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JOURNAL OF CELLULAR PHYSIOLOGY 166:152-169 (1996)

Comparative Analysis of Glucose andClutamine Metabolism in TransformedMammalian Cell Lines, Insect andPrimary Liver Cells

J O R C N E E R M A N N AND R O L A N D W A G N E R *Department for Cell Cu lture Techniques, Gesellschati fu r Biotechnologische Forschungrn.b.H., 0-38124 Braunschweig, Germany

Glucose and glutamine metabolism in several cultured mammalian cell lines(BHK, CHO, an d hybridoma cell lines) were investigated by correlating specificutilization and formation rates with specific maximum activities of regulatoryenzymes involved in glycolysis and glutaminolysis. Results were compared withdata from two insect cell lines and primary liver cells. Flux distribution wasmeasured in a representative mammalian (BHK) and an insect (Spodopfera rugi-perda) cell line using radioactive substrates. A high degree of similarity in manyaspects of glucose and glutamine metabolism was observed among the culturedmammalian cell lines examined. Specific glucose utilization rates were alwaysclose to specific hexokinase activities, indicating that formation of glucose-6-phosphate from glucose (catalyzed by hexokinase) is th e rate limiting step ofglycolysis. No activity of the key enzymes connecting glycolysis with th e tricar-boxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, andphosphoenolpyruvatecarboxykinase,could be detected. Flux distributionin BHKcells showed glycolytic rates very similar to lactate formation rates. No glucose-or pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating thatglucose is mainly metabolized via glycolysis and lactate formation. About 8% ofutilized glucose was metabolized via the pentose phosphate shunt, while 20 to30%of utilized glucose followed pathways other than glycolysis,the tricarboxylicacid cycle, or the pentose phosphate shunt. About 18% of utilized glutaminewas oxidized, consistent with th e notion that glutamine is the majorenergysourcefor mammalian cell lines. Mammalian cells cultured in serum-free low-proteinmedium showed higher utilization rates, flux rates, and enzyme activities thanthe same cells cultured in serum-supplemented medium. Insect cells oxidizedglucose and pyruvate in addition to glutamine. Furthermore, insect cells producedlittle or no lactate and were able to channel glycolytic intermediates into thetricarboxylic acid cycle. Metabolic profiles of the type presented here for a varietyof cell lines may eventually enable one to interfere with the metabolic patternsof cells relevant to biotechnology, with the hope of improving growth rate and/O r ProdLICtiVity. 0 1996 Wiley-Liss, Inc.

As mammalian cell cultu re gains importance in thebiotechnological production of therapeutics, the meta-bolic design of transformed mammalian cell lines be-comes increasingly the focus of process development(Fiechter and Gmiinder, 1989; Stephanopoulos andSinskey, 1993). By means of metabolic improvement,cell lines with increased productivity, substrate spec-trum, or energy yield could be developed, thus reducingproduction costs. Success in attempting a metabolic im-provement of transformed cell lines greatly dependson the availability of detailed information about themetabolic fluxes characteristic of different celllinesandcell types (Sahm, 1993; Kelly et al., 1993). This typeof comparative analys is exposes impo rtant aspects ofmetabolic behaviour an d offers insights into the possi-bilities for metabolic improvement. Recently, Pendse0 1996 WILEY-LISS, INC.

and Bailey (1994)eported a dramatic increase in tis-sue plasminogen activator (tPA) production (up to100%)by CHO cells expressing a recombinant Vitreo-scilla haemoglobin (VHb) in addition to recombinanttPA. The coexpressed haemoglobin provided sufficientoxygen for ATP synthesis even under oxygen-limitedconditions.Considerably more complex than the oxygen-ATP-relationship is the complete primary energy metabo-

Received March 14, 1995; accepted June 19, 1995.*To whom reprint requestdcorrespondence should be addressedat Abteilung Zellkulturtechnik, Gesellschaft fur Biotechnolog-ische Forschung m.b.H., Mascheroder Weg 1, D-38124 Braun-schweig, Germany.


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GLUCOSE AND GLUTAMINE METABOLISM 1N CELL LINES 153lism in mammalian and insect cell lines. It is accom-plished via the major pathways of glutaminolysis, gly-colysis, and TCC, in which certain enzymes function asflux regulators (Glacken, 1988; Lanks and Li, 1988).There are some obvious differences in the energy me-tabolism of primary mammalian cells, insect and trans-formed mammalian cell lines, with regard t o lactateproduction, dissolved oxygen limitation, glucose, andglutamine utilization. Unlike insect and primary mam-malian cells, transformed mammalian cells convert a

ADPAlaATAspATATCCATPBHKCCLCHOcsEBVEDTAEGTA

FCSFrc6PGAPGDHGDPGlcGlclPGlCGPGlnaseGPDHGsGT PHEPESHKIDPIMDMKGALacLDHMDHMOPSOAAPBSPcPCAPDHPDHCPEPPEPCKPFKPGLPKPMSFPPC4.rSFRib5PSEAPSMIFsvTCATCCTrioVHbZK T

2-ME

Abbreviationsadenosine diphosphatealanine aminotransferaseaspartate aminotransferaseAmerican type culture collectionadenosine triphosphateSyrianbaby hamster kidneycontinuous mammalian cell lineChinese hamster ovarycitrate synthaseethylenediaminetetraacetate(ethylenedinitrilo)tetraace-tic acid]ethylene glycol-bis(P-aminoethyl ether N,N,NN-tetra-acetic acid [Ethylene-bis4oxyethylenenitrilo)tetraaceticacid]fetal calf serumfructose-6-phosphateglycerolaldehyde-3-phosphateglutamate dehydrogenaseguanosine diphosphateglucose-1-phosphateglucose-bphosphateglutaminaseglucose-6-phosphate dehydrogenaseglutamine synthetaseguanosine triphosphateN-(2-hydroxyethyl)piperazine-N-(2-ethanesulfoniccid)hexokinaseinosine diphosphateIscoves modified Dulbeccos mediuma-keto-glutaricacid (2-oxopentanedioicacid)lactatelactate dehydrogenase2-mercaptoethanolmalate dehydrogenase3-(N-morpholino)pmpanesulfonic acidoxaloacetic acid (oxobutanedioicacid)phosphate buffered salinepyruvate carboxylaseperchloric acidpyruvate dehydrogenasepyruvate dehydrogenase complexphosphoenolpyruvatephosphoenolpyruvatecarboxykinasephosphofructokinase6-phosphoglucono-6lactonepyruvate kinasephenylmethylsulfonyl fluoridepentose phosphate cyclepyruvateSpoabptera frugiperduribose 5-phosphatesecreted alkaline phosphataseScharfenbergs modified Iscoves-Fl2simian virustrichloroaceticacidtricarboxylic acid cycletrio(hydroxymethy1)aminomethaneVitreoscilla haemoglobinZellkulturtechnik

Epstein-Barrvirus

glucose

large amount of glucose to lactate, with very little glu-cose-derived carbon entering the TCC (Donnelly andScheffler, 1976; Glacken et al., 1986; Bedard et al.,1993; Schrimpf et al., 1994; Crabtree and Newsholme,1972; Lund, 1980). However, a detailed comparison ofthe metabolic patterns of primary mammalian cells,insect and mammalian cell lines has not been pre-viously reported.Apart from glycolysis and TCC there exist other glu-cose-utilizing pathways. For example, a small fractionof the total glucose utilized provides precursors for ri-bose formation via the pentose phosphate shunt, whichplays an essential role in nucleic acid synthesis (Reitzeret al., 1979). In addition to glucose, glutamine servesas a major energy and carbon source in many cell types,since it can be oxidized either completely (to C0.J orpartially via glutaminolysis (Reitzer et al., 1979, 1980;Glacken, 1988; Zielke et al., 1984). The metabolism ofglutamine and glucose is interactive (Newland et al.,1990), with glutamine stimulating glucose utilizationvia glycolysis and the pentose phosphate shunt (OR-ourke and Rider, 1989; Reitzer e t al., 1980), but inhib-iting glucose oxidation in the TCC (Lanks, 1986). Glu-cose, in turn, can influence glutamine utilization inCCLs (Newland et al., 1990; Zielke et al., 1978). Hence,deeper insights into the glucose and glutamine metabo-lism of CCLs relevant t o biotechnology may eventuallylead t o a designed improvement of energy metabolism,and thus t o higher production rates.The objective of the work described here was to inves-tigate pathway fluxesof glucose and glutamine metabo-lism in biotechnologically relevant CCLs with the aimof identifying potentially rate-limiting steps. Cell-spe-c s c utilization and production rates aswellas he max-imum activities of key regulatory enzymes of glucoseand glutamine metabolism were analyzed in Meren tCCLs to determine common characteristics in the me-tabolism of these CCLs. Maximum catalytic activitiesof certain enzymes can serve as quantitative indicatorsof maximal flux through metabolic pathways (Ardawiand Newsholme, 1982, 1983; Newsholme et al., 1986,1987; Fitzpatrick et al., 1993). Metabolic patterns ofCCLs were compared with those of insect cells, whichdisplay a very different metabolic behaviour, especiallywith respect to lactate formation. For further compari-son, the maximum activities of the enzymes of glucoseand glutamine metabolism were also determined forprimary liver cells. Moreover, using radioactively la-beled substrates we measured glucose and glutaminefluxes in representative mammalian (BHK-21 c13) andinsect (Sf-21) cell lines. Analysis of key enzyme activi-ties in different cell lines, combined with a comparativecorrelation of flux and utilizatiodproduction data, indi-cates the rates of maximum flw and suggests rate-limiting steps. This knowledge could serve as a basisto a rational metabolic design.

MATERIALSAND METHODSChemicalsUnless indicated otherwise, all chemicals used wereof highest grade and purchased from Merck (Darm-stadt, Germany), Boehringer Mannheim (Mannheim,Germany), or Sigma Chemical Company (Sigma-Ald-rich, Deisenhofen, Germany). Radioactive materials


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154 NEERMANN AND WAGNERwere obtained from Amersham (Braunschweig, Ger-many).

Cell linesMammalian cell lines. The following mammaliancell lines were used:1. BHK-21 (C-13) (ATCC CCL 10). A baby hamsterkidney cell line purchased from ATCC (Rockville, MD).2. BHK-21 pSVIL2. A recombinant subclone of BHK-21 (C-13), which was manipulated by genetic methodsto produce human interleukin-2 constitutively undercontrol of the SV40early promoter (Conradt et al., 1986).It was provided by the Genetic Engineering Departmentof Gesellschaft fiir Biotechnologische Forschung (GBF).3. BHK-21 BN49/90HK-1. A recombinant subcloneof BHK-21 (C-131, which produces P-galactosidase andalkaline phosphatase. It was created by transfection ofBHK-21 (C-13) cells with the plasmid pHPAPr-l/P-Gal,which contains the Escherichia coli Lac2 gene codingfor P-galactosidase (E.C. 3.2.1.23) under control of thehuman p-actine promoter (Wirth and Schumacher,

unpublished data). The recombinant P-galactosidaseaccumulates in the cytoplasm. The resulting cell linewas additionally transfected by a second plasmid(pRSVSEAP), bearing a mutant of the secreted alka-line phosphatase (SEAP) under control of the SV40promoter (Berger et al., 1988; Racher e t al., 1994).Anartificial termination-translation signal was added atposition 489 resulting in secretion of the expressedprotein into the culture supernatant. The cell line wasprovided by the Genetic Engineering Departmentof GBF.4. CHO-K1 (ATCC CCL 61). A Chinese hamsterovary cell line obtained from ATCC.5. CHO-K1 pMDIIIGPTR. A transfected subclone ofCHO-K1 cells (Motz et al., 1987). It produces two re-combinant Epstein-Barr virus (EBV) surface glycopro-teins of 250 and 350 kd, respectively. This cell linewas provided by Prof. H. Wolf (Institute for MedicalMicrobiology and Hygiene, University of Regensburg,Regensburg, Germany).6. Rat-mouse hybridoma 187.1 (ATCC HB58). Pro-duces a monoclonal antibody (IgGl),specific for mousekappa light chains. It was purchased from ATCC.7. Murine hybridoma MAX16H5. Produces an antiCD-4 monoclonal antibody. The cell line was providedby Prof. F. Emmrich (Insti tute for Clinical Immunologyand Rheumatology, University of Erlangen-Niirnberg,Germany).8. WI-38 (ATCC CCL-75).A cultured diploid cell linefrom human lung tissue purchased from ATCC.

Insect cell lines. The following insect cell lines wereused:1. IPLB-Sf-21AE. Isolated from Spodoptera frugi-perdu (Vaughn et al., 1977). It was provided by Dr.J. Claus (Universidad National del Litoral, Santa Fe,Argentina).2. Sf-9 (ATCC CRL 1711). A subclone of IPLB-Sf-21AE purchased from ATCC.Primary cells. Livers of both pig and mouse weretaken from freshly killed animals and prepared just prior

to the experiments, as described in Cell extraction forenzyme analysis.Culture media

All mammalian cell lines were cultured in mediumbased on a 1:l mixture of IMDM and Ham's F12 (ZKT-I medium; custom-made by Sigma Chemie, catalog no.I-99003), containing 20 mmol L-' glucose, 3 mmol L-'glutamine, and 48 mmol L-' NaHCO,. The mediumwas either supplemented with 2.5% heat-inactivatedfetal calf serum (Sigma) or with 10 mg L-' humantransferrin (Biotest, Dreieich, Germany) and 10 mg L- 'human insulin (Elanco, Indianapolis, IN). CHO-K1pMDIIIGPTR cells were cultured in protein-freeSMIF6-medium (Wolf et al., 1993; Scharfenberg andWagner, 1995) containing a chelator additive as de-scribed previously (Bertheussen, 1993). The differentlysupplemented media will be referred to as FCS-con-taining medium, serum-free medium, o r protein-freemedium, respectively. Insect cells were grown in EX-CELL 401 medium (JRH Bioscience, Lenexa, KS){which contained 15 mmol L-' glucose and 8 mmol L-glutamine. The medium was supplemented with addi-tional 20 mmol L-l glucose.

Cell cultureAll cells were routinely cultured in either 75 cm' T-flasks (Nunc, Roskilde, Denmark) or in spinner flasks(300 mL, 50 rpm) (Techne, Cambridge, UK). The mam-malian cells were incubated at 37C under 90% air/10% COz and 99% humidity (Biocenter 2000, SALVIS,Reussbuhl, Switzerland). Insect cells were incubatedat 27C. For experiments cells were taken from theearly exponential growth phase of the batch cultures.For further comparison, BHK-21 BN49/90HK-1 werealso taken from a continuous chemostat culture (1.6 L,dilution rate: D = 0.022 h-').Viable cell numbers were determined by the trypanblue exclusion method (Boehringer Mannheim) usinga hemocytometer. The numbers were confirmed bycounting nuclei stained with crystal-violet(0.1%crystalviolet in 0.1 mol L ' citrate) using a hemocytometer.

Glucose, lactate, and amino acid analysesGlucose and lactate were routinely determined usinga YSI Model 2000 GlucoseLactate analyzer (YellowSprings Instruments, Yellow Springs, OH). Glutamineand other amino acids were assayed by reverse-phaseHPLC with fluorometric detection after derivatisationwith ortho-phthaldialdehydeas described in detail else-where (Larsen and West, 1981; Ryll et al., 1990).

Enzymes and extraction buffersAll enzymes analyzed are listed in Table 1. For en-zyme extraction three Merent buffers were used basedon buffers previously described (Ardawi and News-holme, 1982), but with slight modifications as follows:The extraction buffer for HK, PDH, PK, LDH, GDH,AspAT, AlaAT, MDH, and GS consisted of 50 mmol L-'triethanolamine (2,2'2"-nitrilotriethanol) pH adjustedwith 1mol L-' NaOH), 1mmol L-' EDTA, 2 mmol L-'MgC1, ,and 30 mmol L-' 2-mercaptoethanol(2-hydroxy-ethylmercaptan). The extraction buffer for PFK and CScontained 50 mmol L-' Tris/HCl, 1 mmol L-' EDTA,and 5 mmol L-l MgS04. The extraction buffer for PC


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GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES 155TABLE 1. Enzvmes analyzed in this workEnzymeHexokinaseGlucose-6-phosphate dehydrogenasePhosphofiuctokinasePyruvate kinaseLactate dehydrogenaseGlutaminaseGlutamate dehydrogenaseAlanine aminotransferaseAspartate aminotransferaseGlutamine synthetaseMalate dehydrogenaseCitrate synthasePyruvate carboxylasePhosphoenolpyruvate carboxykinase

Pyruvate dehydrogenase complexPyruvate dehydrogenaseDihydrolipoyl transacetylaseLkhydrolipoyl dehydrogenase

Composed of

AbbreviationHKGPDHPFKPKLDHGlnaseGDHAlaATAspATGSMDHcsPCPEPCK

PDHCPDH

Rational nameATP: D-hexose 6-phosphotransferaseD-glucose-6-phosphate: NADP' oxidoreductaseAT P D-fructose-6-phosphate 1-phosphotransferaseATP pyruvate 2-0-phosphotransferase(S)-lactate: NAD' oxidoreductaseGlutamine amidohydrolaseLgl utamate : NAD' oxidoreductaseL-alanine: 2-oxoglutarate amino-transaminaseL-aspartate: 2-oxoglutarate amino-transaminaseL-glutamate: ammonia ligase [ADP forming]@)-malate: NAD' oxidoreductase; Wm al at e; NADP'oxidoreductase [oxaloacetate-decarboxylatinglCitrate: oxaloacetate-lyase [(pro-3S)-CHzCOO-+acetyl-CoAlPyruvate: carbon-dioxide ligase [ADP-forming]GTP: oxaloacetate carhoxy-lyase[transphosphorylatinglAT P oxaloacetate carboxy-lyase[ ransphosphorylatingl

Pyruvate: lipoamide 2-ox ido red uae [demrbxylatingAcetyl-COA: dihydrolipamide S-acetyltransferaseDihydrolipoamide: NAD' oxidoreductase

and acceptor-acetylatingl

E.C. number'2.7.1.11.1.1.492.7.1.112.7.1.401.1.1.273.5.1.21.4.1.22.6.1.22.6.1.16.3.1.21.1.1.371.1.1.404.1.3.76.4.1.14.1.1.324.1.1.49

1.2.4.12.3.1.121.8.1.4

'E.C. = enzyme classification.

and PEPCK consisted of 50 mmol L-' triethanolamine(pH adjusted with 1mol L-'NaOH), 300 mmol L-l su-crose, and 1mmol L-' EDTA. For extraction of PDHCa 20 mmol L-l HEPES buffer (pH adjusted with 1molL-' NaOH), containing 3 mmol L-' MgCl,, 3 mmol L-'EGTA, 0.5mmol L-' EDTA, and 3 mmolL-' dithiothrei-tol, was used. The extraction buffer for Glnase contained50 mmol L-' Tris/HCl, 150 mmol L-' KH,P0&.J3PO4(pH 7.41, and 1mmol L-' EDTA. All extraction bufferscontained 0.1%(v/v) TRITON-X-100 and were adjustedto pH 7.4 for mammalian and pH 6.5 for insect cells.

Cell extraction fo r enzyme analysisCell lines.Cells were removed from plastic or spinnerflasks, counted and centrifuged at 200g for 5 minutes(RotantaRP, Hettich, Tuttlingen, Germany) at 4C. Thecell pellet was washed twice with 20 mL phosphate-buffered saline (PBS, pH 7.4 for mammalian cells, pH6.2 for insect cells) and resuspended in 3 mL of theappropriate enzyme-extraction buffer at 4C. Completecell lysis was accomplished by a freeze-thaw cycle fol-lowed by 20 strokes in a homogenizer (Dounce, Braun-Diessel Biotech, Melsungen, Germany). The lysateswere centrifuged at 3,OOOg for 5 minutes and the re-

sulting supernatanb were assayed for enzyme activity.Primary cells.Fresh livers from both pig and mousewere first perfused with 10 and 1L, res ectively, of ammol L- NaCl, 5 mmol L-' KCl, 8 mmol L-' MgCl,,0,l mmol L-' PMSF, 5 mmol L-' 2-ME) and thereafterwith equal volumes of PBS (10 and 1L). A know n massof the tissue (about 2 g) was lysed as described abovefor the various cell lines.

Enzyme activity measurementsEnzyme activities were measured for all mammaliancells at 37"C, pH 7.4, and for insect cells at 27"C, pH

suitable perfusion buffer (20 mmol L-P MOPS, 130

6.5, to obtain standardized conditions which resemblethe natural environment as closely as possible. Thespectrophotometric assays were performed using anLKB Ultrospec K kinetic spectrophotometer (Phar-macia, Freiburg, Germany). Enzyme activities werecalculated from the ra te of change of absorbance withina linear range at 340 nm, except for CS, PC, andPEPCK, which were measured at 412 nm, and PDHC.PDHC activity was determined only qualitatively by acolorimetric change within the visible spectrum, sincequantitative assays (Schwartz and Reed, 1970) moni-toring the NADH increase a t 340 nm are not applicablein crude extracts with high LDH-activity competingfo rthe substrate pyruvate.Each assay was validated by showing a linear corre-lation between enzyme addition and spectrophotomet-ric response. Each measurement was repeated at leastfive times and the mean value was calculated. Theassays were based on methods described previously asshown in Table 2.

Metabo l i t e poolsIntracellular glycolytic metabolite concentrations(glucose, glucose-6-phosphate, fructose-6-phosphate,phosphoenolpyruvate, pyruvate, lactate) were deter-mined for BHK-21 BN49/90HK-1 cells and insect Sf-21cells either by spectrophotometric assays (Hugo et al.,1992) or using the YSI Model 2000 Glucose/Lactateanalyzer (Yellow Springs Instruments). Extractions ofchilled cells (5 x lo8) were performed as described(Schmid and Blanch, 1992). The metabolite pools weredetermined from triplicate extracts.

Radioactive measurementsRadioactive samples were counted with 2 mLQuicksafe A scintillator (Zinsser, Frankfurt, Germany)


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156 NEERMANN AND WAGNERTABLE 2. References for methods of enzyme analysis'Metabolism Enzyme ReferenceGlucose metabolism Hexokinase Fornaini et al. (1982)Glucose-6-phosphatedehydrogenase Julian and Reithel(1975)Phosphofi-uctokinase Massey and Deal (1982)Pyruvate kinase Kahn and Mane (1982)Lactate dehydrogenase Zammit and Newsholme (1976)Pyruvate carboxylase Ballard and Hanson (1967)Pyruvate dehydrogenase complex Reed and Willms (1966)Phosphoenolpyruvatecarboxykinase Zammit and Newsholme (1976)

Glutaminase (phosphate-activated) Kvamme et al. (1985)Glutamine synthetase Fbwe et al. (1970)Alanine transaminase Sugden an d Newsholme (1975)Aspartate transaminase Sugden and Newsholme (1975)Malate dehydrogenase (NAD(P)+-linked) Newsholme and Williams (1978)

Glutamine metabolism Glutamate dehydrogenase Fisher (1985)

Tricarbonic acid cycle Citrate synthase Alp et al. (1976)'We could not discriminate between the two isoenzymes of PEPCK that bind GDP and ADP, respectively, because thethree nucleotidesGDP, ADP, and IDP were present in the assay.

using a Beckman LS 5000 CE scintillation counter(Beckman Instruments, Palo Alto, CA).Metabolic flux

Metabolic flux rates were examined for BHK-21BN49/90HK-1 cells and IPLB-Sf-21AE cells. Incuba-tion temperature, pH and media conditions were main-tained as stated above, except for pyruvate enteringthe TCC, where the medium was replaced by pyruvate(5 g L-l) solubilized in PBS buffer (pH 7.4 or 6.5), asdescribed previously (Bartos et al., 1993). Replicatetests were performed in all experiments.The glucose membrane transport was determined aspreviously described in detail (Fitzpatrick e t al., 1993).Briefly, to determine the glucose transport rate inde-pendently from any further influences of cellular me-tabolism, a radiolabelled glucose analogue 2-deoxy-D-[l-3Hl-glucose 0.1 pCi A 3.7 x l o 3Bq) was used, whichis not a substrate for HK and cannot be metabolized.The glucose transport rate was examined for BHK-21BN49/90HK-1 cells (1 x lo7 mL-') growing in FCS-containing and serum-free medium.Glycolytic flux was determined as described by Fitz-patrick et al. (19931, based on the methods of Ham-merstedt (1973) and Bontemps et al. (1978). Briefly,the glycolytic flux was determined by the release oftritiated water from the metabolism of D-[3-3Hl-glucoseby measuring the flux of glucose metabolites throughthe aldolase and triose phosphate isomerase reactions.The rates of released tritiated water were linear for theinvestigated mammalian and insect cell lines during a3.5 hour observation. Cells (5 x lo6)were incubated in1mL medium containing D-[3-3Hl-glucose(0.5 pCi P18.5 x lo3 Bq).Glucose carbon entering the TCC was measured bythe rate of 14C02 elease from 6-14C-glucose Petch andButler, 1994).Pentose phosphate pathwa flux was calculated fromthe difference in the rate of CO, released from l-14C-glucose and 6-14C-glucose Katz and Wood, 1963).Pyruvate carbon entering the TC C via pyruvate dehy-drogenase was measured by the rate of 14C02 eleasefrom l-14C-pmvate Bartos et al.. 1993).

L

14C02 eleased from L-[U-'4Cl-glutamine (Brand et al.,1984).The 14C02 ormation was determined essentially fol-lowing the methods of Bartos et al. (1993): Cells weresuspended in growth medium, or yruvate containingPBS, at a concentration of 5 x 10PmL-1. A respectiveradioactive substrate was added to the suspensions infollowing amounts: l-14C-glucose 0.5pCi f 18.5 x lo3Bq), 6-14C-glucose 0.5 pCi f 18.5 X lo3By) L-[U-14C]-glutamine (0.5pCi & 18.5 x lo3Bq), or 1- C-pyruvate(1 FCi A 3.7 x lo 4 Bq). Aliquots (1.5 mL) were dis-pensed into Petri dishes (21 cm2, Nunc) modified asfollows: The Petri dish cover had a small injection portsealed with adhesive tape (Beiersdorf, Hamburg, Ger-many). A small polyethylene tube (Eppendorf, Ham-burg, Germany) containing300pL of a 1:l(v/v) mixtureof phenylethylamine and methanol to trap 14C02wasattached t o the inside of the cover by an adhesive tape.The dishes were airsealed with rubber bands and incu-bated a t the appropriate temperature up to 4 hours. Atregular intervals of about 30 min 200 pL of 10% (w/v>TCA were injected into the cell suspension. Followingthe incubation period the phenylethylamine was al-lowed to assimilate 14C02completely (1 hour) beforeradioactivity was measured.

RESULTSCell-specific substrate utilization, formation,and mitotic ratesAt first, the initial cell-specific glucose and glutamineutilization and lactate formation rates were investigated,

since they are of particular importance for later compari-son with measured flux rates and enzyme activities.Cells started their exponential growth phase withvery high specific glucose (Fig. 1)and glutamine (Fig.2) utilization rates, and in the case of mammalian cells,very high lactate formation rates. With increasing cul-tivation time these specific utilization and formationrates decreased as glucose and glutamine concentra-tions declined. This phenomenon has been observed invarious batch cultures (Ljunggren and Haggstrom,1994; Fitzpatrick et al., 1993; Ogata e t al., 1993).In our experiments the initial specific glucose andGlutami;; oxidation was measured by the rate of glutamine utilization rates of the examined cell lines


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GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES 157

0.251 ,b] ;.;. l.,...l , I , I , I , ,.*0.30 0 . .0 20 40 60 80 100 120 140a cultivation time I (h)

Fig. 1. Cell specific glucose consumption and lactate formation ratefor BHK (a) s well as for insect, CHO, and hybridoma cell lines (b).With increasing cultivation time the cell specific glucose utilizationand lactate formation rates decreased for all cell lines. The initialglucose utilization rates for most mammalian cell lines were found to

have been determined over relatively short time peri-ods (5 hours to 10 hours). This was necessary t o com-pare, qualitatively as well as quantitatively, the spe-cific utilization and formation rates with flux data ac-quired from radioactive labeling experiments, whichwere carried out for practical reasons over short timeperiods (up to 5 hours). For the same reasons the en-zyme activities were determined in the early exponen-tial phase, since enzyme activities can change withchanging culture conditions (Lind et al., 1991; Fitzpa-trick e t al., 1993; Kratje e t al., 1994a,b).Glucose utilization and actate formation rates

Cell-specific substrate utilization and byproduct for-mation rates vary during batch cultures. When cellswere resuspended in fresh growth medium they nor-mally had to adapt to the new culture conditions byreplenishing the intracellular pools depleted during thepreceding starvation phase. A s a result, the cell-specificglucose utilization and lactate formation rates werehighest at the initial phase of culture and then de-creased constantly (Fig.1).For most mammalian batchcultures examined, the initial cell-spec& glucose up-take rates were found to be between 0.15 and 0.21 nmol

-A-CHO-KI,serum-free-+-Hybridoma MAX16H5. serum-free-X-Hybridoma 187.1,3%FCS

1._=p 0.30- 0 20 40 60 80 100 120 140b cultivation time I (h")

range between 0.15and 0.22 nmol s-l They were lower for bothinsect and hybridoma lines, with 0.08 and 0.11 nmol s-l lo-" cells to0.13 nmol 8-' cells, respectively. While all mammalian cells ini-tially produced between 0.17 and 0.32nmol s- ' lactate, the insectcells secreted no or extremely little lactate.

5-l depending on culture supplements and cellconcentration. For both insect cell lines and hybridomacultures the initial cell-specificglucose utilization rateswere lower, a t about 0.08 and 0.11-0.13 nmol s-lrespectively (Table 3).For continuous mammalian cell lines, the initial cell-specific lactate formation rates varied between 0.17nmol s-' lop6 or the murine hybridoma MAX16H5 and0.32 nmol s-l for the BHK-21 c13 grown underserum-free culture conditions. The calculated lactate/glucose molar ratios differed among the cell lines andchanged slightly with increasing cultivation time, butwere always clearly >1 Wagneret al., 1988).This ndi-cates th at for the most par t glucose was transformedinto lactate in all cultured mammalian cells. In thecase of BHK cells the lactatdglucose molar quotientwas greater for cells cultured in serum-free mediumthan fo r those grown in FCS-supplemented medium. Arepresentative lactate/glucose quotient for each cellline at the initial phase of culture is given in Table 3.The cell specific lactate production in Sf-9 and Sf-21insect cells differed significantly from that found forcultured continuous mammalian cells. The Sf-9 and Sf-21 insect cells produced very lit tle or no lactate with a


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158

$- 0.35-0-rr'v )- 0.30-0:..25-a,-2.- 0.20-K.--, 0.15-K.-E$ 0.10--cn0% 0.05-a,av)-R 0.00

NEERMANN AND WAGNER

+ nsect 5 1 - 2 1 , ~ mol L-' glutamine.- - nsect SI-21, 12 rnrnolC' glutarnine. ., ., .... .,, .:

0 ................... d:/// ....................... ... ........I I I I I -

a- 0.091.ca, 0.065 0.055 0.04.-c.-

5Sa,.s 0.03

I 1-A- CHO-K1, serum-free-X- Hybridoma 167.1, with 3% FCS4- HK-21 c13. With 2.5% FCS- -O . .BHK-21 c13, serum-free.-V-HK-21 BN49/90HK-l. with 2.5% FCS- A- BHK-21 BN 49MOHK-1, serum-free

P) 0.01 -P.. .......LA. . . . . 'V5 0.00-

0l , . ~ . ~ . ~ ~ l . ~ . l80 100 120 140 1600 40 60a o

v 0 20 40 60 80 100 120 140cultivation time / (h-')

Fig. 2. Cell specific glutamine utilization rated for different mamma-lian (a) nd insect (b) cell lines. The specific glutamine utilizationrates were determined to range between 0.05 and 0.08 nmol s-' lo-'for mammalian cells at culture start,while the utilization rates forthe insectSf-21ine were significantly higher. However, the glutamine

concentrations n the insectmedium were higher than in the mamma-lian cell culture medium. The increase in the cell specific glutamineutilization of the CHO-K1 cells might be due to an initia l lag-phasein the glutamine metabolism.

LadGlc molar ratio of 0.02 a s shown in Figure 1andTable 3.Glutamine utilization

The cell-specific glutamine utilization rates werehighest during the initial culture phase fo r BHK and

hybridoma cell lines. In contrast, other cells-espe-cially the insect lines -showed increasing cell-specificglutamine utilization rates during the first 50 hoursof culture (Fig. 2). The initial cell-specific glutamineutilization rates were found to range between 0.05 and0.08 nmol s- l for cultured mammalian cells and


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GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES 159TABLE 3. Cell specific substrate utilization, byproduct formation, molar quotient of the LadGlc rates ( Q b d d , and mitotic rates ( u ) at theinitial ahases of batch cultures for a number of cell lines'Cell line Glc-rate[nmol s- ' 10-YInsect Sf-9, serum-freeInsect Sf-21, serum-freeCHO-K1, serum-freeBHK-21 c13. 2.5% FCS- - ~-~~ ~ ~ ~ - -~BHK-21 c13, serum-freeBHK-21 BN49/90HK-1, serum-freeBHK-21 BN49/90HK-1, 2.5% FCSHybridoma MAX16HB5, serum-freeHybridoma 187.1 3% FCS

-0.088-0.093-0.169-0.168-0.205-0.159-0.168-0.110-0.131

Lac-rate0.0020.0020.2550.2210.3150.2080.2600.1690.204

[nmol s- l 10-61 QLadGk0.020.021.511.311.541.311.551.541.56

Gln-rate[nmol s-' 10-61-

-0.12(-0.18)-0.068-0.054-0.081-0.050-0.062--0.053

Urh-70.0320.0300.0280.0370.0270.0370.0280.0300.037

'After error propagation a standard deviation of 7-12% for each value was calculated.The minus expresses consumption of the metabolite. Value in parenthesesrefersto 12 mmol L-' lutamine in the medium.

TABLE 4. Cell snecific flux rates throueh Dathwavs of glucose and elutamine metabolism in BHK BN49/90HK-1and insect Sf-21 cells'~~

Glc flux ratedcell lines BHK-21 BN49/90HK-1 BHK-21 BN49/90HK-1 Insect SF-21(2.5% FCS) (serum-free) (serum-free)Glc membrane transportGlycolytic fluxPPC-fluxFlux of Glc-metabolites into the TCC&-flux into TCC

0.31000.09800.0076n.d. < 0.0006n.d. < 0.0006

0.29000.12700.0082n.d. < 0.0006n.d. < 0.0006

-0.06700.00690.05500.0950

Gln flux ratesGln oxidation to CO, 0.0090 0.0115 0.0171'The flux rates were expressed as cell specific values in nmol s- 'Methods. n.d. = not detected. and determined by using radioactively labeled substrates as described in Materials and

between 0.12 and 0.17 nmol 5- l cells for Sf-21insect cells (Table 3). The higher utilization rate of in-sect cells correlated with a more than two times higherglutamine concentration in insect cell medium in com-parison to the mammalian cell medium formulation.Cell-specific utilization rates linearly increased withincreasing glutamine concentration in the medium forinsect cells. The initial glutamine utilization rates ofmammalian cells cultured in serum-free medium were1.2-1.5 times higher than those of cells cultivated inFCS-supplemented medium.

Mitotic ratesThe average specific mitotic rates [v = (In X2 - InXl)/ln2 (t2- tJ; X,= cell number at cultivation time61 were similar for all cell lines during the first 50hours of cultivation. Differences in specific mitotic rateswithin a cell line apparently depended on FCS-supple-mentation of the culture medium. In FCS-supple-mented medium about 1.4-fold higher specific mitoticrate were observed than in serum-free medium. Aver-age specific mitotic rates for the first40 hours of cultureare presented in Table 3.

Metabolic fluxesThe metabolic flux measurements were performedwith BHK-21 BN49/90HK-1 cells cultured in freshserum-free as well as serum-supplemented (2.5%FCS) medium and insect Sf-21 cells cultivated infresh serum-free medium. All flux rate s are presentedin Table 4.

Glucose metabolismGlycolysis. The cell specific flux rates were deter-mined as 0.098 and 0.127 nmol s-l for BHK-21

BN49/90HK-1 cultured in FCS-containing medium andthose grown in serum-free medium, respectively (Table4). The cell specific flux rate for the insect Sf-21 cellswas 0.067 nmol s-' lop6, nd thus clearly lower thanin BHK cells.TCC. In BHK-21 BN49/90HK-1 cells, neither a cellspecific flux of glucose metabolites nor a pyruvate fluxinto the TCC was detectable above the detection limitof 0.0006 nmol s-l lop6.This indicates tha t if there wasany flux at all of glucose metabolites or pyruvate intothe TCC of BHK cells, it accounted for less than 0.64%of the total glycolyticflux, thus contributing no signifi-cant amount of energy. In addition, this result suggeststhat the PDHC activity is very low due to repressionor inactivation, o r does not exist at all in continuousmammalian cell lines.In insect Sf-21 cells the cell specific glucose metabo-lite flux into the citric acid cycle was constant durinthe 4-hour period of observation at 0.055 nmol s 10 .Additionally, the cell specific pyruvate flux into theTCC was measured for 1.5 hours at 0.095 nmol s-'Therefore, in these cells the conversion of glucosemetabolites and pyruvate (via PFHC) into TCC inter-mediates is far more extensive than in BHK cells.Pentose phosphate pathway. The cell specific flux ofglucose metabolites through the pentose phosphateshunt was determined as 0.0076and 0.0082 nmol 5-lfor BHK-21 BN40/90HK-1 cells cultured in 2.5%

B


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NEERMANN AND WAGNER160TABLE 5a. Cell specific enzyme activities in nmol s- lo- of enzymes involved in glucose metabolism for a number of cell lines (see alsoFig. 3)Celldenzymes HK GPDH PKF PK LDH PEPCK PC PDHC If,BHK-21 c13,2.5%FCS 0.24 0.31 0.81 12.80 19.33 n.d. n.d. n.d. 53.3BHK-21 c13, serum-free 0.30 0.45 1.05 22.10 26.20 n.d. n.d. n.d. 73.7BHK-21 pSVIL2, serum-free 0.31 0.43 1.13 15.10 10.20 -

CHO-K1 pMDIIIGPTR, 2.5%FCS 0.22 0.29 0.88 23.30 16.30 -CHO-K1 pMDIIIGPTR, protein-free 0.25 0.27 0.69 15.00 10.60 - -Hybridoma MAX16H5, serum-free 0.12 0.12 0.49 9.20 10.30 -Insect Sf-21, serum-free 0.43 0.45 1.23 4.26 5.57 0.10 0.55 ad. 9.9Insect Sf-9, serum-free 0.45 0.55 1.42 2.44 4.90 0.11 0.65 act. 5.4Primary WI-38, 5% FCS 0.13 0.23 0.58 13.7 20.60 -Primary mouse liver cells 0.96 0.30 3.85 4.52 7.10 0.08 0.17 act. 4.7

BHK-21 BN49/90HK-1, 2.5%FCS 0.22 0.25 0.76 14.60 16.80 n.d. n.d. n.d. 66.4BHK-21 BN49/90HK-1, serum-free 0.24 0.26 0.82 20.50 22.40 n.d. n.d. n.d. 85.4- - 48.7chemostate culture, steady state 0.19 0.17 0.45 13.50 13.80 n.d. n.d. n.d. 71.1

- - 105.9CHO-K1 pMDIIIGPTR, serum-free 0.30 0.42 1.24 24.70 17.50 n.d. n.d. n.d. 82.3- 60.0Hybridoma 187.1, 3% FCS 0.13 0.14 0.61 8.80 10.40 n.d. n.d. - 67.7- - 76.7

BHK BN49/90HK-1, 2.5%FCS

- - 105.4Primary pig liver cells 0.52 0.29 1.87 2.12 3.37 0.09 0.22 act. 4.1The enzyme activities were determined spectrophotometrically from crude cell extracts as described in Materials and Methods. Values for mouse and pig liverexpressed as tissue mass specific activities innmol s- lo- mg. Each value represents the mean of at least five determinations. The standard deviation after e m rpropagationis 10-14% in each case. n.d. = not detected; act. = active.

FCS-containing and serum-free medium, respectively(Table 4). These rates account for 8.1 and 6.5% of therespective glycolytic rates.For insect Sf-21 cells the cell specific pentose phos-phate pathway flux ra te was estimated at 0.0069 nmols- accounting for 10.3%of the glycolytic flux. Incontrast to other fluxes of the oxidative metabolism,the PPC flux was in the same order of magnitude inboth insect and BHK cells.Glucose transport. The cell specific transport rateswere linear during a 10-minute period of measurementa t 0.34 and 0.32 nmol s-l for BHK-21 BN49190HK-1cells grown in 2.5% FCS-containing medium and se-rum-free medium, respectively.Glutamine metabolism

Glutumine oxidation. The cell specific rate of lutamine oxidation was found to be 0.0090 nmol s- 10for BHK-21 BN 49/90HK-1 cells cultured in 2.5% FCS-supplemented medium and 0.0115 nmol s-l l op6 forthe same cell line grown under serum-free conditions(Table 4). This accounts for 18.0 and 18.5% respec-tively, of the total glutamine utilized.For insect Sf-21 cells the cell-specific rate of gluta-mine oxidation was 0.0171 nmol s- low6,epresenting14.3% of the overall glutamine utilization (Table 4).Enzyme activities

The maximal activities of a number of key enzymesof glycolysis, glutaminolysis and the TCC are given inTables 5a and b for the cell lines examined.Glucose metabolism. It is striking that HK, theglycolytic entrance enzyme, exhibited in all cell linesthe lowest cell specific activity (0.12-0.31 nmol s-10-7 among all glycolytic enzymes (Table 5a).Only GPDH as the branching enzyme into the pen-tose phosphate shunt displayed a maximum cell spe-cific activity of 0.12-0.43 nmol s-l lop6,which is withinthe low range of HK.Moving along the glycolytic pathway the maximumenzyme activities increased, with PK and LDH showing

F -6

the highest activities. However, it should be noted tha tthe divergence between HK, PFK, and PK was deci-sively smaller in insect and primary liver tissue cellsthan in CCLs. This divergences can be expressed bythe pyruvate kinasehexokinase (PWHK) ratio, servingas a flux regulation index (Ifl):While Ifl ell between 49and 105 in CCLs, it ranged only from 4 t o 10 in allinsect cells and primary liver tissue cells investigated.Thus, the importance of HK for flux regulation seemsto be higher in CCLs than in insect or primary livertissue cells.It is interesting t o note that also the WI-38 diploidcell had undergone relatively few passages (17 pas-sages, 32-39 generations), it exhibited an enzyme ac-tivity pattern and a PWHK ratio (Ifl= 105)characteris-tic of cell lines which had been in culture much longer.Most important for the physiological behaviour ofcells in culture seems to be the activity and/or presenceof mediator enzymes between glycolysis and TCC,namely PDHC, PC, and PEPCK. In various culturedmammalian cells no activity of these three enzymescould be detected by the spectrophotometric assay,while in insect and primary liver tissue cells the sameenzymes were clearly active (Table 5a).Gluta mine metabolism. As was the case for glycol-ysis, the activity of Glnase, the entrance enzyme ofglutamine metabolism, was lower in all cell lines exam-ined by a fador of 3 to 5 compared to the activity ofGDH, which operates immediately downstream ofGlnase (Table 5b). This result suggests that glutami-nase rather than glutamate dehydrogenase regulatesthe glutaminolytic flux. The activity of AlaAT, whichconverts glutamate into alanine, was comparablet o theGlnase-activity and lower than the GDH-activity inmost cells investigated. Only in primary liver tissuecells AlaAT-activity was found to be distinctivelyhigher than Glnase-activity (Table 5b). AspAT repre-sented in all cell lines investigated the enzyme withthe highest activity in the glutaminolytic pathway. Inprimary liver tissue cells the GDH activity appearedto be more dominant when compared t o CCLs. At the


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GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES 161TABLE 5b. Cell specific enzym e activity in nmol s - '(see also Fig. 3)' of enzymes involved in glutamine an d TC C metabolism for a numb er of cell linesCelldenzymes cs M DH (NAD') GDH Glnase GS &PAT AlaATBHK-21 BN49/90HK-l, 2.5% FCS 0.60 24.50 0.80 0.29 0.17 1.58 0.20BHK-21 BN49/90HK-1, serum-free 1.19 25.30 1.16 0.32 0.25 2.43 0.22

BHK-21~13,.5% FCS 0.57 27.70 1.21 0.44 0.31 1.68 0.16BHK-21 c13, serum-free 0.84 33.70 2.05 0.83 - 2.78 0.15BHK-21 pSVIL2,2.5% FCS 0.67 20.60 0.83 - - 1.42 0.16BHK-21 BN49/90, 2.5% FCS chemostateculture 0.70 16.90 0.34 0.06 0.05 0.95 0.30CHO-K1,2.5% FCS 0.69 31.50 0.69 0.13 - 1.45 0.15CHO-K1, serum-free 0.86 28.60 0.82 0.15 - 1.68 0.08Hybridoma 187.1,3% FCS 0.42 14.20 0.54 0.15 0.09 1.11 0.12Insect Sf-21, serum-free 2.20 4.12 3.10 0.68 0.39 0.64 0.20Insect Sf-9, serum-free 2.60 3.72 1.54 0.34 0.36 1.29 0.25Primary WI-38,5% FCS 0.30 12.50 0.35 0.12 - 0.57 0.18Primary mouse liver cells 1.12 8.50 5.67 0.52 2.31 13.67 5.58Primary pig liver cells 1.07 11.31 11.72 0.73 2.18 5.81 3.54'The enzyme activities were determined spectrophotometrically from crude cell extracts as described in Materia ls and Methods. Values for mouse and pig livercells are expressed as tissue mass specific activities in nmol s-' lo - ' mg. Each value represents the mean of at least five determinations. The standa rd deviationafter error propagation is 10-14% in each case.

TABLE 6. Intracellular concentra tions of glucose me tabolites in th e glycolytic pathwa y'Celldmetabolites Glc Glc6P Frc6P PEP PVr LacBHK-21 BN49mOHK-1~~~~~ . - 18.7 % 7.2.5% FCS 3.2 ? 2.8 0.04 2 0.02 0.34 ? 0.14 0.06 ? 0.05 0.51 ? 0.30BHK-21 BN49/90HK-l,serum-free 2.9 2 2.6 0.04 -+ 0.02 0.32 ? 0.15 0.02 ? 0.02 0.40 ? 0.26 19.5 ? 8.0

free 1.1? 1.0 0.12 -+ 0.80 0.12 2 0.10 0.08 ? 0.08 0.53 2 0.35 2.8 ? 1.7'All values are cell specific pools expressed in nmol 10 '. Despite high standard deviations it can be noted tha t glucose and lactate form the highest intracellularpools of the glycolytic pathway in both insect and BHK cells, with the lacta te concentration higher than the glucose concentration. The intracellular lactate poolis significantly lower in insect than in BHK cells.

Insect Sf-21, serum-

same time the GS activity in primary liver tissue was4 times higher than the respective Glnase-activity, in-dicating a flux from the TCC towards glutamine ra therthan vice versa. In contrast, CCLs and insect cells didnot show a high GS-activity.TCC.The activities of two representative TCC en-zymes, CS and NAD+-linkedMDH, were determined.The NAD+-linked malate dehydrogenase activity wasfound to be very high in all cells investigated. In addi-tion, CS activity, ensuring sufficient TCC flux, waspresent in all cells. A NADP+-linked MDH-activitycould not be detected in any CCL investigated.

Intracellular metabolite poolsThese results should be interpreted with caution.The measurement of intracellular metabolite pools is

not very sensitive, it often results in high standarddeviations and is prone to artifacts during sample-han-dling. Despite these limitations, however, the measure-ment of intracellular metabolite pools affords a qualita-tive picture of a cell's metabolic patterns.The results shown in Table 6 reflect that in bothBHK and insect cells, glucose and lactate made up thegreatest intracellular pools of all glycolytic metabolites.In BHK cells the cell-specific lactate concentration (20nmol was about 6 times higher th an the cell spe-cific glucose concentration (3 nmol Consideringthe low lactate production rate of insect cells it is notsurprising that the cell specific intracellular lactate

concentration was more than 6-fold lower in insect cellsthan in CCLs.

DISCUSSIONGeneral aspectsGlucose and glutamine serve as major energy sourcesfor most in vitro cultivated cell lines (Lanks and Li,1988; Zielke et al., 1984; Reitzer et al., 1979; Wagneret al., 1988). The glucose and glutamine metabolism ofdifferent cultivated mammalian and insect cell lineswere investigated at the early exponential growthphase in batch cultivation. Cell-specific glucose andglutamine utilization and lactate formation rates weredetermined over relatively short time periods (5-10hours).Due to the described decrease of those rates withincreasing cultivation time these short time determina-tions might be one reason t ha t the specific glucose andglutamine utilization and lactate formation rates wereslightly higher than cited in the literature for mamma-lian cell batch cultures (Lin and Miller, 1992; Ogata e tal., 1993; Fitzpatrick et al., 1993; Sharfstein et al.,1994). Higher utilization and formation rates may alsobe due to higher overall glucose and glutamine concen-trations, different media composition, or culture sys-tems. Regarding these points the differences of 0.01-0.06 nmol s-l in the cell specific utilization andformation rates are in good agreement with the specific


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162

Fig. 3. Summary of metabolic pathways and enzyme activities involved which have been investigatedin this work. Arrows with dotted line indicate that the respective metabolic way does not exist in CCLsor is active at very low rates below the sensitivity of the tests used.


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GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES 163glucose and glutamine utilization and lactate formationrates reported by other authors.Furthermore, most enzyme activities determined inour work (see Fig. 3)were slightly higher than thosereported by Fitzpatrick et al. (1993).However, theseinvestigators performed their assays at 25"C, whereaswe performed our experiments at physiological temper-atures (37"C, pH 7.4,or mammalian and 27"C, pH 6.5,for insect cells).

Glucose metabolismUtilization, production, and flux rates

Utilization and production rates.The cell specificglu-cose utilization rates were very similar in BHK andCHO cell lines (0.15-0.21 mol s-l BHK cellscultured in serum-free medium exhibited slightlyhigher glucose utilization rates than in serum-con-taining medium. The cell-specific glucose utilizationrates for both hybridoma cell lines were determined a t0.11and 0.13nmol s-l and respectively, and areconsistent with maximum glucose utilization ratescited in the literature for other hybridomas (Petch andButler, 1994; itzpatrick e t al., 1993). n contrast, theinitial cell-specificglucose utilization rates in insect celllines grown under serum-free culture conditions weredetermined at 0.08 mol s-' and were significantlylower than those of mammalian cell lines. Since insectcells usually produced very little amounts of lactate,whereas mammalian cells characteristically displayeda high conversion ratio of glucose to lactate (Bedard etal., 1993) t already can be assumed that a high amountof glucose was completely oxidized. Initial cell-specificlactate roduction rates between 0.17and 0.32nmolmolar LadGlc-ratio between 1.31 and 1.56,which isconsistent with data cited elsewhere (Hansen and Em-borg, 1994;Ljunggren and Haggstrom, 1994;Lin andMiller, 1992).Moreover, this ratio was slightly higherfor BHK cells cultivated under serum-free conditions(1.5)han for those cultured in serum-containing me-dium (1.3).The high cell-specific lactate production rates inmammalian cells indicate a high glycolytic flux withvery li ttle conversion of glycolytic metabolites into TCCintermediates. Furthermore, the specific molar lactatelglucose ratios point towards a higher glycolytic flux incells cultured under serum-free conditions compared t othose cultivated in serum-supplemented medium. How-ever, according to the findings of Zielke et al. (19801,up to 13% of the lactate production may be attributedt o glutamine utilization. The amount of lactate derivedfrom glutamine in serum-free and serum-supple-mented cultures was not determined in our studies.Insect cells on the other hand only produced verylittle lactate. Therefore glucose must be convertedmainly into other metabolites (Deutschmann andJager, 1994).Thus, some different pictures of glucosemetabolism and enzyme activities are expected toevolve for mammalian and insect cells.Flux rates. The cell-specific glycolyticflux in BHK-21 B N49 /9OHK-1 cells cultured in FCS-supplementedmedium was measured at 0.098 nmol s-' Thisvalue is very consistent with the cell specific-lactateformation rate of 0.208nmol s-l considering a

s-l 10-Bwere measured for mammalian cells, giving a

10% standard deviation and the formation of a smallfraction of lactate by glutamine o r other substrates(Zielke et al., 1980).Assuming complete lactate forma-tion via glucose,a glycolytic flux of 0.104 mol s-'cells should have been measured. In conclusion, theglycolytic flux accounted for about 62% of the total glu-cose utilized. At the same time, no conversion of glucosemetabolites o r pyruvate into the TCC could be detected,indicating that less than 0.4% of the glucose metabo-lized was transformed to TCC intermediates. Thisfinding is not only consistent with the rates of glycolyticflux and lactate formation described above, but alsowith results obtained for hybridoma cell lines by otherinvestigators (Fitzpatrick et al., 1993; etch and But-ler, 1994).Estimation of the pentose phosphate path-way flux revealed a cell-specific rate of 0.0076 nmols-l This corresponds to 4.8% of the total glucoseutilized. This pathway supplies important precursors(Rib5P) of nucleic acids and is therefore absolutely es-sential for cell replication (Reitzer et al., 1980; tryer,1988). t was estimated that about 32% of the utilizedglucose was metabolized in pathways different fromglycolysis, TCC, and oxidative PPC (corresponding toa cell specific flux of 0.051 nmol s-' e.g., lipidmetabolism and protein glycosylation were not investi-gated here.The cell-specific glycolytic flux in BHK-21 BN49/9OHK-1 cells cultivated under serum-free conditionswas 0.127nmol s-l (Table 4), which was consistentwith the observed cell specific lactate formation rate of0.260nmol s-l low6. he glycolytic flux accounted for76% of the total glucose utilized. As was the case forcells in FCS-containing medium, no transformation ofglucose metabolites o r pyruvate into the TCC could bedetected (less than 0.36% of the utilized lucose). Thecell specific PPC-flux was 0.0082nmol s - ~ whichcorresponds to 4.9% of the total glucose utilized. Thesedata show that in BHK cells grown under serum-freeconditions around 18% of the glucose consumed (cell-specificflux of 0.030 mol s-' was metabolized inpathways different from glycolysis, TCC, and oxidativepentose-phosphate shunt (e.g., lipid metabolism, pro-tein glycosylation).Comparison of BHK cells cultivated under serum-free and serum-containing conditions shows th at glyco-lytic flux was higher under serum-free conditions, asindicated by the higher LadGlc-ratio. However, theconversion rates of glucose metabolites into the TCCas well as the oxidative pentose phosphate pathwayflux rates were very similar in both cases. This meansthat BHK cells in serum-supplemented culture utilizedglucose at a higher rate in pathways other than theones addressed here, which may be one reason forhigher specific mitotic rates observed in serum-con-taining media. The exact roles which FCS on the onehand and insulin and transfenin on the other may playin flux distribution remain unknown at this point, al-though a higher glucose utilization rate has been re-ported when insulin was present in growth media (Ryllet al., 1990). n increase of the PFK activity has beenproposed for chicken embryo fibroblasts in the presenceof insulin (Van Schaftingen, 1987).The flux data presented here are in good agreementwith flux rates for hybridoma cell lines reported byFitzpatrick e t al. (1993) nd Petch and Butler (19941,


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164 NEERMANN AND WAGNERbut differ to some degree from the flux data publishedby Sharfstein et al. (1994), who applied 13C-NMR spec-troscopy. For a murine B-lymphocyte hybridoma(CC9ClO) cell line a cell specific glycolytic flux of 0.127nmol s-lhas been estimated by other authors (Petch and Butler,1994). In contrast to our results, however, they addi-tionally reported a cell specific glucose metabolite fluxinto the TCC of 0.0008 nmol 5-l Fitzpatrick etal. (1993) investigated another murine B-lymphocytehybridoma (PCQXB1/2) cell line and estimated signifi-cantly lower cell s ecific flux rates (glycolytic flux of0.063 nmol s-' 10- ,PPC-flux of 0.0022 nmols-1They also reported a glucose metabolite flux into theTCC, although at a cell specificrate of only 0.0001 nmols-' These values are within the range of the fluxrates obtained in our experiments for BHK-21 BN49190HK-1 cells. In contrast, a hybridoma cell line exam-ined by Sharfstein e t al. (1994) by 13C-NMR-spectros-copy showed a cell-specific glycolytic ra te of only 0.027nmol s-l lop6and a PPC-flux of 0.0025 nmol s-' lop6.These values are considerably lower than those mea-sured in our experiments. It should be noted, however,that I3C-NMRspectroscopy analysis requires very highcell densities and hence culture conditions which differfrom those traditionally employed in radioactive la-belling experiments. This may explain the discrepan-cies between the metabolic rates measured bySharfstein et al. (1994) and those described here o rreported previously. In previous publications (Fitzpa-trick et al., 1993; Petch and Butler, 1994; Sharfsteinet al., 1994) the sum of glycolytic flux, PPC-flux, andglucose metabolite flux into the TCC accounted forroughly 92 to 100% of the total glucose utilized. A pre-viously investigated CHO cell line converted more then97%of the consumed glucose into lactate (Donnelly andScheffler, 1976). These relative values are higher t hanour findings for BHK-21 BN49/90HK-1 cells. The differ-ence might be explained by the fact that in these stud-ies, in contrast to our experiments, cells were probablytaken from the midexponential or late-exponentialgrowth phases and medium supplemented with 10%FCS was used. I t has been observed that glucose metab-olism drastically changes with increasing culture timeas evidenced by a n increasing molar lactate/glucose ra-tio (Ljunggren and Haggstrom, 1994), ndicating an in-creasing glycolytic flux.Comparison of insect and mammalian celllines. The cell-specific glycolytic flux ra te in insect Sf-21 cells (serum-free conditions) was 0.067 nmol s- lrepresenting 72% of the total glucose utilized. Despitethis glycolytic flux extremely little lactate was pro-duced. In addition, a relatively high turnover of glucosemetabolites into TCC intermediates was detected byCOz evolution. The cell specific ra te was determined as0.055 nmol s which is close t o the gl colyticflux rate. The difference of 0.012 nmol s-l 10- can bepartially explained by a standard error of around 10%and the channelling of glycolytic metabolites to otherpathways, e.g., the production of alanine from pyruvatein the presence of an alanine dehydrogenase or alaninetransaminase activity. Furthermore, small differencesin the physiological states of the cells during the differ-ent experiments cannot be excluded. In conclusion, theresults obtained show that the majority of glucose con-

and a PPC flux of 0.0048 nmol s-l 10

2

2

sumed (60%)was converted into TCC intermediates.This significant difference between insect cells andCCLs was confirmed by measuring the C0 2 evolutionfrom high amounts of radiolabelled pyruvate added tothe culture medium ( 5 g L-l). Using this method thecell-specificflux ra te of yruvate into the TCC was esti-the pyruvate converting enzymes (probably PDHC) didnot work a t their V,,, (maximum velocity) during glu-cose utilization.The lower glucose utilization and glycolytic flux ratesin insect cells, compared to BHK cells, appear to be theeconomical rationale, when considering the high fluxof glucose metabolites into the TCC and the resultingenergy yield. Thus, the flux of glucose metabolites intothe TCC is actually the most significant difference inmetabolism between continuous insect and mamma-lian cells. Insect cells should be able to generate ordersof magnitude more energy derived from glucose thanCCLsl (expressed in ATP units), considering an energyyield of 36 mol ATP produced per mol glucose oxidizedvia TCC compared to only 2 mol ATP per mol glucoseoxidized via glycolysis.The glucose flux via the oxidative PPC was deter-mined t o a cell specific rate of 0.0069 nmol s-' ininsect cells, accounting for 7.4% of the total glucoseconsumed (Table 4). In summary, about 20% of theutilized glucose (0.019 n m ~ l s - ~ is metabolized inpathways not investigated here, such as lipid metabo-lism and protein glycosylation. The absolute flw rateof glucose into the oxidative branch of the PPC in insectcells was very similar to those found in BHK cells, indi-cating that both cell types need to regenerate about thesame amount of NADPH or require about the sameamount of Rib5P-units as nucleic acid precursors. Thisassumption is supported by similar mitotic rates esti-mated for both cell lines, indicating that the PPC mightdetermine the ra te of the cell division. Insect Sf-21 cellsshowed a mitotic rate of 0.030 h-l, which is similar tothat of the BHK-21 BN49/90HK-1 cells cultured underserum-free conditions (0.028 h-').However, the mitotic rates of insect cells were belowthe mitotic rate of BHK cells cultured in FCS-supple-mented medium (0.037 h-'). This indicates that therate of cell division was not limited by the rate of ATPproduction, which is in accordance to results recentlypublished by Renner e t al. (1994) who reported a 2-foldhigher mitotic rate of CHO cells expressing recombi-nant cell division promoting proteins such as cyclinE. It can be expected, therefore, that a higher ATPproduction rate results in a better productivity of cellsa t a stable mitotic rate.

Interestingly, both insect and BHK cells cultivatedunder serum-free conditions metabolized only about20% of the total glucose consumed in pathways notinvestigated here (lipid metabolism, protein glycosyla-tion), while for BHK cells grown in serum-containingmedium this part accounted for 32%of the glucose uti-lized. However, to what extent glucose influences cellgrowth via biomass production or pathways other thanglycolysis, PPC, or TCC, remains to be determined.Rate l imiting steps: enzyme activities

In order t o identify possible rate-limiting steps in glu-cose metabolism, the maximum in vitro activities of key

mated at 0.095 nmol s- Y -0 (Table 4), indicating that


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GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES 165enzymes and the rate of glucose transport into cells weremeasured. The maximum activities of key enzymes canindicate a maximum flux for a desired pathway (Ardawiand Newsholme, 1982,1983).All enzymes and pathwaysinvestigated are summarized in Figure 3.Hexokinase.By comparing all glycolytic enzyme ac-tivities (Table 5a), it becomes obvious that in all celllines analyzed here, HK exhibited the lowest cell spe-cific activity. For all CCLs investigated in this work,the maximum specific HK activity was quite close tothe relevant specific glucose consumption rates (Tables3 and 5a).It has been previously suggested for in vitro culti-vated primary r at thymocytes tha t PFK is the regula-tory enzyme of glycolysis (Brand, 1985; Newsholme etal., 1987).However, PFK activities determined here forCCLs and insect cells were significantly above the HKactivities (2.5-4-fold, Table 5a) and above the calcu-lated glycolytic flux (Table 41, which is in agreementwith data reported for a murine hybridoma line (Fitz-patrick et al., 1993). Based on literature values of KMfor HK and PFK, it can be concluded that HK ratherthan PFK does not work at its maximum velocity, duet o a relatively low ATP affinity (KM[ATP]= 0.2-1.2mmol L-', KMIGlc]= 0.02 - 0.1 mmol L-l; Wilson,1985) compared with PFK (KM[ATP] 0.01 mmol L-l,KM[Frc6Pl= 0.02 mmol L-l; Massey and Deal, 1982).This assumption is supported by findings tha t hexoki-nase can easily be inhibited by GlcGP, with %-valuesin the low micromolar range (Wilson, 1985; Fiechterand Gmiinder, 1989). The ra te of glucose transport intothe BHK-21 BN49/90HK-1 cells for both serum-supple-mented (0.34 nmol s- * and serum-free culture(0.32 nmol s- l modes was found to be distinctivelyhigher than glucose utilization or the glycolytic fluxrates determined. Although glucose utilization and gly-colytic flux rates were measured over several hours,while glucose transport was determined over 10 mi-nutes, it appears very unlikely that glucose transport israte limiting. This assumption is confirmed by pre-viously reported glucose transport rates for murine B-lymphocyte hybridomas (cell-specific glucose import:0.267 nmol s-l Fitzpatrick et al., 1993)and chickenfibroblasts (cell-specific glucose import: 0.357 nmol s-'Kleitzien and Perdue, 1975, assuming 0.55mg pro-tein per lo6 cells; Fitzpatrick et al., 1993), which aresimilar to the data obtained here for BHK cells.These data point towards a bottleneck in glucose me-tabolism at the hexokinase reaction particularly forCCLs and were supported by qualitative trends fromintracellular metabolite pool measurements (Table 6).Glucose and lactate exhibited higher intracellular con-centrations than the other intermediates of glycolysis.Another striking similarity of all cell lines examinedwa s a general increase in cell-specific enzyme activity,the closer the enzymes are disposed towards the end-point of glycolysis, indicating a metabolic sink. For ex-ample, PK andLDH howed significantly higher activi-ties than HK and PFK in CCLs. However, these differ-ences in activity between enzymes of early and latesteps of glycolysis were not as large in insect and pri-mary cells. This becomes evident when examining thePWHK activity ratios termed flux indices (Ifl):Whilethe Iflof insect and primary liver cells ranges between4 and 10, the Ifl of CCLs ranges between 49 and 106

(Table 5a). Previously reported PWHK ratios were near13for lymphocytes (Ardawi and Newsholme, 1982) andbetween 26 and 560 for a murine hybridoma cell linedepending on the physiological state of the cells (Fitz-patrick et al., 1993).The dramatic differences in IRndi-cate t ha t glycolysis in insect and primary cells is betterbalanced and provides more regulative possibilitiesthan in continuous mammalian cells. Indeed, PWHKactivity ratios have already been shown to be useful asan indication of the relative importance of these twoenzymes in glycolytic regulation (Ardawi and News-holme, 1982; Fitzpatrick et al., 1993). Hence, hexoki-nase seems to play a far more important role for ad-justing the glycolytic rate in CCLs than in insect orprimary liver cells.Taken together, these data indicate very stronglythat the hexokinase activity may determine the overallra te of glucose metabolism in most CCLs. Similar find-ings o r conclusions have been previously described fo ra murine B-lymphocyte hybridoma line (Fitzpatrick e tal., 1993), for Aspergillus niger (Torres, 1994) and fora breast cancer cell line (Hugo et al., 1992).

Mediation between glycolysis and TCC. A deci-sive difference between insect and primary liver cellson the one hand and CCLs on the other is the activityand presence of branching enzymes from glycolysis intothe TCC, such as PDHC, PC, and PEPCK. While theseenzymes were active in insect and primary liver cells,they were not in CCLs. The results here fully confirmthe findings of the flux experiments stated above. Aspecific protein kinase has been reported to be part ofthe PDHC, which can phosphorylate PDH at a serineresidue. The phosphorylation of PDH can inhibit itsactivity leading to the accumulation of pyruvate (Ran-dle et al., 1984;Fiechter and Gmiinder, 1989). A lackof PC activity was also confirmed for a murine hybrid-oma cell line by employing 13C-Nh4R spectroscopy(Mancuso et al., 1994). In conclusion, insect and pri-mary liver cells can convert at least a considerable partof their glucose metabolites into TCC intermediates,while transformed mammalian cells probably can not.This would explain, at least to some extent, the differ-ent metabolic behaviour between these two cell typesas far as lactate production is concerned. However, itis not clear why insect cells produce hardly any lactate.Indeed, the activity of the three branching enzymes,PDHC, PC, and PEPCK (Table 5a) is higher than theglycolytic flux observed in insect cells (Table 4) , but theactivities of PK and LDH, which are potential substratecompetitors, are still higher. It is not k n o w n whethera regulated glycolysis or a well-balanced NADH/NADratio in insect or primary cells inhibits lactate forma-tion and promotes flux towards the TCC. It has beenhypothesised that a high NADH/NAD ratio prevails inthe cytoplasmic space of in vitro cultivated mammaliancells, leading glycolysis towards lactate production inorder to regenerate NAD (Lanks, 1986).To what extentthe NADH/NAD ratio influences the activities of theenzymes PDHC, PC, or LDH remains unknown.PPC. The cell-specific GPDH activity was alwaysnearly identical to the respective HK, except for pri-mary liver cells (Table 5a). This activity is most likelyhigh enough not t o limit the PPC flux, since the mea-sured flux via PPC was significantly lower than theGPDH activity (Table 4).


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166 NE ERM A" AND WAGNERChange of enzyme activities. An additional inter-esting aspect of the enzyme activities measured hereis that all glycolytic enzymes in BHK and CHO cellscultivated under serum-free conditions exhibited ahigher cell specific activity than in the same cells cul-tured in serum-supplemented medium. This could ex-plain the higher glucose consumption rates observed inall BHK and CHO cells grown under serum-free condi-tions (Table 3) and the higher glycolytic flux in BHKcells cultivated under serum-free conditions (Table 4)compared t o the same cells cultured in the presence ofserum. However, it is not known whether the absenceof certain FCS components, cell stress, or the presenceof insulin and transferrin were responsible for theslightly higher enzyme activities under serum-freeculture.All glycolytic enzyme activities determined in BHKcells grown under serum-containing conditions inchemostat culture were lower at steady state thanthose of the same cells during early exponential growthin batch fermentations (Table 5a). However, the rela-tive enzymatic activities remained nearly the same, in-

dicating that the cell-specific enzyme activities changewith the physiological state of the cell and depend onthe culture conditions (see also Fitzpatrick e t al., 1993).The fundamental metabolic patterns, however, re-mained stable during nutrient limitation. Enzyme ac-tivities, therefore, may to some extent reflect the physi-ological state of the cells.Glutamine metabolism

In addition to its function a s an essential amino acid,glutamine acts as an important energy source whendeaminated into the TCC intermediate a-ketoglutarate(2-oxoglutarate) (Reitzer et al., 1979; Glacken, 1988).a-Ketoglutarate can be completely oxidized to COz viaTCC or partially oxidized to aspartate, alanine, or lac-tate, which are often secreted into the medium (Wagneret al., 1988). Hence, glutamine metabolism comprisesa special metabolic network termed glutaminolysis andconsisting of up t o eight different pathways (Haggs-trom, 1991).Utilization rates. The initial cell-specific glutamineutilization rates of cultured mammalian cells rangedbetween 0.05 and 0.08 nmol s-* (Table 3). As wasthe case for glucose, the cell-specific glutamine utiliza-tion rate of cells grown under serum-free conditionswas higher than those for the same cells grown in se-rum-supplemented medium. Insect cells showed initialcell-specific lutamine utilization rates of 0.12 to 0.18mine in the medium, which was adjusted to 8 or 12mmol L-', respectively. This reveals that the cells canadapt their glutamine utilization rate to the mediumsupply and indicates that there is a t least no apparentinternal rate limitation within the concentration rangecommonly used in insect cell culture.Flux rates. Experiments using radiolabelled [U-''C]-glutamine showed that 18.0 and 18.5%of the uti-lized glutamine were oxidized toC O , in BHK cells culti-vated under serum-containing and serum-free condi-tions, respectively (Table4). Insect cells oxidized to CO,14.3% of the consumed glutamine. However, the abso-lute amount of glutamine oxidized was higher in insectthan in BHK cells, since the cell-specific glutamine uti-

nmol s- ll 0-8,depending on the concentration of gluta-

lization rate was about 2 times higher (Table3).Hence,insect cells should be able t o produce far more ATPthan mammalian cells, since they channel significantamounts of glucose and glutamine metabolites intothe TCC.When using [U-14Cl-glutamineit is not possible todetermine the exact pathway of glutamine metabolism.This is because glutamine can only be partially oxidizedin the TCC. Nevertheless, the glutamine oxidationrates in both BHK and insect cells demonstrate theimportance of glutamine for energy supply in both celltypes. Jenkins et al. (1992) reported a cell specific glu-tamine oxidation rate of 0.0064 nmol s-l for amurine B-lymphocyte hybridoma line, accounting for36% of the total glutamine utilized. This observationis within the range of our results, considering that intheir medium the glutamine concentration was only 2mmol L-', resulting in a lower cell-specific glutamineconsumption rate. About the same extent of glutamineoxidation was also reported for Chinese hamster fibro-blasts (Donnelly and Scheffler, 1976).Rate limiting steps: enzyme activity. With re-spect to the cell-specific enzyme activities involved inglutaminolysis it can be concluded that in all mamma-lian cells examined here, AspAT showed the highestactivity followed by GDH, supporting the suggestionthat glutamine is partially oxidized via TCC and subse-quently metabolized through the aspartate transami-nation pathway, branching out of the TCC via transam-ination of oxaloacetate. This hypothesis is supportedby MDH and CS activities. While MDH exhibited ex-tremely high activities, CS, which operates immedi-ately downstream of MDH, showed 2- to 3-fold loweractivity than AspAT (Table 5b). Therefore, it is highlyprobable tha t a significant portion of glutamine carbonis directed through the aspartate transaminase path-way in CCLs. Sharfstein et al. (1994) concluded fromdata obtained from analysis of the metabolism of hy-bridoma cells that the TCC flux from KGA towardsOAA is higher than that from citrate to KGA, whichwould be consistent with the obtained enzyme pattern.Moreover, besides being essential for protein synthesis,aspartate may also be an essential intermediate fo rpyrimidine synthesis and of the malate/aspartate shut-tle, which is important for the transfer of reducingequivalents from cytosol t o mitochondria.In insect cells GDH exhibited the highest activity,followed by AspAT. Furthermore MDH activity was notnearly as high as in CCLs and CS activity was higherthan AspAT activity, suggesting that, compared tomammalian cells, a lower amount of glutamine carbonpasses through the aspartate transaminase pathwayin comparison to mammalian cells. In summary, theenzyme pattern seems t o be better balanced in insectcells than in CCLs.In all cell lines investigated the Glnase activity waslower than GDH and AspAT activity, This stands incontrast to results reported by Jenkins et al. (1992),who found the Glnase activity to be higher than therespective GDH activity in a murine hybridoma line.However, the Glnase activity was at least threefoldhigher than the specific glutamine utilization rate inall cell lines examined, which is confirmed by resultsreported from other authors (Jenkins et al., 1992). Ithas been postulated that glutamine transport rather


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GLUCOSE AND GLUTAMINE M ETABOLISM IN CELL LINES 16 7than enzyme activity is rate-limiting in a murine hy-bridoma cell line (Fitzpatrick et al., 1993; Jenkins etal., 1992). However, Jenkins et al. (1992) admit tha tglutaminase may be the subject of additional intracel-lular regulation asdescribed by Zielke et al. (1984) andArdawi and Newsholme (1982), and a likely candidatefor regulating on the rate of glutaminolysis.

The lowest activities of the enzymes of glutamino-lysis examined in CCLs and insect cells are exhibitedby GS and AlaAT. GS in particular was shown to bestimulated by using glutamine-deficient media (Gawlit-zek et al., 1995). In contrast, during the early phasesof the growth cycle of mammalian and insect cells, glu-tamine is found in high concentrations in the mediumand is mainly consumed and not synthesized, thus re-quiring only a low GS activity. This becomes clear whenGS activity is examined in primary liver cells. Althoughliver cells must have a high capacity for glutamine syn-thesis due to their specific function in the intact organ-ism, glutamine must also be produced as a result ofa likely insufficient supply of external glutamine. Ittherefore is no surprise that GS activity was found t obe higher than Glnase activity in primary liver cells.It can be assumed tha tAlaATmainly works in a regula-tive way, disposing a surplus of glutamate o r TCC me-tabolites.It has been hypothesized tha t the glutamine carbonexists in the mitochondria1TCC as the metabolite ma-late, which can be converted into alanine, lactate, oraspartate in the cytoplasm (McKeehan, 1982).Thispro-cess could increase the cytoplasmic NADWNAD ratio,resulting in an important contribution to drive lactateproduction via glycolysis for NAD estoration (Lanks,1986; Petch and Butler, 1994). This theory could beapplied presuming that an active malate shunt existsand an electron transfer system-like the NAD+-linked MDH-is present in the cytosol, as describedin the literature (Lanks, 1986; Mancuso et al., 1994;Sharfstein et al., 1994; Zimmerle and Alter, 1993). Atthe same time, the glycerol-phosphateor malate/aspar-tate shuttles have t o show very low or no activity. Theextremely high MDH activity observed in all mamma-lian cells compared to that in insect cells could be anindicator for its cytoplasmicor even malate shunt activ-ity, causing a high cytoplasmicNADWNAD ratio. Thiscould partially explain why the lactate formation viaglycolysis in CCLs is higher than in insect cells. Thishypothesis, however, depends on two assumptions anddoes not take into account the activities of enzymesconnecting glycolysis and TCC.Activities of glutaminolytic enzymes were higher inmammalian cells cultivated under serum-free condi-tions than in the same cells cultured in the presenceof FCS. Thiscorrelates with a higher specific glutamineconsumption and glutamine oxidation rates in serum-free cultured cells and indicates that enzyme activitiescan be influenced by the culture conditions.

ConclusionsA general point emerging from this study is the simi-larity in many aspects of the glucose and glutaminemetabolism in many cultured mammalian cell lines.Hexokinase appears to determine the rate of glycolyticflux n various CCLs. While in insect cell lines bothglucose and glutamine a re metabolized via the TCC, in

transformed mammalian cells only glutamine contri-butes to the TCC significantly. Based on the data pre-sented here this is prevented by very low or totallyrepressed PDHC, PC, and PEPCK activities and a highLDH activity. Activities of all enzymes are higher whenmammalian cells are cultured under serum-free condi-tions compared to serum-supplemented culture.TCC intermediates can be converted into manyamino acids, which certainly requires a constant re-plenishment of TCC metabolites. However, an increasein TCC oxidation of glucose metabolites in CCLs wouldpromote a more efficient glucose utilization and a si-multaneous lactate reduction. Furthermore, an interre-lation between glutaminolysis and glycolytic lactateformation via cytoplasmic NADWNAD pools is sug-gested.In conclusion, a better understanding of the intersec-tion of glucose and glutamine metabolism, as well asflux determination of NADH-transfer between cytosoland mitochondria may be necessary for a successfulattempt to alter the metabolism of mammalian cells bydirected metabolic management or design.

ACKNOWLEDGMENTSThe authors thank their colleagues of the Cell Cul-ture Techniques department of GBF, M. Ackermann,Dr. V. Jager, Dr.M. Patole, and A. Kobold for supplyinginsect cells, J. Hammer for amino acid analysis, andDr. K Scharfenberg for providing recombinant CHOcells. We thank Dr. Manfred Wirth for kindly contribut-ing the recombinant BHK-21 BN49/90HK-1 cell lineand Dr. Stephanos Grammatikos for critically readingthe manuscript.

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