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

Comparative Analysis of Glucose and Clutamine Metabolism in Transformed Mammalian Cell Lines, Insect and Primary Liver Cells
J O R C NEERMANN AND R O L A N D WAGNER* Department for Cell Culture Techniques, Gesellschati fur Biotechnologische Forschung rn.b.H., 0-38124 Braunschweig, Germany

Glucose and glutamine metabolism in several cultured mammalian cell lines (BHK, CHO, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. Results were compared with data from two insect cell lines and primary liver cells. Flux distribution was measured in a representative mammalian (BHK) and an insect (Spodopfera frugiperda) cell line using radioactive substrates. A high degree o similarity in many f aspects of glucose and glutamine metabolism was observed among the cultured mammalian cell lines examined. Specific glucose utilization rates were always f close to specific hexokinase activities, indicating that formation o glucose-6phosphate from glucose (catalyzed by hexokinase) is the rate limiting step of glycolysis. No activity of the key enzymes connecting glycolysis with the tricarboxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvatecarboxykinase,could be detected. Flux distribution in BHK cells showed glycolytic rates very similar to lactate formation rates. No glucoseor pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating that glucose is mainly metabolized via glycolysis and lactate formation. About 8% of utilized glucose was metabolized via the pentose phosphate shunt, while 20 to 30% of utilized glucose followed pathways other than glycolysis, the tricarboxylic acid cycle, or the pentose phosphate shunt. About 18% of utilized glutamine was oxidized, consistent with the notion that glutamine is the major energy source for mammalian cell lines. Mammalian cells cultured in serum-free low-protein medium showed higher utilization rates, flux rates, and enzyme activities than the same cells cultured in serum-supplemented medium. Insect cells oxidized glucose and pyruvate in addition to glutamine. Furthermore, insect cells produced little or no lactate and were able to channel glycolytic intermediates into the tricarboxylic acid cycle. Metabolic profiles of the type presented here for a variety of cell lines may eventually enable one to interfere with the metabolic patterns of cells relevant to biotechnology, with the hope of improving growth rate and/ Or ProdLICtiVity. 0 1996 Wiley-Liss, Inc.

As mammalian cell culture gains importance in the biotechnological production of therapeutics, the metabolic design of transformed mammalian cell lines becomes increasingly the focus of process development (Fiechter and Gmiinder, 1989; Stephanopoulos and Sinskey, 1993). By means of metabolic improvement, cell lines with increased productivity, substrate spectrum, or energy yield could be developed, thus reducing production costs. Success in attempting a metabolic improvement of transformed cell lines greatly depends on the availability of detailed information about the metabolic fluxes characteristic of different cell lines and cell types (Sahm, 1993; Kelly e t al., 1993). This type of comparative analysis exposes important aspects of metabolic behaviour and offers insights into the possibilities for metabolic improvement. Recently, Pendse
0 1996 WILEY-LISS, INC.

and Bailey (1994) reported a dramatic increase in tissue plasminogen activator (tPA) production (up to 100%)by CHO cells expressing a recombinant Vitreoscilla haemoglobin (VHb) in addition to recombinant tPA. The coexpressed haemoglobin provided sufficient oxygen for ATP synthesis even under oxygen-limited conditions. Considerably more complex than the oxygen-ATPrelationship is the complete primary energy metaboReceived March 14, 1995; accepted June 19, 1995. *To whom reprint requestdcorrespondence should be addressed at Abteilung Zellkulturtechnik, Gesellschaft fur Biotechnologische Forschung m.b.H., Mascheroder Weg 1, D-38124 Braunschweig, Germany.

GLUCOSE AND GLUTAMINE METABOLISM 1N CELL LINES

153

lism in mammalian and insect cell lines. It is accomplished via the major pathways of glutaminolysis, glycolysis, and TCC, in which certain enzymes function as flux regulators (Glacken, 1988; Lanks and Li, 1988). There are some obvious differences in the energy metabolism of primary mammalian cells, insect and transformed mammalian cell lines, with regard to lactate production, dissolved oxygen limitation, glucose, and glutamine utilization. Unlike insect and primary mammalian cells, transformed mammalian cells convert a

ADP AlaAT AspAT ATCC ATP BHK CCL CHO

cs

EBV EDTA EGTA FCS Frc6P GAP GDH GDP Glc GlclP GlCGP Glnase GPDH

Gs
GTP HEPES HK IDP IMDM KGA Lac LDH 2-ME MDH MOPS OAA PBS

Pc

PCA PDH PDHC PEP PEPCK PFK PGL PK PMSF PPC

large amount of glucose to lactate, with very little glucose-derived carbon entering the TCC (Donnelly and Scheffler, 1976; Glacken et al., 1986; Bedard et al., 1993; Schrimpf et al., 1994; Crabtree and Newsholme, 1972; Lund, 1980). However, a detailed comparison of the metabolic patterns of primary mammalian cells, insect and mammalian cell lines has not been previously reported. Apart from glycolysis and TCC there exist other glucose-utilizing pathways. For example, a small fraction of the total glucose utilized provides precursors for ribose formation via the pentose phosphate shunt, which plays an essential role in nucleic acid synthesis (Reitzer et al., 1979). In addition to glucose, glutamine serves as a major energy and carbon source in many cell types, Abbreviations since it can be oxidized either completely (to C0.J or adenosine diphosphate partially via glutaminolysis (Reitzer et al., 1979, 1980; alanine aminotransferase Glacken, 1988; Zielke et al., 1984). The metabolism of aspartate aminotransferase glutamine and glucose is interactive (Newland et al., American type culture collection 1990), with glutamine stimulating glucose utilization adenosine triphosphate via glycolysis and the pentose phosphate shunt (ORS r a baby hamster kidney yin continuous mammalian cell line ourke and Rider, 1989; Reitzer et al., 1980), but inhibChinese hamster ovary iting glucose oxidation in the TCC (Lanks, 1986). Glucitrate synthase cose, in turn, can influence glutamine utilization in Epstein-Barr virus ethylenediaminetetraacetate [(ethylenedinitrilo)tetraace- CCLs (Newland et al., 1990; Zielke et al., 1978). Hence, tic acid] deeper insights into the glucose and glutamine metaboethylene glycol-bis(P-aminoethyl ether N,N,NN-tetra- lism of CCLs relevant to biotechnology may eventually acetic acid [Ethylene-bis4oxyethylenenitrilo)tetraacetic lead to a designed improvement of energy metabolism, acid] fetal calf serum and thus t o higher production rates. fructose-6-phosphate The objective of the work described here was to invesglycerolaldehyde-3-phosphate tigate pathway fluxes of glucose and glutamine metaboglutamate dehydrogenase lism in biotechnologically relevant CCLs with the aim guanosine diphosphate glucose of identifying potentially rate-limiting steps. Cell-speglucose-1-phosphate c s c utilization and production rates as well as the maxglucose-bphosphate imum activities of key regulatory enzymes of glucose glutaminase and glutamine metabolism were analyzed in M e r e n t glucose-6-phosphatedehydrogenase glutamine synthetase CCLs to determine common characteristics in the meguanosine triphosphate tabolism of these CCLs. Maximum catalytic activities N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) of certain enzymes can serve as quantitative indicators hexokinase of maximal flux through metabolic pathways (Ardawi inosine diphosphate Iscoves modified Dulbeccos medium and Newsholme, 1982, 1983; Newsholme et al., 1986, a-keto-glutaricacid (2-oxopentanedioicacid) 1987; Fitzpatrick et al., 1993). Metabolic patterns of lactate CCLs were compared with those of insect cells, which lactate dehydrogenase display a very different metabolic behaviour, especially 2-mercaptoethanol malate dehydrogenase with respect to lactate formation. For further compari3-(N-morpholino)pmpanesulfonic acid son, the maximum activities of the enzymes of glucose oxaloacetic acid (oxobutanedioicacid) and glutamine metabolism were also determined for phosphate buffered saline primary liver cells. Moreover, using radioactively lapyruvate carboxylase perchloric acid beled substrates we measured glucose and glutamine pyruvate dehydrogenase fluxes in representative mammalian (BHK-21c13) and pyruvate dehydrogenase complex insect (Sf-21) cell lines. Analysis of key enzyme activiphosphoenolpyruvate ties in different cell lines, combined with a comparative phosphoenolpyruvatecarboxykinase phosphofructokinase correlation of flux and utilizatiodproduction data, indi6-phosphoglucono-6lactone cates the rates of maximum flw and suggests ratepyruvate kinase limiting steps. This knowledge could serve as a basis phenylmethylsulfonyl fluoride to a rational metabolic design. pentose phosphate cycle
pyruvate Spoabptera frugiperdu ribose 5-phosphate secreted alkaline phosphatase Scharfenbergs modified Iscoves-Fl2 simian virus trichloroaceticacid tricarboxylic acid cycle trio(hydroxymethy1)aminomethane Vitreoscilla haemoglobin Zellkulturtechnik

4.r

SF Rib5P

SEAP
SMIF

MATERIALS AND METHODS Chemicals

Unless indicated otherwise, all chemicals used were of highest grade and purchased from Merck (Darmstadt, Germany), Boehringer Mannheim (Mannheim, Germany), or Sigma Chemical Company (Sigma-Aldrich, Deisenhofen, Germany). Radioactive materials

sv

TCA TCC Trio VHb ZKT

154

NEERMANN AND WAGNER

were obtained from Amersham (Braunschweig, Germany).

to the experiments, as described in Cell extraction for

enzyme analysis.

Cell lines Mammalian cell lines. The following mammalian cell lines were used:
1. BHK-21 (C-13) (ATCC CCL 10). A baby hamster kidney cell line purchased from ATCC (Rockville, MD). 2. BHK-21 pSVIL2. A recombinant subclone of BHK21 (C-13), which was manipulated by genetic methods to produce human interleukin-2 constitutively under control of the SV40 early promoter (Conradt et al., 1986). It was provided by the Genetic Engineering Department of Gesellschaft fiir BiotechnologischeForschung (GBF). 3. BHK-21 BN49/90HK-1. A recombinant subclone of BHK-21 (C-131, which produces P-galactosidase and alkaline phosphatase. It was created by transfection of BHK-21 (C-13)cells with the plasmid pHPAPr-l/P-Gal, which contains the Escherichia coli Lac2 gene coding for P-galactosidase (E.C. 3.2.1.23) under control of the human p-actine promoter (Wirth and Schumacher, unpublished data). The recombinant P-galactosidase accumulates in the cytoplasm. The resulting cell line was additionally transfected by a second plasmid (pRSVSEAP), bearing a mutant of the secreted alkaline phosphatase (SEAP) under control of the SV40 promoter (Berger et al., 1988; Racher et al., 1994). An artificial termination-translation signal was added at position 489 resulting in secretion of the expressed protein into the culture supernatant. The cell line was provided by the Genetic Engineering Department of GBF. 4. CHO-K1 (ATCC CCL 61). A Chinese hamster ovary cell line obtained from ATCC. 5. CHO-K1 pMDIIIGPTR. A transfected subclone of CHO-K1 cells (Motz et al., 1987). It produces two recombinant Epstein-Barr virus (EBV) surface glycoproteins of 250 and 350 kd, respectively. This cell line was provided by Prof. H. Wolf (Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany). 6. Rat-mouse hybridoma 187.1 (ATCC HB58). Produces a monoclonal antibody (IgGl),specific for mouse kappa light chains. It was purchased from ATCC. 7. Murine hybridoma MAX16H5. Produces an anti CD-4 monoclonal antibody. The cell line was provided by Prof. F. Emmrich (Institute for Clinical Immunology and Rheumatology, University of Erlangen-Niirnberg, Germany). 8. WI-38 (ATCC CCL-75).A cultured diploid cell line from human lung tissue purchased from ATCC.

Culture media All mammalian cell lines were cultured in medium based on a 1:l mixture of IMDM and Ham's F12 (ZKTI 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 medium was either supplemented with 2.5% heat-inactivated fetal calf serum (Sigma) or with 10 mg L-' human transferrin (Biotest, Dreieich, Germany) and 10 mg Lhuman insulin (Elanco, Indianapolis, IN). CHO-K1 pMDIIIGPTR cells were cultured in protein-free SMIF6-medium (Wolf et a . 1993; Scharfenberg and l, Wagner, 1995) containing a chelator additive as described previously (Bertheussen, 1993). The differently supplemented media will be referred to as FCS-containing medium, serum-free medium, or protein-free medium, respectively. Insect cells were grown in EXCELL 401 medium (JRH Bioscience, Lenexa, KS){ which contained 15 mmol L-' glucose and 8 mmol Lglutamine. The medium was supplemented with additional 20 mmol L-l glucose.

'

Cell culture All cells were routinely cultured in either 75 c' Tm flasks (Nunc, Roskilde, Denmark) or in spinner flasks (300 mL, 50 rpm) (Techne, Cambridge, UK). The mammalian cells were incubated at 37C under 90% air/ 10% COz and 99% humidity (Biocenter 2000, SALVIS, Reussbuhl, Switzerland). Insect cells were incubated a t 27C. For experiments cells were taken from the early exponential growth phase of the batch cultures. For further comparison, BHK-21 BN49/90HK-1 were also taken from a continuous chemostat culture (1.6 L, dilution rate: D = 0.022 h-'). Viable cell numbers were determined by the trypan blue exclusion method (Boehringer Mannheim) using a hemocytometer. The numbers were confirmed by counting nuclei stained with crystal-violet (0.1%crystal violet in 0.1 mol L ' citrate) using a hemocytometer. Glucose, lactate, and amino acid analyses Glucose and lactate were routinely determined using a YSI Model 2000 GlucoseLactate analyzer (Yellow Springs Instruments, Yellow Springs, OH). Glutamine and other amino acids were assayed by reverse-phase HPLC with fluorometric detection after derivatisation with ortho-phthaldialdehyde as described in detail elsewhere (Larsen and West, 1981; Ryll et al., 1990).

Enzymes and extraction buffers All enzymes analyzed are listed in Table 1. For enInsect cell lines. The following insect cell lines were zyme extraction three M e r e n t buffers were used based used: on buffers previously described (Ardawi and Newsholme, 1982), but with slight modifications as follows: 1. IPLB-Sf-21AE. Isolated from Spodoptera frugi- The extraction buffer for HK,GPDH, PK, LDH, GDH, perdu (Vaughn et al., 1977). It was provided by Dr. AspAT, AlaAT, MDH, and GS consisted of 50 mmol L-' J. Claus (Universidad National del Litoral, Santa Fe, triethanolamine (2,2'2"-nitrilotriethanol)(pH adjusted Argentina). with 1mol L-' NaOH), 1mmol L-' EDTA, 2 mmol L-' 2. Sf-9 (ATCC CRL 1711). A subclone of IPLB-Sf- MgC1, , and 30 mmol L-' 2-mercaptoethanol(2-hydroxy21AE purchased from ATCC. ethylmercaptan). The extraction buffer for PFK and CS Primary cells. Livers of both pig and mouse were contained 50 mmol L-' Tris/HCl, 1 mmol L-' EDTA, taken from freshly killed animals and prepared just prior and 5 mmol L-l MgS04. The extraction buffer for PC

GLUCOSE AND GLUTAMINE METABOLISM I CELL LINES N TABLE 1 Enzvmes analyzed in this work . Enzyme Hexokinase Glucose-6-phosphate dehydrogenase Phosphofiuctokinase Pyruvate kinase Lactate dehydrogenase Glutaminase Glutamate dehydrogenase Alanine aminotransferase Aspartate aminotransferase Glutamine synthetase Malate dehydrogenase Citrate synthase Pyruvate carboxylase Phosphoenolpyruvate carboxykinase Abbreviation HK GPDH PFK PK LDH Glnase GDH AlaAT AspAT GS MDH Rational name ATP: D-hexose 6-phosphotransferase D-glucose-6-phosphate: NADP' oxidoreductase ATP D-fructose-6-phosphate 1-phosphotransferase ATP pyruvate 2-0-phosphotransferase (S)-lactate: NAD' oxidoreductase Glutamine amidohydrolase Lglutamate: NAD' oxidoreductase L-alanine: 2-oxoglutarate amino-transaminase L-aspartate: 2-oxoglutarate amino-transaminase L-glutamate: ammonia ligase [ADP forming] @)-malate: NAD' oxidoreductase; Wmalate; NADP' oxidoreductase [oxaloacetate-decarboxylatingl Citrate: oxaloacetate-lyase [(pro-3S)-CHzCOO-+ acetyl-CoAl Pyruvate: carbon-dioxide ligase [ADP-forming] GTP: oxaloacetate carhoxy-lyase [transphosphorylatingl ATP oxaloacetate carboxy-lyase [transphosphorylatingl Pyruvate: lipoamide 2-oxidoreduae [demrbxylating and acceptor-acetylatingl Acetyl-COA: dihydrolipamide S-acetyltransferase Dihydrolipoamide: NAD' oxidoreductase

155
E.C. number' 2.7.1.1 1.1.1.49 2.7.1.11 2.7.1.40 1.1.1.27 3.5.1.2 1.4.1.2 2.6.1.2 2.6.1.1 6.3.1.2 1.1.1.37 1.1.1.40 4.1.3.7 6.4.1.1 4.1.1.32 4.1.1.49

cs
PC PEPCK

Pyruvate dehydrogenase complex Composed of Pyruvate dehydrogenase Dihydrolipoyl transacetylase Lkhydrolipoyl dehydrogenase
'E.C.
=

PDHC PDH 1.2.4.1 2.3.1.12 1814 ...

enzyme classification.

and PEPCK consisted of 50 mmol L-' triethanolamine (pH adjusted with 1mol L-' NaOH), 300 mmol L-l sucrose, and 1mmol L-' EDTA. For extraction of PDHC a 20 mmol L-l HEPES buffer (pH adjusted with 1mol L-' NaOH), containing 3 mmol L-' MgCl,, 3 mmol L-' EGTA, 0.5 mmol L-' EDTA, and 3 mmol L-' dithiothreitol, was used. The extraction buffer for Glnase contained 50 mmol L-' Tris/HCl, 150 mmol L-' KH,P0&.J3PO4 (pH 7.41, and 1mmol L-' EDTA. All extraction buffers contained 0.1% (v/v) TRITON-X-100 and were adjusted to pH 7.4 for mammalian and pH 6.5 for insect cells.

Cell extraction for enzyme analysis Cell lines. Cells were removed from plastic or spinner flasks, counted and centrifuged at 200g for 5 minutes (RotantaRP, Hettich, Tuttlingen, Germany) at 4C. The cell pellet was washed twice with 20 mL phosphatebuffered saline (PBS, pH 7.4 for mammalian cells, pH 6.2 for insect cells) and resuspended in 3 mL of the appropriate enzyme-extraction buffer at 4C. Complete cell lysis was accomplished by a freeze-thaw cycle folMetabolite pools lowed by 20 strokes in a homogenizer (Dounce, BraunDiessel Biotech, Melsungen, Germany). The lysates Intracellular glycolytic metabolite concentrations were centrifuged at 3,OOOg for 5 minutes and the resulting supernatanb were assayed for enzyme activity. (glucose, glucose-6-phosphate, fructose-6-phosphate, phosphoenolpyruvate, pyruvate, lactate) were deterPrimary cells. Fresh livers from both pig and mouse insect Sf-21 were first perfused with 10 and 1L, res ectively, of a mined for BHK-21 BN49/90HK-1 cells and(Hugo et al., suitable perfusion buffer (20 mmol L- P MOPS, 130 cells either by spectrophotometric assays 1992) or using the YSI mmol L- NaCl, 5 mmol L-' KCl, 8 mmol L-' MgCl,, analyzer (Yellow Springs Model 2000 Glucose/Lactate Instruments). 0,l mmol L-' PMSF, 5 mmol L-' 2-ME) and thereafter chilled cells (5 x lo8) were performedExtractions of as described with equal volumes of PBS (10 and 1L). A known mass (Schmid and Blanch, 1992). The metabolite pools were of the tissue (about 2 g) was lysed as described above determined from triplicate extracts. for the various cell lines. Radioactive measurements Enzyme activity measurements Enzyme activities were measured for all mammalian cells at 37"C, pH 7.4, and for insect cells at 27"C, pH Radioactive samples were counted with 2 mL Quicksafe A scintillator (Zinsser, Frankfurt, Germany)

6.5, to obtain standardized conditions which resemble the natural environment as closely as possible. The spectrophotometric assays were performed using an LKB Ultrospec K kinetic spectrophotometer (Pharmacia, Freiburg, Germany). Enzyme activities were calculated from the rate of change of absorbance within a linear range at 340 nm, except for CS, PC, and PEPCK, which were measured at 412 nm, and PDHC. PDHC activity was determined only qualitatively by a colorimetric change within the visible spectrum, since quantitative assays (Schwartz and Reed, 1970) monitoring the NADH increase at 340 nm are not applicable in crude extracts with high LDH-activity competing for the substrate pyruvate. Each assay was validated by showing a linear correlation between enzyme addition and spectrophotometric response. Each measurement was repeated at least five times and the mean value was calculated. The assays were based on methods described previously as shown in Table 2.

156
Metabolism Glucose metabolism

NEERMANN AND WAGNER

TABLE 2. References for methods of enzyme analysis'

Enzyme Hexokinase Glucose-6-phosphatedehydrogenase Phosphofi-uctokinase Pyruvate kinase Lactate dehydrogenase Pyruvate carboxylase Pyruvate dehydrogenase complex Phosphoenolpyruvatecarboxykinase Glutamate dehydrogenase Glutaminase (phosphate-activated) Glutamine synthetase Alanine transaminase Aspartate transaminase Citrate synthase Malate dehydrogenase (NAD(P)+-linked) Reference Fornaini et al. (1982) Julian and Reithel(1975) Massey and Deal (1982) Kahn and Mane (1982) Zammit and Newsholme (1976) Ballard and Hanson (1967) Reed and Willms (1966) Zammit and Newsholme (1976) Fisher (1985) l Kvamme et a . (1985) Fbwe et al. (1970) Sugden and Newsholme (1975) Sugden and Newsholme (1975) Alp et al. (1976) Newsholme and Williams (1978)

Glutamine metabolism

Tricarbonic acid cycle

'We could not discriminate between the two isoenzymes of PEPCK that bind GDP and ADP, respectively, because the three nucleotides GDP, 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-21 BN49/90HK-1 cells and IPLB-Sf-21AE cells. Incubation temperature, pH and media conditions were maintained as stated above, except for pyruvate entering the TCC, where the medium was replaced by pyruvate (5 g L-l) solubilized in PBS buffer (pH 7.4 or 6.5), as described previously (Bartos et al., 1993). Replicate tests were performed in all experiments. The glucose membrane transport was determined as previously described in detail (Fitzpatrick et al., 1993). Briefly, to determine the glucose transport rate independently from any further influences of cellular metabolism, a radiolabelled glucose analogue 2-deoxy-D[l-3Hl-glucose(0.1 pCi A 3.7 x lo3 Bq) was used, which is not a substrate for HK and cannot be metabolized. The glucose transport rate was examined for BHK-21 BN49/90HK-1 cells (1 x lo7 mL-') growing in FCScontaining and serum-free medium. Glycolytic flux was determined as described by Fitzpatrick et al. (19931, based on the methods of Hammerstedt (1973) and Bontemps et al. (1978). Briefly, the glycolytic flux was determined by the release of tritiated water from the metabolism of D-[3-3Hl-glucose by measuring the flux of glucose metabolites through the aldolase and triose phosphate isomerase reactions. The rates of released tritiated water were linear for the investigated mammalian and insect cell lines during a 3.5 hour observation. Cells (5 x lo6) were incubated in 1 mL medium containing D-[3-3Hl-glucose(0.5 pCi P 18.5 x lo3 Bq). Glucose carbon entering the TCC was measured by the rate of 14C02 release from 6-14C-glucose(Petch and Butler, 1994). Pentose phosphate pathwa flux was calculated from the difference in the rate of LCO, released from l-14Cglucose and 6-14C-glucose(Katz and Wood, 1963). Pyruvate carbon entering the TCC via pyruvate dehydrogenase was measured by the rate of 14C02release from l-14C-pmvate(Bartos et a. 1993). l. Glutami;; oxidation was measured by the rate of

14C02 released from L-[U-'4Cl-glutamine (Brand et al., 1984). The 14C02 formation was determined essentially following the methods of Bartos et al. (1993): Cells were suspended in growth medium, or yruvate containing PBS, at a concentration of 5 x 10PmL-1. A respective radioactive substrate was added to the suspensions in following amounts: l-14C-glucose(0.5 pCi f 18.5 x lo3 Bq), 6-14C-glucose(0.5 pCi f 18.5 X lo3 By) L-[U-14C]glutamine (0.5 pCi & 18.5 x lo3 Bq), or 1- C-pyruvate (1 FCi A 3.7 x lo4 Bq). Aliquots (1.5 mL) were dispensed into Petri dishes (21 cm2, Nunc) modified as follows: The Petri dish cover had a small injection port sealed with adhesive tape (Beiersdorf, Hamburg, Germany). A small polyethylene tube (Eppendorf, Hamburg, Germany) containing 300 pL of a 1:l(v/v) mixture of phenylethylamine and methanol to trap 14C02was attached t o the inside of the cover by an adhesive tape. The dishes were airsealed with rubber bands and incubated a t the appropriate temperature up to 4 hours. At regular intervals of about 30 min 200 pL of 10% (w/v> TCA were injected into the cell suspension. Following the incubation period the phenylethylamine was allowed to assimilate 14C02completely (1 hour) before radioactivity was measured.

RESULTS Cell-specificsubstrate utilization, formation, and mitotic rates At first, the initial cell-specific glucose and glutamine utilization and lactate formation rates were investigated, since they are of particular importance for later comparison with measured flux rates and enzyme activities. Cells started their exponential growth phase with very 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 cultivation time these specific utilization and formation rates decreased as glucose and glutamine concentrations declined. This phenomenon has been observed in various batch cultures (Ljunggren and Haggstrom, 1994; Fitzpatrick et al., 1993; Ogata et al., 1993). In our experiments the initial specific glucose and glutamine utilization rates of the examined cell lines

GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES

157

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

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20

80

100

120

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40

60

80

100

120

140

cultivation time I (h)

cultivation time I (h")

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

range between 0.15 and 0.22 nmol s-l They were lower for both insect and hybridoma lines, with 0.08 and 0.11 nmol s-l lo-" cells to cells, respectively. While all mammalian cells ini0.13 nmol 8-' tially produced between 0.17 and 0.32 nmol s-' lactate, the insect cells secreted no or extremely little lactate.

have been determined over relatively short time periods (5 hours to 10 hours). This was necessary to compare, qualitatively as well as quantitatively, the specific utilization and formation rates with f u data aclx quired from radioactive labeling experiments, which were carried out for practical reasons over short time periods (up to 5 hours). For the same reasons the enzyme activities were determined in the early exponential phase, since enzyme activities can change with changing culture conditions (Lind et al., 1991; Fitzpatrick et al., 1993; Kratje et al., 1994a,b).

depending on culture supplements and cell concentration. For both insect cell lines and hybridoma cultures the initial cell-specificglucose utilization rates were lower, a t about 0.08 and 0.11-0.13 nmol s-l respectively (Table 3). For continuous mammalian cell lines, the initial cellspecific lactate formation rates varied between 0.17 nmol s-' lop6for the murine hybridoma MAX16H5 and for the BHK-21 c13 grown under 0.32 nmol s-l serum-free culture conditions. The calculated lactate/ glucose molar ratios differed among the cell lines and changed slightly with increasing cultivation time, but Glucose utilization and lactate formation rates were always clearly >1 (Wagner et al., 1988).T i indihs Cell-specific substrate utilization and byproduct for- cates that for the most part glucose was transformed mation rates vary during batch cultures. When cells into lactate in all cultured mammalian cells. In the were resuspended in fresh growth medium they nor- case of BHK cells the lactatdglucose molar quotient mally had to adapt to the new culture conditions by was greater for cells cultured in serum-free medium replenishing the intracellular pools depleted during the than for those grown in FCS-supplemented medium. A preceding starvation phase. As a result, the cell-specific representative lactate/glucose quotient for each cell glucose utilization and lactate formation rates were line a t the initial phase of culture is given in Table 3. highest at the initial phase of culture and then deThe cell specific lactate production in Sf-9 and Sf-21 creased constantly (Fig. 1). most mammalian batch insect cells differed significantly from that found for For cultures examined, the initial cell-spec& glucose up- cultured continuous mammalian cells. The Sf-9 and Sftake rates were found to be between 0.15 and 0.21 nmol 21 insect cells produced very little or no lactate with a
5-l

158

NEERMANN AND WAGNER

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CHO-K1, serum-free Hybridoma 167.1, with 3% FCS 4 BHK-21 c13. With 2.5% FCS - - O . . BHK-21 c13, serum-free .-VBHK-21 BN49/90HK-l. with 2.5% FCS A- BHK-21 BN 49MOHK-1, serum-free

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concentrations in the insect medium were higher than in the mammalian cell culture medium. The increase in the cell specific glutamine utilization of the CHO-K1 cells might be due to an initial lag-phase in the glutamine metabolism.

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

LadGlc molar ratio of 0.02 as shown in Figure 1and Table 3. Glutamine utilization The cell-specific glutamine utilization rates were highest during the initial culture phase for BHK and

hybridoma cell lines. In contrast, other cells-especially the insect lines -showed increasing cell-specific glutamine utilization rates during the first 50 hours of culture (Fig. 2). The initial cell-specific glutamine utilization rates were found to range between 0.05 and 0.08 nmol s-l for cultured mammalian cells and

GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES

159

TABLE 3. Cell specific substrate utilization, byproduct formation, molar quotient of the LadGlc rates ( Q b d d , and mitotic rates ( u ) a t the initial ahases of batch cultures for a number of cell lines'

Cell line Insect Sf-9, serum-free Insect Sf-21, serum-free CHO-K1, serum-free BHK-21 c13. 2.5% ~-~ FCS BHK-21 c13, serum-free BHK-21 BN49/90HK-1, 2.5% FCS BHK-21 BN49/90HK-1, serum-free Hybridoma MAX16HB5, serum-free Hybridoma 187.1 3% FCS
~ ~ -~ ~ 7
-

Glc-rate [nmol s-' 10-Y -0.088 -0.093 -0.169 -0.168 -0.205 -0.159 -0.168 -0.110 -0.131

Lac-rate [nmol s-l 10-61 0.002 0.002 0.255 0.221 0.315 0.208 0.260 0.169 0.204

QLadGk

Gln-rate [nmol s-' 10-61

rh-7 0.032 0.030 0.028 0.037 0.027 0.037 0.028 0.030 0.037

0.02 0.02 1.51 1.31 1.54 1.31 1.55 1.54 1.56

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

-

-0.053

'After error propagation a standard deviation of 7-12% for each value was calculated. The minus expresses consumption of the metabolite. Value in parentheses refers to 12 mmol L-'glutamine 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 Glc membrane transport Glycolytic flux PPC-flux Flux of Glc-metabolites into the TCC &-flux into TCC Gln flux rates Gln oxidation to CO,

BHK-21 BN49/90HK-1 (2.5% FCS) 0.3100 0.0980 0.0076 n.d. < 0.0006 n.d. < 0.0006

BHK-21 BN49/90HK-1 (serum-free) 0.2900 0.1270 0.0082 n.d. < 0.0006 n.d. < 0.0006 0.0115

Insect SF-21 (serum-free)

0.0670 0.0069 0.0550 0.0950

0.0171

0.0090

'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

Glycolysis. The cell specific flux rates were determined as 0.098 and 0.127 nmol s-l for BHK-21 BN49/90HK-1cultured in FCS-containing medium and those grown in serum-free medium, respectively (Table 4). The cell specific flux rate for the insect Sf-21 cells was 0.067 nmol s-' lop6,and thus clearly lower than in BHK cells. TCC. In BHK-21 BN49/90HK-1 cells, neither a cell specific flux of glucose metabolites nor a pyruvate flux into the TCC was detectable above the detection limit of 0.0006 nmol s-l lop6.This indicates that if there was Mitotic rates any flux a t all of glucose metabolites or pyruvate into The average specific mitotic rates [v = (In X2 - In the TCC of BHK cells, it accounted for less than 0.64% Xl)/ln2 (t2- tJ; X, = cell number at cultivation time of the total glycolytic flux, thus contributing no signifi61 were similar for all cell lines during the first 50 cant amount of energy. In addition, this result suggests hours of cultivation. Differences in specific mitotic rates that the PDHC activity is very low due to repression within a cell line apparently depended on FCS-supple- or inactivation, or does not exist at all in continuous mentation of the culture medium. In FCS-supple- mammalian cell lines. mented medium about 1.4-fold higher specific mitotic In insect Sf-21 cells the cell specific glucose metaborate were observed than in serum-free medium. Aver- lite flux into the citric acid cycle was constant durin age specificmitotic rates for the first 40 hours of culture the 4-hour period of observation at 0.055 nmol s 10 . are presented in Table 3. Additionally, the cell specific pyruvate flux into the TCC was measured for 1.5 hours at 0.095 nmol s-' Metabolic fluxes Therefore, in these cells the conversion of glucose The metabolic flux measurements were performed metabolites and pyruvate (via PFHC) into TCC interwith BHK-21 BN49/90HK-1 cells cultured in fresh mediates is far more extensive than in BHK cells. Pentose phosphate pathway. The cell specific flux of serum-free as well as serum-supplemented (2.5% FCS) medium and insect Sf-21 cells cultivated in glucose metabolites through the pentose phosphate fresh serum-free medium. All flux rates are presented shunt was determined as 0.0076 and 0.0082 nmol 5-l for BHK-21 BN40/90HK-1 cells cultured in 2.5% in Table 4.

between 0.12 and 0.17 nmol 5-l cells for Sf-21 insect cells (Table 3). The higher utilization rate of insect cells correlated with a more than two times higher glutamine concentration in insect cell medium in comparison to the mammalian cell medium formulation. Cell-specific utilization rates linearly increased with increasing glutamine concentration in the medium for insect cells. The initial glutamine utilization rates of mammalian cells cultured in serum-free medium were 1.2-1.5 times higher than those of cells cultivated in FCS-supplemented medium.

Glucose metabolism

160
TABLE 5a. Cell specific enzyme activities in nmol s- Fig. 3) Celldenzymes BHK-21 c13,2.5%FCS BHK-21 c13, serum-free BHK-21 BN49/90HK-1, 2.5%FCS BHK-21 BN49/90HK-1, serum-free BHK-21 pSVIL2, serum-free BHK BN49/90HK-1, 2.5%FCS chemostate culture, steady state CHO-K1 pMDIIIGPTR, 2.5%FCS CHO-K1 pMDIIIGPTR, serum-free CHO-K1 pMDIIIGPTR, protein-free Hybridoma 187.1, 3% FCS Hybridoma MAX16H5, serum-free Insect Sf-21, serum-free Insect Sf-9, serum-free Primary WI-38, 5% FCS Primary mouse liver cells Primary pig liver cells HK 0.24 0.30 0.22 0.24 0.31 0.19 0.22 0.30 0.25 0.13 0.12 0.43 0.45 0.13 0.96 0.52

NEERMANN AND WAGNER

lo- of enzymes involved in glucose metabolism for a number of cell lines (see also
PKF
0.81 1.05 0.76 0.82 1.13 0.45 0.88 1.24 0.69 0.61 0.49 1.23 1.42 0.58 3.85 1.87 PK 12.80 22.10 14.60 20.50 15.10 13.50 23.30 24.70 15.00 8.80 9.20 4.26 2.44 13.7 4.52 2.12 LDH 19.33 26.20 16.80 22.40 10.20 13.80 16.30 17.50 10.60 10.40 10.30 5.57 4.90 20.60 7.10 3.37 PEPCK n.d. n.d. n.d. n.d.
-

GPDH 0.31 0.45 0.25 0.26 0.43 0.17 0.29 0.42 0.27 0.14 0.12 0.45 0.55 0.23 0.30 0.29

PC n.d. n.d. n.d. n.d.

PDHC n.d. n.d. n.d. n.d.

If,
53.3 73.7 66.4 85.4 48.7 71.1 105.9 82.3 60.0 67.7 76.7 9.9 5.4 105.4 4.7 4.1

n.d.
-

n.d. n.d.

n.d.

n.d.
-

n.d.
-

n.d.
-

n.d.

ad.

0.10 0.11
-

0.55 0.65

act.

0.17 0.22

act. act.

0.08 0.09

The enzyme activities were determined spectrophotometrically from crude cell extracts as described in Materials and Methods. Values for mouse and pig liver expressed as tissue mass specific activities i nmol s- lo- mg. Each value represents the mean of at least five determinations. The standard deviation after e m r n propagation is 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 the respective glycolytic rates. For insect Sf-21 cells the cell specific pentose phosphate pathway flux rate was estimated at 0.0069 nmol s- accounting for 10.3%of the glycolytic flux. In contrast to other fluxes of the oxidative metabolism, the PPC flux was in the same order of magnitude in both insect and BHK cells. Glucose transport. The cell specific transport rates were linear during a 10-minute period of measurement a t 0.34 and 0.32 nmol s-l for BHK-21BN49190HK1cells grown in 2.5% FCS-containing medium and serum-free medium, respectively. Glutamine metabolism Glutumine oxidation. The cell specific rate of luta mine oxidation was found to be 0.0090 nmol s- F 10-6 for BHK-21 BN 49/90HK-1 cells cultured in 2.5% FCSsupplemented medium and 0.0115 nmol s-l lop6 for the same cell line grown under serum-free conditions (Table 4). This accounts for 18.0 and 18.5% respectively, of the total glutamine utilized. For insect Sf-21 cells the cell-specific rate of glutamine oxidation was 0.0171 nmol s- low6, representing 14.3% of the overall glutamine utilization (Table 4). Enzyme activities The maximal activities of a number of key enzymes of glycolysis, glutaminolysis and the TCC are given in Tables 5a and b for the cell lines examined. Glucose metabolism. It is striking that HK, the glycolytic entrance enzyme, exhibited in all cell lines the 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 pentose phosphate shunt displayed a maximum cell specific activity of 0.12-0.43 nmol s-l lop6,which is within the low range of HK. Moving along the glycolytic pathway the maximum enzyme activities increased, with PK and LDH showing

the highest activities. However, it should be noted that the divergence between HK, PFK, and PK was decisively smaller in insect and primary liver tissue cells than in CCLs. This divergences can be expressed by the pyruvate kinasehexokinase (PWHK) ratio, serving as a flux regulation index (Ifl): While Iflfell between 49 and 105 in CCLs, it ranged only from 4 to 10 in all insect cells and primary liver tissue cells investigated. Thus, the importance of HK for flux regulation seems to be higher in CCLs than in insect or primary liver tissue cells. It is interesting to note that also the WI-38 diploid cell had undergone relatively few passages (17 passages, 32-39 generations), it exhibited an enzyme activity pattern and a PWHK ratio (Ifl= 105)characteristic of cell lines which had been in culture much longer. Most important for the physiological behaviour of cells in culture seems to be the activity and/or presence of mediator enzymes between glycolysis and TCC, namely PDHC, PC, and PEPCK. In various cultured mammalian cells no activity of these three enzymes could be detected by the spectrophotometric assay, while in insect and primary liver tissue cells the same enzymes were clearly active (Table 5a). Glutamine metabolism. As was the case for glycolysis, the activity of Glnase, the entrance enzyme of glutamine metabolism, was lower in all cell lines examined by a fador of 3 to 5 compared to the activity of GDH, which operates immediately downstream of Glnase (Table 5b). This result suggests that glutaminase rather than glutamate dehydrogenase regulates the glutaminolytic flux. The activity of AlaAT, which converts glutamate into alanine, was comparable t o the Glnase-activity and lower than the GDH-activity in most cells investigated. Only in primary liver tissue cells AlaAT-activity was found to be distinctively higher than Glnase-activity (Table 5b). AspAT represented in all cell lines investigated the enzyme with the highest activity in the glutaminolytic pathway. In primary liver tissue cells the GDH activity appeared to be more dominant when compared to CCLs. At the

GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES TABLE 5b. Cell specific enzyme activity in nmol s - ' (see also Fig. 3)'
Celldenzymes

161

of enzymes involved in glutamine a n d TCC metabolism for a number of cell lines

cs
0.57 0.84 0.60 1.19 0.67 0.70 0.69 0.86 0.42 2.20 2.60 0.30 1.12 1.07

MDH (NAD')
27.70 33.70 24.50 25.30 20.60 16.90 31.50 28.60 14.20 4.12 3.72 12.50 8.50 11.31

GDH 1.21 2.05 0.80 1.16 0.83 0.34 0.69 0.82 0.54 3.10 1.54 0.35 5.67 11.72

Glnase
0.44 0.83 0.29 0.32

GS
0.31

&PAT
1.68 2.78 1.58 2.43 1.42 0.95 1.45 1.68 11 . 1 06 .4 1.29 0.57 13.67 5.81

AlaAT
0.16 0.15 0.20 0.22 0.16 0.30 0.15 0.08 0.12 0.20 0.25 0.18 5.58 3.54

BHK-21~13, 2.5% FCS BHK-21 c13, serum-free BHK-21 BN49/90HK-l, 2.5% FCS BHK-21 BN49/90HK-1, serum-free BHK-21 pSVIL2,2.5% FCS BHK-21 BN49/90, 2.5% FCS chemostate
culture

0.17 0.25

CHO-K1,2.5% FCS CHO-K1, serum-free Hybridoma 187.1,3% FCS Insect Sf-21, serum-free Insect Sf-9, serum-free Primary WI-38,5% FCS Primary mouse liver cells Primary pig liver cells

0.06 0.13 0.15 0.15 0.68 03 .4 0.12 0.52 0.73

0.05

0.09 0.39 0.36

2.31 2.18

'The enzyme activities were determined spectrophotometrically from crude cell extracts as described in Materials and Methods. Values for mouse and pig liver cells are expressed as tissue mass specific activities in nmol s-' lo-' mg. Each value represents the mean of at least five determinations. The standard deviation after error propagation is 10-14% in each case.

TABLE 6. Intracellular concentrations of glucose metabolites in t h e glycolytic pathway'

Celldmetabolites

Glc

Glc6P

Frc6P

PEP

PVr

Lac

BHK-21 BN49mOHK-1 . 2.5% FCS BHK-21 BN49/90HK-l, serum-free Insect Sf-21, serumfree
-~~~~ -~

3.2

2.8

0.04 2 0.02

0.34 0.32

? ?

0.14 0.15

0.06 ? 0.05 0.02 0.08

? ?

0.51 ? 0.30

18.7 % 7.2 19.5 ? 8.0 2.8

?

2.9 2 2.6
1.1? 1.0

0.04 -+ 0.02
0.12 -+ 0 8 .0

0.02 0.08

0.40

0.26

0.12 2 0.10

0.53 2 0.35

1.7

'All values are cell specific pools expressed in nmol 10 '. Despite high standard deviations it can be noted that glucose and lactate form the highest intracellular pools of the glycolytic pathway in both insect and BHK cells, with the lactate concentration higher than the glucose concentration. The intracellular lactate pool is significantly lower in insect than in BHK cells.

same time the GS activity in primary liver tissue was

4 times higher than the respective Glnase-activity, in-

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

dicating a flux from the TCC towards glutamine rather than vice versa. In contrast, CCLs and insect cells did DISCUSSION not show a high GS-activity. General aspects TCC. The activities of two representative TCC enzymes, CS and NAD+-linkedMDH, were determined. Glucose and glutamine serve as major energy sources The NAD+-linked malate dehydrogenase activity was for most in vitro cultivated cell lines (Lanks and Li, found to be very high in all cells investigated. In addi- 1988; Zielke et al., 1984; Reitzer et al., 1979; Wagner tion, CS activity, ensuring sufficient TCC flux, was et al., 1988). The glucose and glutamine metabolism of present in all cells. A NADP+-linked MDH-activity different cultivated mammalian and insect cell lines could not be detected in any CCL investigated. were investigated a t the early exponential growth phase in batch cultivation. Cell-specific glucose and Intracellular metabolite pools glutamine utilization and lactate formation rates were These results should be interpreted with caution. determined over relatively short time periods (5-10 The measurement of intracellular metabolite pools is hours). Due to the described decrease of those rates with not very sensitive, it often results in high standard deviations and is prone to artifacts during sample-han- increasing cultivation time these short time determinadling. Despite these limitations, however, the measure- tions might be one reason that the specific glucose and ment of intracellular metabolite pools affords a qualita- glutamine utilization and lactate formation rates were slightly higher than cited in the literature for mammative picture of a cell's metabolic patterns. The results shown in Table 6 reflect that in both lian cell batch cultures (Lin and Miller, 1992; Ogata et BHK and insect cells, glucose and lactate made up the al., 1993; Fitzpatrick et al., 1993; Sharfstein et al., greatest intracellular pools of all glycolytic metabolites. 1994). Higher utilization and formation rates may also In BHK cells the cell-specific lactate concentration (20 be due to higher overall glucose and glutamine concennmol was about 6 times higher than the cell spe- trations, different media composition, or culture syscific glucose concentration (3 nmol Considering tems. Regarding these points the differences of 0.01the low lactate production rate of insect cells it is not 0.06 nmol s-l in the cell specific utilization and surprising that the cell specific intracellular lactate formation rates are in good agreement with the specific

162

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

GLUCOSE AND GLUTAMINE METABOLISM IN CELL LINES

163

glucose and glutamine utilization and lactate formation rates reported by other authors. Furthermore, most enzyme activities determined in our work (see Fig. 3) were slightly higher than those reported by Fitzpatrick et al. (1993).However, these investigators performed their assays at 25"C, whereas we performed our experiments at physiological temperatures (37"C, pH 7.4, mammalian and 27"C, pH 6.5, for for insect cells).

Glucose metabolism Utilization, production, and flux rates Utilization and production rates. The cell specificglucose utilization rates were very similar in BHK and CHO cell lines (0.15-0.21nmol s-l BHK cells cultured in serum-free medium exhibited slightly higher glucose utilization rates than in serum-containing medium. The cell-specific glucose utilization rates for both hybridoma cell lines were determined a t 0.11 and 0.13 nmol s-l and respectively, and are consistent with maximum glucose utilization rates cited in the literature for other hybridomas (Petch and Butler, 1994;Fitzpatrick et al., 1993).In contrast, the initial cell-specificglucose utilization rates in insect cell lines grown under serum-free culture conditions were determined at 0.08nmol s-' and were significantly lower than those of mammalian cell lines. Since insect cells usually produced very little amounts of lactate, whereas mammalian cells characteristically displayed a high conversion ratio of glucose to lactate (Bedard et al., 1993)it already can be assumed that a high amount of glucose was completely oxidized. Initial cell-specific lactate roduction rates between 0.17 and 0.32 nmol s-l 10- were measured for mammalian cells, giving a B molar LadGlc-ratio between 1.31 and 1.56,which is consistent with data cited elsewhere (Hansen and Emborg, 1994;Ljunggren and Haggstrom, 1994;Lin and Miller, 1992).Moreover, this ratio was slightly higher for BHK cells cultivated under serum-free conditions (1.5) than for those cultured in serum-containing medium (1.3). The high cell-specific lactate production rates in mammalian cells indicate a high glycolytic flux with very little conversion of glycolytic metabolites into TCC intermediates. Furthermore, the specific molar lactatel glucose ratios point towards a higher glycolytic flux in cells cultured under serum-free conditions compared t o those cultivated in serum-supplemented medium. However, according to the findings of Zielke et al. (19801, up to 13% of the lactate production may be attributed to glutamine utilization. The amount of lactate derived from glutamine in serum-free and serum-supplemented cultures was not determined in our studies. Insect cells on the other hand only produced very little lactate. Therefore glucose must be converted mainly into other metabolites (Deutschmann and Jager, 1994).Thus, some different pictures of glucose metabolism and enzyme activities are expected to evolve for mammalian and insect cells. Flux rates. The cell-specific glycolytic flux in BHK21 BN49 /9OHK-1 cells cultured in FCS-supplemented medium was measured at 0.098 nmol s-' This value is very consistent with the cell specific-lactate considering a formation rate of 0.208 nmol s-l

10% standard deviation and the formation of a small fraction of lactate by glutamine or other substrates (Zielke et al., 1980).Assuming complete lactate formation via glucose, a glycolytic flux of 0.104nmol s-' cells should have been measured. In conclusion, the glycolytic flux accounted for about 62% of the total glucose utilized. At the same time, no conversion of glucose metabolites or pyruvate into the TCC could be detected, indicating that less than 0.4% of the glucose metabolized was transformed to TCC intermediates. This finding is not only consistent with the rates of glycolytic flux and lactate formation described above, but also with results obtained for hybridoma cell lines by other investigators (Fitzpatrick et a . 1993;Petch and Butl, ler, 1994).Estimation of the pentose phosphate pathway flux revealed a cell-specific rate of 0.0076 nmol s-l This corresponds to 4.8% of the total glucose utilized. This pathway supplies important precursors (Rib5P) of nucleic acids and is therefore absolutely essential for cell replication (Reitzer et al., 1980;Stryer, 1988).It was estimated that about 32% of the utilized glucose was metabolized in pathways different from glycolysis, TCC, and oxidative PPC (corresponding to a cell specific flux of 0.051 nmol s-' e.g., lipid metabolism and protein glycosylation were not investigated here. The cell-specific glycolytic flux in BHK-21 BN49/ 9OHK-1 cells cultivated under serum-free conditions was 0.127 nmol s-l (Table 4), which was consistent with the observed cell specific lactate formation rate of 0.260 nmol s-l low6. The glycolytic flux accounted for 76% of the total glucose utilized. As was the case for cells in FCS-containing medium, no transformation of glucose metabolites or pyruvate into the TCC could be detected (less than 0.36% of the utilized lucose). The cell specific PPC-flux was 0.0082nmol s - ~ which corresponds to 4 9 of the total glucose utilized. These .% data show that in BHK cells grown under serum-free conditions around 18% of the glucose consumed (cellspecific flux of 0.030nmol s-' was metabolized in pathways different from glycolysis, TCC, and oxidative pentose-phosphate shunt (e.g., lipid metabolism, protein glycosylation). Comparison of BHK cells cultivated under serumfree and serum-containing conditions shows that glycolytic flux was higher under serum-free conditions, as indicated by the higher LadGlc-ratio. However, the conversion rates of glucose metabolites into the TCC as well as the oxidative pentose phosphate pathway flux rates were very similar in both cases. This means that BHK cells in serum-supplemented culture utilized glucose a t a higher rate in pathways other than the ones addressed here, which may be one reason for higher specific mitotic rates observed in serum-containing media. The exact roles which FCS on the one hand and insulin and transfenin on the other may play in flux distribution remain unknown at this point, although a higher glucose utilization rate has been reported when insulin was present in growth media (Ryll et al., 1990).An increase of the PFK activity has been proposed for chicken embryo fibroblasts in the presence of insulin (Van Schaftingen, 1987). The flux data presented here are in good agreement with flux rates for hybridoma cell lines reported by Fitzpatrick et al. (1993) and Petch and Butler (19941,

164

NEERMANN AND WAGNER

but differ to some degree from the flux data published by Sharfstein et al. (1994), who applied 13C-NMRspectroscopy. For a murine B-lymphocyte hybridoma (CC9ClO)cell line a cell specific glycolytic flux of 0.127 and a PPC flux of 0.0048 nmol s-l 10 nmol s-l has been estimated by other authors (Petch and Butler, 1994). In contrast to our results, however, they additionally reported a cell specific glucose metabolite flux into the TCC of 0.0008 nmol 5-l Fitzpatrick et al. (1993) investigated another murine B-lymphocyte hybridoma (PCQXB1/2) cell line and estimated significantly lower cell s ecific flux rates (glycolytic flux of 0.063 nmol s-' 10-2 PPC-flux of 0.0022 nmols-1 , They also reported a glucose metabolite flux into the TCC, although at a cell specific rate of only 0.0001 nmol s-' These values are within the range of the flux rates obtained in our experiments for BHK-21 BN491 90HK-1 cells. In contrast, a hybridoma cell line examined by Sharfstein et al. (1994) by 13C-NMR-spectroscopy showed a cell-specific glycolytic rate of only 0.027 nmol s-l lop6and a PPC-flux of 0.0025 nmol s-' lop6. These values are considerably lower than those measured in our experiments. It should be noted, however, that I3C-NMRspectroscopy analysis requires very high cell densities and hence culture conditions which differ from those traditionally employed in radioactive labelling experiments. This may explain the discrepancies between the metabolic rates measured by Sharfstein et al. (1994) and those described here or reported previously. In previous publications (Fitzpatrick et al., 1993; Petch and Butler, 1994; Sharfstein et al., 1994) the sum of glycolytic flux, PPC-flux, and glucose metabolite flux into the TCC accounted for roughly 92 to 100% of the total glucose utilized. A previously investigated CHO cell line converted more then 97% of the consumed glucose into lactate (Donnelly and Scheffler, 1976). These relative values are higher than our findings for BHK-21 BN49/90HK-1cells. The difference might be explained by the fact that in these studies, in contrast to our experiments, cells were probably taken from the midexponential or late-exponential growth phases and medium supplemented with 10% FCS was used. It has been observed that glucose metabolism drastically changes with increasing culture time as evidenced by an increasing molar lactate/glucose ratio (Ljunggren and Haggstrom, 1994), indicating an increasing glycolytic flux. Comparison of insect and mammalian cell lines. The cell-specific glycolytic flux rate in insect Sf21 cells (serum-free conditions) was 0.067 nmol s-l representing 72% of the total glucose utilized. Despite this glycolytic flux extremely little lactate was produced. In addition, a relatively high turnover of glucose metabolites into TCC intermediates was detected by COz evolution. The cell specific rate was determined as which is close to the gl colytic 0.055 nmol s flux rate. The difference of 0.012 nmol s-l 10- 2can be partially explained by a standard error of around 10% and the channelling of glycolytic metabolites to other pathways, e.g., the production of alanine from pyruvate in the presence of an alanine dehydrogenase or alanine transaminase activity. Furthermore, small differences in the physiological states of the cells during the different experiments cannot be excluded. In conclusion, the results obtained show that the majority of glucose con-

sumed (60%)was converted into TCC intermediates.

T i significant difference between insect cells and hs

CCLs was confirmed by measuring the C02 evolution from high amounts of radiolabelled pyruvate added to the culture medium ( 5 g L-l). Using this method the cell-specificflux rate of yruvate into the TCC was estimated at 0.095 nmol s- 10 (Table 4), indicating that the pyruvate converting enzymes (probably PDHC) did not work a t their V,,, (maximum velocity) during glucose utilization. The lower glucose utilization and glycolytic flux rates in insect cells, compared to BHK cells, appear to be the economical rationale, when considering the high flux of glucose metabolites into the TCC and the resulting energy yield. Thus, the flux of glucose metabolites into the TCC is actually the most significant difference in metabolism between continuous insect and mammalian cells. Insect cells should be able to generate orders of magnitude more energy derived from glucose than CCLsl (expressed in ATP units), considering an energy yield of 36 mol ATP produced per mol glucose oxidized via TCC compared to only 2 mol ATP per mol glucose oxidized via glycolysis. The glucose flux via the oxidative PPC was determined to a cell specific rate of 0.0069 nmol s-' in insect cells, accounting for 7.4% of the total glucose consumed (Table 4). In summary, about 20% of the utilized glucose (0.019 n m ~ l s - ~ is metabolized in pathways not investigated here, such as lipid metabolism and protein glycosylation. The absolute flw rate of glucose into the oxidative branch of the PPC in insect cells was very similar to those found in BHK cells, indicating that both cell types need to regenerate about the same amount of NADPH or require about the same amount of Rib5P-units as nucleic acid precursors. This assumption is supported by similar mitotic rates estimated for both cell lines, indicating that the PPC might determine the rate of the cell division. Insect Sf-21 cells showed a mitotic rate of 0.030 h-l, which is similar to that of the BHK-21 BN49/90HK-1 cells cultured under serum-free conditions (0.028 h-'). However, the mitotic rates of insect cells were below the mitotic rate of BHK cells cultured in FCS-supplemented medium (0.037 h-'). This indicates that the rate of cell division was not limited by the rate of ATP production, which is in accordance to results recently published by Renner et al. (1994) who reported a 2-fold higher mitotic rate of CHO cells expressing recombinant cell division promoting proteins such as cyclin E. It can be expected, therefore, that a higher ATP production rate results in a better productivity of cells a t a stable mitotic rate. Interestingly, both insect and BHK cells cultivated under serum-free conditions metabolized only about 20% of the total glucose consumed in pathways not investigated here (lipid metabolism, protein glycosylation), while for BHK cells grown in serum-containing medium this part accounted for 32% of the glucose utilized. However, to what extent glucose influences cell growth via biomass production or pathways other than glycolysis, PPC, or TCC, remains to be determined.

Y -

Rate limiting steps: enzyme activities In order to identify possible rate-limiting steps in glucose metabolism, the maximum in vitro activities of key

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165

enzymes and the rate of glucose transport into cells were measured. The maximum activities of key enzymes can indicate a maximum flux for a desired pathway (Ardawi and Newsholme, 1982,1983).All enzymes and pathways investigated are summarized in Figure 3. Hexokinase. By comparing all glycolytic enzyme activities (Table 5a), it becomes obvious that in all cell lines analyzed here, HK exhibited the lowest cell specific activity. For all CCLs investigated in this work, the maximum specific HK activity was quite close to the relevant specific glucose consumption rates (Tables 3 and 5a). It has been previously suggested for in vitro cultivated primary rat thymocytes that PFK is the regulatory enzyme of glycolysis (Brand, 1985; Newsholme et al., 1987).However, PFK activities determined here for CCLs and insect cells were significantly above the HK activities (2.5-4-fold, Table 5a) and above the calculated glycolytic flux (Table 41, which is in agreement with data reported for a murine hybridoma line (Fitzpatrick et al., 1993). Based on literature values of K M for HK and PFK, it can be concluded that HK rather than PFK does not work at its maximum velocity, due t o a relatively low ATP affinity (KM[ATP]= 0.2-1.2 mmol 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 that hexokinase can easily be inhibited by GlcGP, with %-values in the low micromolar range (Wilson, 1985; Fiechter and Gmiinder, 1989).The rate of glucose transport into the BHK-21 BN49/90HK-1 cells for both serum-supplemented (0.34 nmol s-* and serum-free culture (0.32 nmol s-l modes was found to be distinctively higher than glucose utilization or the glycolytic flux rates determined. Although glucose utilization and glycolytic flux rates were measured over several hours, while glucose transport was determined over 10 minutes, it appears very unlikely that glucose transport is rate limiting. This assumption is confirmed by previously reported glucose transport rates for murine Blymphocyte hybridomas (cell-specific glucose import: 0.267 nmol s-l Fitzpatrick et al., 1993)and chicken fibroblasts (cell-specific glucose import: 0.357 nmol s-' Kleitzien and Perdue, 1975, assuming 0.55 mg protein per lo6 cells; Fitzpatrick et al., 1993), which are similar to the data obtained here for BHK cells. These data point towards a bottleneck in glucose metabolism at the hexokinase reaction particularly for CCLs and were supported by qualitative trends from intracellular metabolite pool measurements (Table 6). Glucose and lactate exhibited higher intracellular concentrations than the other intermediates of glycolysis. Another striking similarity of all cell lines examined was a general increase in cell-specific enzyme activity, the closer the enzymes are disposed towards the endpoint of glycolysis, indicating a metabolic sink. For example, PK and LDH showed significantly higher activities than HK and PFK in CCLs. However, these differences in activity between enzymes of early and late steps of glycolysis were not as large in insect and primary cells. This becomes evident when examining the PWHK activity ratios termed flux indices (Ifl): While the Iflof insect and primary liver cells ranges between 4 and 10, the Ifl of CCLs ranges between 49 and 106

(Table 5a). Previously reported PWHK ratios were near 13 for lymphocytes (Ardawi and Newsholme, 1982)and between 26 and 560 for a murine hybridoma cell line depending on the physiological state of the cells (Fitzpatrick et al., 1993).The dramatic differences in IR indicate that glycolysis in insect and primary cells is better balanced and provides more regulative possibilities than in continuous mammalian cells. Indeed, PWHK activity ratios have already been shown to be useful as a n indication of the relative importance of these two enzymes in glycolytic regulation (Ardawi and Newsholme, 1982; Fitzpatrick et al., 1993). Hence, hexokinase seems to play a far more important role for adjusting the glycolytic rate in CCLs than in insect or primary liver cells. Taken together, these data indicate very strongly that the hexokinase activity may determine the overall rate of glucose metabolism in most CCLs. Similar findings or conclusions have been previously described for a murine B-lymphocyte hybridoma line (Fitzpatrick et al., 1993), for Aspergillus niger (Torres, 1994) and for a breast cancer cell line (Hugo et al., 1992). Mediation between glycolysis and TCC. A decisive difference between insect and primary liver cells on the one hand and CCLs on the other is the activity and presence of branching enzymes from glycolysis into the TCC, such as PDHC, PC, and PEPCK. While these enzymes were active in insect and primary liver cells, they were not in CCLs. The results here fully confirm the findings of the flux experiments stated above. A specific protein kinase has been reported to be part of the PDHC, which can phosphorylate PDH at a serine residue. The phosphorylation of PDH can inhibit its activity leading to the accumulation of pyruvate (Randle et al., 1984; Fiechter and Gmiinder, 1989). A lack of PC activity was also confirmed for a murine hybridoma cell line by employing 13C-Nh4R spectroscopy (Mancuso et al., 1994). In conclusion, insect and primary liver cells can convert a t least a considerable part of their glucose metabolites into TCC intermediates, while transformed mammalian cells probably can not. This would explain, a t least to some extent, the different metabolic behaviour between these two cell types as far as lactate production is concerned. However, it is 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 the glycolytic flux observed in insect cells (Table 4), but the activities of PK and LDH, which are potential substrate competitors, are still higher. It is not known whether a regulated glycolysis or a well-balanced NADH/NAD ratio in insect or primary cells inhibits lactate formation and promotes flux towards the TCC. It has been hypothesised that a high NADH/NAD ratio prevails in the cytoplasmic space of in vitro cultivated mammalian cells, leading glycolysis towards lactate production in order to regenerate NAD (Lanks, 1986). To what extent the NADH/NAD ratio influences the activities of the enzymes PDHC, PC, or LDH remains unknown. PPC. The cell-specific GPDH activity was always nearly identical to the respective HK, except for primary liver cells (Table 5a). This activity is most likely high enough not to limit the PPC flux, since the measured flux via PPC was significantly lower than the GPDH activity (Table 4).

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Change of enzyme activities. An additional interesting aspect of the enzyme activities measured here is that all glycolytic enzymes in BHK and CHO cells cultivated under serum-free conditions exhibited a higher cell specific activity than in the same cells cultured in serum-supplemented medium. This could explain the higher glucose consumption rates observed in all BHK and CHO cells grown under serum-free conditions (Table 3) and the higher glycolytic flux in BHK cells cultivated under serum-free conditions (Table 4) compared t o the same cells cultured in the presence of serum. However, it is not known whether the absence of certain FCS components, cell stress, or the presence of insulin and transferrin were responsible for the slightly higher enzyme activities under serum-free culture. All glycolytic enzyme activities determined in BHK cells grown under serum-containing conditions in chemostat culture were lower at steady state than those of the same cells during early exponential growth in batch fermentations (Table 5a). However, the relative enzymatic activities remained nearly the same, indicating that the cell-specific enzyme activities change with the physiological state of the cell and depend on the culture conditions (see also Fitzpatrick et al., 1993). The fundamental metabolic patterns, however, remained stable during nutrient limitation. Enzyme activities, therefore, may to some extent reflect the physiological state of the cells. Glutamine metabolism In addition to its function as an essential amino acid, glutamine acts as an important energy source when deaminated into the TCC intermediate a-ketoglutarate (2-oxoglutarate) (Reitzer et al., 1979; Glacken, 1988). a-Ketoglutarate can be completely oxidized to COz via TCC or partially oxidized to aspartate, alanine, or lactate, which are often secreted into the medium (Wagner et al., 1988). Hence, glutamine metabolism comprises a special metabolic network termed glutaminolysis and consisting of up to eight different pathways (Haggstrom, 1991). Utilization rates. The initial cell-specificglutamine utilization rates of cultured mammalian cells ranged between 0.05 and 0.08 nmol s-* (Table 3). As was the case for glucose, the cell-specific glutamine utilization rate of cells grown under serum-free conditions was higher than those for the same cells grown in serum-supplemented medium. Insect cells showed initial cell-specific lutamine utilization rates of 0.12 to 0.18 nmol s-ll0- 8, depending on the concentration of glutamine in the medium, which was adjusted to 8 or 12 mmol L-', respectively. This reveals that the cells can adapt their glutamine utilization rate to the medium supply and indicates that there is a t least no apparent internal rate limitation within the concentration range commonly used in insect cell culture. Flux rates. Experiments using radiolabelled [U''C]-glutamine showed that 18.0 and 18.5%of the utilized glutamine were oxidized to CO, in BHK cells cultivated under serum-containing and serum-free conditions, respectively (Table 4). Insect cells oxidized to CO, 14.3% of the consumed glutamine. However, the absolute amount of glutamine oxidized was higher in insect than in BHK cells, since the cell-specific glutamine uti-

lization rate was about 2 times higher (Table 3). Hence, insect cells should be able t o produce far more ATP than mammalian cells, since they channel significant amounts of glucose and glutamine metabolites into the TCC. When using [U-14Cl-glutamine it is not possible to determine the exact pathway of glutamine metabolism. This is because glutamine can only be partially oxidized in the TCC. Nevertheless, the glutamine oxidation rates in both BHK and insect cells demonstrate the importance of glutamine for energy supply in both cell types. Jenkins et al. (1992) reported a cell specific glutamine oxidation rate of 0.0064 nmol s-l for a murine B-lymphocyte hybridoma line, accounting for 36% of the total glutamine utilized. This observation is within the range of our results, considering that in their medium the glutamine concentration was only 2 mmol L ' resulting in a lower cell-specific glutamine -, consumption rate. About the same extent of glutamine oxidation was also reported for Chinese hamster fibroblasts (Donnelly and Scheffler, 1976). Rate limiting steps: enzyme activity. With respect to the cell-specific enzyme activities involved in glutaminolysis it can be concluded that in all mammalian cells examined here, AspAT showed the highest activity followed by GDH, supporting the suggestion that glutamine is partially oxidized via TCC and subsequently metabolized through the aspartate transamination pathway, branching out of the TCC via transamination of oxaloacetate. This hypothesis is supported by MDH and CS activities. While MDH exhibited extremely high activities, CS, which operates immediately downstream of MDH, showed 2- to 3-fold lower activity than AspAT (Table 5b). Therefore, it is highly probable that a significant portion of glutamine carbon is directed through the aspartate transaminase pathway in CCLs. Sharfstein et al. (1994) concluded from data obtained from analysis of the metabolism of hybridoma cells that the TCC flux from KGA towards OAA is higher than that from citrate to KGA, which would be consistent with the obtained enzyme pattern. Moreover, besides being essential for protein synthesis, aspartate may also be an essential intermediate for pyrimidine synthesis and of the malate/aspartate shuttle, which is important for the transfer of reducing equivalents from cytosol t o mitochondria. In insect cells GDH exhibited the highest activity, followed by AspAT. Furthermore MDH activity was not nearly as high as in CCLs and CS activity was higher than AspAT activity, suggesting that, compared to mammalian cells, a lower amount of glutamine carbon passes through the aspartate transaminase pathway in comparison to mammalian cells. In summary, the enzyme pattern seems t o be better balanced in insect cells than in CCLs. In all cell lines investigated the Glnase activity was lower than GDH and AspAT activity, This stands in contrast to results reported by Jenkins et al. (1992), who found the Glnase activity to be higher than the respective GDH activity in a murine hybridoma line. However, the Glnase activity was at least threefold higher than the specific glutamine utilization rate in all cell lines examined, which is confirmed by results reported from other authors (Jenkins et al., 1992). It has been postulated that glutamine transport rather

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than enzyme activity is rate-limiting in a murine hybridoma cell line (Fitzpatrick et al., 1993; Jenkins et al., 1992). However, Jenkins et al. (1992) admit that glutaminase may be the subject of additional intracellular regulation as described by Zielke et al. (1984) and Ardawi and Newsholme (1982), and a likely candidate for regulating on the rate of glutaminolysis. The lowest activities of the enzymes of glutaminolysis examined in CCLs and insect cells are exhibited by GS and AlaAT. GS in particular was shown to be stimulated by using glutamine-deficient media (Gawlitzek et al., 1995). In contrast, during the early phases of the growth cycle of mammalian and insect cells, glutamine is found in high concentrations in the medium and is mainly consumed and not synthesized, thus requiring only a low GS activity. This becomes clear when GS activity is examined in primary liver cells. Although liver cells must have a high capacity for glutamine synthesis due to their specific function in the intact organism, glutamine must also be produced as a result of a likely insufficient supply of external glutamine. It therefore is no surprise that GS activity was found t o be higher than Glnase activity in primary liver cells. It can be assumed that AlaAT mainly works in a regulative way, disposing a surplus of glutamate or TCC metabolites. It has been hypothesized that the glutamine carbon exists in the mitochondria1TCC as the metabolite malate, which can be converted into alanine, lactate, or aspartate in the cytoplasm (McKeehan, 1982).This process could increase the cytoplasmic NADWNAD ratio, resulting in an important contribution to drive lactate production via glycolysis for NAD restoration (Lanks, 1986; Petch and Butler, 1994). This theory could be applied presuming that an active malate shunt exists and an electron transfer system-like the NAD+linked MDH-is present in the cytosol, as described in the literature (Lanks, 1986; Mancuso et al., 1994; Sharfstein et al., 1994; Zimmerle and Alter, 1993). At the same time, the glycerol-phosphate or malate/aspartate shuttles have t o show very low or no activity. The extremely high MDH activity observed in all mammalian cells compared to that in insect cells could be an indicator for its cytoplasmic or even malate shunt activity, causing a high cytoplasmic NADWNAD ratio. This could partially explain why the lactate formation via glycolysis in CCLs is higher than in insect cells. This hypothesis, however, depends on two assumptions and does not take into account the activities of enzymes connecting glycolysis and TCC. Activities of glutaminolytic enzymes were higher in mammalian cells cultivated under serum-free conditions than in the same cells cultured in the presence of FCS. This correlates with a higher specific glutamine consumption and glutamine oxidation rates in serumfree cultured cells and indicates that enzyme activities can be influenced by the culture conditions.

transformed mammalian cells only glutamine contributes to the TCC significantly. Based on the data presented here this is prevented by very low or totally repressed PDHC, PC, and PEPCK activities and a high LDH activity. Activities of all enzymes are higher when mammalian cells are cultured under serum-free conditions compared to serum-supplemented culture. TCC intermediates can be converted into many amino acids, which certainly requires a constant replenishment of TCC metabolites. However, an increase in TCC oxidation of glucose metabolites in CCLs would promote a more efficient glucose utilization and a simultaneous lactate reduction. Furthermore, an interrelation between glutaminolysis and glycolytic lactate formation via cytoplasmic NADWNAD pools is suggested. In conclusion, a better understanding of the intersection of glucose and glutamine metabolism, as well as flux determination of NADH-transfer between cytosol and mitochondria may be necessary for a successful attempt to alter the metabolism of mammalian cells by directed metabolic management or design.

ACKNOWLEDGMENTS
The authors thank their colleagues of the Cell Culture Techniques department of GBF, M. Ackermann, Dr. V. Jager, Dr. Patole, and A. Kobold for supplying M. insect cells, J. Hammer for amino acid analysis, and Dr. K Scharfenberg for providing recombinant CHO cells. We thank Dr. Manfred Wirth for kindly contributing the recombinant BHK-21 BN49/90HK-1 cell line and Dr. Stephanos Grammatikos for critically reading the manuscript.

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Conclusions
A general point emerging from this study is the similarity in many aspects of the glucose and glutamine metabolism in many cultured mammalian cell lines. Hexokinase appears to determine the rate of glycolytic flux in various CCLs. While in insect cell lines both glucose and glutamine are metabolized via the TCC, in

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