6,288Nautilus #2 (667 g total weight)Kidney 28.7 494 14,187Pericardia1 gland 30.2 2,274 68,684Gill 0.7 352 254Systemic heart 44.2 2,355 104,072Retractor muscle 5.7 2,459 14,040Liver 3.4 1,014 3,396separately (Tables 3, 4). As expected from thein vitro experiments, in vivo incorporationrates of 14C-glucose into tissue glycogen poolsgreatly exceed the rates of 14C-proline incor-poration. For proline, highest rates of incor-poration appear in kidney, gill, and systemicheart (Table 3). In comparison, highest ratesofglucose incorporation appear in pericardialgland, kidney, systemic heart, and retractormuscle; the results for the gill are equivocalsince very high rates of 14C-glucose incorpo-ration into glycogen occurred in one indi-vidual, but very low rates in the other (Table4). Although the basis for this discrepancy is• """'-' , , ~ r, "" ."" , .•_ ., '" " ,Nautilus Metabolism-FIELDS AND HOCHACHKAnot understood, it may be worth emphasizingthat the level of endogenous glycogen in thegill varied in the two cases.CONCLUSIONSEven if these experiments must be consi-dered as preliminary, they are instructive inestablishing a rather marked difference in therates of utilization of proline and glucose bothin oxidation and in replenishing glycogendepots in Nautilus tissues. In contrast to thesituation in the octopus, glucose appears to bea particularly useful substrate for both thesemetabolic functions. In view of these results, itwould be surprising if glucose were not themain carrier of carbohydrate carbon in bloodand the primary immediate precursor of gly-cogen in most tissues of the Nautilus body.From the in vivo experiments, a high clear-ance rate for both substrates implies the ex-istence of an effective circulation and mecha-nisms for the rapid clearance of metabolitessuch as glucose and amino acids. However,the kinetics of exchange between tissues andblood have not been clarified, and this re-mains a subject for further research into meta-bolic biochemistry not only of Nautilus but ofother cephalopods.341LITERATURE CITEDBALLANTYNE, J. S., P. W. HOCHACHKA, andT. P. MOMMSEN. 1981. Studies on the meta-bolism of migratory squid, Loligo opales-cens: Enzymes of tissues and heart mito-chondria. Mar. BioI. Lett. 2: 75-85.HOCHACHKA, P. W., C. J. FRENCH, andJ. MEREDITH. 1978. Metabolic and ultra-structural organization in Nautilus muscles.J. Exp. Zool. 205:51-62.JOHANSEN, K., J. R. REDMOND, and G. B.BOURNE. 1978. Respiratory exchange andtransport of oxygen in Nautilus pompilius.J. Exp. Zool. 205: 27-36.MOMMSEN, T. P., and P. W. HOCHACHKA.1981. Respiratory and enzymatic proper-ties of squid heart mitochondria. Eur.J. Biochem. 120: 345-350.STOREY, K. B., and J. M. STOREY. 1978.Energy metabolism in the mantle muscle ofthe squid, Loligo pealeii. J. Compo Physio!.123:169-175.STOREY, K. B., J. M. STOREY, K. JOHANSEN,and P. W. HOCHACHKA. 1979. Octopinemetabolism in Sepia officinalis: Effect ofhypoxia and metabolite loads on the bloodlevels of octopine and related compounds.Can. J. Zool. 57: 2331-2336." ~ • ~ _. ". f ,.. " ., ,," N ~.. _ , • "".., _. • r " ,,",'" , , .. • , •

Glucose and Proline Metabolism in Nautilus

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Glucose and Proline Metabolism in Nautilus

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