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Bioscience Reports I, 669-686 (1981) Printed in Great Britain 669 C a r b o h y d r a t e m e t a b o l i s m in c u l t u r e d a n i m a l c e l l s Review Michael 3. MORGAN and Pelin FAIK Department of Biochemistry, University of Leicester, Leicester LEI 7RH, U.K. Carbohydrate metabolism has been extensively studied in plants, animals, and micro-organism% and the pathways are well established. Animal cell culture techniques have improved dramatically during the past two decades such that animal ceils may now be manipulated almost as readily as micro-organisms. This review will c o n c e n t r a t e on those areas of carbohydrate metabolism in which the study of ceils in c u l t u r e is leading to fresh insights and new horizons. Particular emphasis will be placed on exploring the relationships between cell g r o w t h and t h e role of carbohydrates as energy sources and biosynthetic precursors. Carbohydrate metabolism may be regulated rapidly in response to the concentration of enzyme substrates, e f f e c t o r s , and inhibitor% or more slowly by events at the level of gene expression. A thesis will ]be develped that our comprehension of such processes will em anat e from and be f a c i l i t a t e d by an investigation of carbohydrate metabolism J[n cultured animal cells. T h e U t i l i z a t i o n o~ Carbohydrates In order to determine which carbohydrates can be utilized it is essential to use c a r b o h y d r a t e - f r e e media which can be supplemented with the carbohydrate under investigation (Faik & Morgan~ 1977a). T h e e a r l y l i t e r a t u r e contains a number of reports in which these c r i t e r i a were not met and which are, t herefore, unreliable. A furt her complication is the definition of growth: cells continue to divide for some time even a f t e r transfer to medium without added carbohydrate. (Sell division ceases (presumably) when energy stores and biosynthetic precursors become exhausted. Growth should, t h e r e f o r e , be measured through a number of cell divisions and be compared with appropriate c a r b o h y d r a t e - d e f i c i e n t control cultures. Only a limited number of carbohydrates support the growth of most cells in culture (Table 1). Glucose, mannose~ fructose, and galactose are utilized by most cell types. Glucose and mannose appear to be metabolized rapidly and permit similar rapid growth rates, whereas growth on fructose and galactose is much slower. Growth on disaccharides such as maltose and on polysaccharides such as starch and glycogen has been reported (Rheinwald & Green, 197#; Dahl et al., 1976; Faik & Morgan, 1976), but serum contains a number of saccharidases and when the activity of these has been removed the growth-supporting activity of the saccharide has been lost (Rheinwald & Green, 197#; 5cannell & Morgan, 1980). 9 1981 The Biochemical Society 670 MORGAN Table i. & FAIK Growth substrates of cultured cells A. Compounds supporting growth D-fructose (i-395-799) ; D-galactose D-glucose (i-i0); D-mannose (1-3,5-7,9) Hexoses: Pentoses: (1-3,5-7,9); D-ribose, D-talose (2); D-xylose (2,8) eellobiose, melibiose, turanose (2); maltose (5); Disaccharides: trehalose (2,5) maltotriose (5) Polysaccharides: amylose, dextrin, glycogen (5) Trisaccharides: Alcohols: sorbitol (2) G-I-P (2,6); G-6-P (2,5,7,9) Sugar phosphates: B. Compounds not supporting growth Heptoses: glucoheptose, mannoheptulose, sedoheptulose (5) D-galactose (1,2,5,6); D- or L-fucose, L-sorbose (2,5); L-glucose (1,5); L-rhamnose (2,5,6); D-gulose, D-allose, D-altrose (2); 2-DOG (2-deoxyglueose), 3-O-MeG (3-O-methylglueose) (3); D-fructose, D-mannose (6); D-rhamnose (5) Hexoses: D- or L-arabinose (2,5,6); D-lyxose (2,6); D-ribose (],2,5,7-9); D-xylose (1,5,6,9); L-xylose (5,8); 2-deoxy-D-ribose (5); D-talose (5) Disaccharides: cellobiose, mellibiose, turanose (5,6); lactose (1,5-7,9); maltose (1,4,6,7,9,10); palatinose (5); sucrose (1,2,5-7,9); trehalose (6,10) Pentoses: Trisaccharides: melizitose (5,6); raffinose (1,5,6) arabic acid, arabinogalactan, lactobionic acid, mucic acid, stachyose (5); glycogen (i); inulin (6); starch (4,10) Polysaccharides: D- or L-arabitol, m-inositol, mannoheptitol, ribitol, sorbitol (5); i-erythritol, galactitol (5,6); mannitol (2,5,6); xylitol (2,5) Alcohols: ~- or 8-methylglucoside (2,5); ~- or a-methylxyloside, 8-methylgalactoside (5); e-methylmannoside (5,6); eseulin, salicin (6) Sugar acids: L-ascorbic acid, D-gluearic acid, D-glucoheptonic acid (5); D-galacturonic acid, D-glucuronic acid (2,5,6); D-gluconic acid (2,5) Glycosides: D-galactolactone (5,6); D-glucuronolactone, ribonolactone (5) Lactones: CDP-mannose (6) D-glucosamine (3,5); mannosamine, acetylmannosamine Nucleotide sugars: Hexosamines: (6) glucoheptuno-l,4-1actone, CARBOHYDRATE METABOLISM IN ANIMAL CELLS 671 Gal-6-P, mannitol 6-P (6); G-6-P, DL-~-glycerophosphate (5,6); 8-glycerophosphate (5); phosphoglyceric acid (2) Phosphates: 6-carbon compounds: citric acid (2,5,7,8) 5-carbon compounds:, ~-ketoglutaric acid (5) dihydroxymaleic acid, dihydroxytartaric acid, oxaloacetic acid, tartaric acid (5); fumaric acid, malic acid, succinic acid (2,5,7,9); D-erythrose (1,2,5) 4-carbon compounds: compounds: dihydroxyacetone, glyceraldehyde (5); glycerol (2,5,9); lactic acid (2,5,7,9); pyruvic acid (2,5-7,9) 3-carbon 2-carbon compounds: Amino acids: acetic acid (2,5) Ala, Arg, Asp, Glu, GIu-NH2, His, Isoleu, Ser, Thr, Val (7,9) Misce22aneous: glucuronamide, triacetylglucal (5) Numbers in parentheses refer to references: (i) Harris & Kutsky, 1953; (2) Eagle et al., 1958; (3) Melnykovych & Bishop, 1972; (4) Rheinwald & Green, 1974; (5) Burns et al., 1976; (6) Dahl et al., 1976; (7) Faik & Morgan, 1976; (8) Demetrakopoulos et al., i[977; (9) Faik & Morgan, 1977a; (i0) Scannell & Morgan, 1980. Thus, most cells in culture have a very limited ability to utilize c a r b o h y d r a t e s and do not display the metabolic versatility of the animals from which they originate. A number of laboratories have reported the isolation of variants which are able to metabolize a wider range of carbohydrates than their parents and which provide a useful system for studying regulation (see below). The initial steps in the metabolism of any carbohydrate convert it 1:o an intermediate of the main metabolic pathways (Fig. 1), and it is appropriate next to consider the regulation of these pathways. G1 ycol ysi s Changes in the rate of glucose utilization following the application of various stimuli have frequently been observed in cultured cells. Thus an increase in aerobic glycolysis has been observed following lymphocyte proliferation (Wang et al., 1976) and when fibroblasts are exposed to agents known to stimulate proliferation, such as serum, g r o w t h f a c t o r s , and virus t r a n s f o r m a t i o n (Fodge & Rubin, 1973; Diamond et al,, 1978). These changes have been ascribed to alterations in the activity of either the glucose transport system and/or enzymes of glycolysis, and are considered to play an important role in the regulation of cell growth. 672 MORGAN & ,.g & & FAIK ~ iJ w w <: .-I o o ,_ [ ILl (n >X rr m =< := ,.- o ~ m o u ~- >. m z o == i z =< =< CARBOHYDRATE METABOLISM IN ANIMAL CELLS 673 Transport Information about the transport of carbohydrates is largely limited to hexose t r a n s p o r t ( m a i n l y g l u c o s e and its analogues) and in p a r t i c u l a r to c h a n g e s in the rates of hexose transport following t r e a t m e n t with various stimuli. A number of the earlier studies are difficult tO assess since the overall rate of metabolism (rather than t r a n s p o r t ) was m e a s u r e d and f r e q u e n t l y sub-optimum conditions applied. Discussion will be restricted to t h o s e reports where the f o l l o w i n g m i n i m a l r e q u i r e m e n t s applied: l) Rates of uptake were m e a s u r e d over short time intervals. 2) Substrates were added at s a t u r a t i n g concentrations and/or kinetic constants were determined. 3) Transport was distinguished from the further metabolism of the substrate. A distinction should be drawn between 'uptake' and 'transport'. 'Uptake' will be defined as the transfer of substrate into the cell and its metabolism, and 'transport' as the transfer of unmodified substrate a c r o s s the cell m e m b r a n e . Thus the uptake of 2-deoxyglucose (2-DOG) is the sum of its transport and phosphorylation by kinase. The two most frequently studied glucose analogues are 2-DOG, which is subject to phosphorylation and further, but slower, metabolism ( S c h m i d t et al., 1974), and 3-O-methylglucose (3-O-MeG), which appears not to be metabolized, and has been shown to compete with glucose for the same carrier system (Venuta & Rubin, 1973). The Km for 2-DOG uptake has been reported by a number of laboratories (Table 2) and there is good agreement for a value of approximately 2 raM. Values reported for 3-O-MeG uptake are rather higher, around 5-10 mM (Table 2). Colby and Romano (197#) reported differences in the kinetics of glucose inhibition of 2-DOG uptake and 3-O-MeG uptake which indicate that whereas glucose and 2-DOG share the same transport system, which can transport 3-O-MeG, 3-O-MeG may also be t r a n s p o r t e d by a s t e r e o s p e c i f i c a l l y similar, but different, system. Mullin et al. (19g0) have also concluded that 3-O-MeG and glucose are transported by different systems. Most reports agree that there are no significant changes in the Km for glucose transport following a variety of stimuli, including serum (Kletzien & Perdue, 1974c), transformation by chemicals (Oshiro & DiPaolo, 1973) or tumour viruses (Romano & Colby, 1973; Venuta & Rubin, 1973), or changes in glucose concentration (Salter & Cook, Fig. I. Major metabolic reactions of carbohydrate metabolism. Abbreviations used: PRPP synthetase, phosphoribosyl pyrophosphate synthetase, EC 2.7.6.1; HK, hexokinase, EC 2.7.1.1; GPI, glucose-phosphate isomerase, EC 5.3.1.9; PFK, phosphofructokinase, EC 2.7.1.11; FDPase, fructose diphosphatase, EC 3.1.3.11; GPK, 3-phosphoglycerate kinase, EC 2.7.2.3; TOK~ triokinase~ EC 2.7.1.28; PK, pyruvate kinase, EC 2.7.1.40; PC, pyruvate carboxylase, EC 6.4.1.1; PEPCK, phosphoenoipyruvat~ carboxykinase, EC 4.1.1.32; RK, ribokinase, EC 2.7.1.15. 67# MORGAN Table 2. & FAIK Rates of glucose utilization and uptake of glucose analogues Glucose used* Reference I0.3 70.8 0.75 2.0 10 (1)C (1)S (2) (3)C (3)S 2~DOG uptake Km~ Vmax* Reference 3-O-MeG uptake Km# Vmax* Reference Chick cells 2.0 16.7 33.2 14 90 (4)N (4)T (5)C (5)S 3.5 5.0 1.0 19 37.5 0.6 1.9 (4)N (4)T (6)N (6)T Primate cells (including human cells) 15.7 19.7 11.3 (7)C (7)S (8) 2.0 3.3 4.7 1.9 1.9 0.57 4.4 18 26 (9) (10)N (10)T (II)C (II)S 10.0 5.2 5.3 (13)N (13)T (16)N (16)T (22)S (22)C (23)N (23)T 4.7 5.3 4.8 14 23 (10) (II)C (II)S 10.4 12.5 11.2 (24)T (24) (24)N Rodent cells 24 11.6 77.8 70 1oo 18.8 34.7 1.1 4.9 29.4 139 406 5.4 19.2 100 (12) (13) (14) (15) (15) (16)N (16)T (17)c (17)S (18) (19) (19) (20)N (20)T (21) 0.75 0.75 0.8 0.9 0.8 4.9 2.0 2.5 1.0 7.0 4.7 124 18.3 9.1 71 125 *nmol/min/mg cell protein (recalculated where necessary on the assumption cells equal 1 mg c~tosolic protein) %ms C, control cells; S, stimulated cells; N, normal cells; T, transformed cells that 10 7 References: (i) Bissell et al., 1972; (2) Fodge & Rubin, 1975; (3) Rubin & Koide, 1975; (4) Kletzlen & Perdue, 1974a; (5) Kletzien & Perdue (1976); (6) Venuta & Rubln, 1973; (7) Carlson & Suttie, 1967; (8) Reitzer et al.~ 1980; (9) Salter & Cook, 1976; (i0) Corkey et al.~ 1981; (II) Reward et al. 9 1979; (12) Rolland & Hongslo, 1978; (13) Bose & Zlotnick, 1973; (14) Bustamante & Pedersen~ 1977; (15) Morgan~ Bowness & Faik, unpublished d a t a ; (16) Gregory & Bose, 1977; (17) Diamond et al., 1978; (18) Bailey et al., 1959; (19) Schwartz & Johnson, 1976; (20) Pouyssegur et al., 1980; (21) Ratanaka, 1976; (22) Walum & Edstrom~ 1976; (23) Plagemann et al., 1975; (24) Colby & Romano~ 1974. 1976) or during the cell cycle (Plagemann et al., 1975). This indicates that there is no qualitative change in the nature of the glucose transporter. There is disagreement about whether or not there are changes in Vma x following such stimuli (which would suggest changes in the number of glucose carriers) and in particular whether or not such changes are an essential feature of the transformed cell. The controversy really concerns the interpretation of the experimental data, whatever the initiating stimulus. Experimentally one observes an CARBOHYDRATE METABOLISM IN ANIMAL CELLS 675 increase in the uptake of glucose or its analogues following transiormation. This could be due to changes in either transport per se, or m e t a b o l i s m , or both. C ol by and R o m a n o (1974) measured the i n t r a c e l l u l a r c o n c e n t r a t i o n of 2-DOG, 2-deoxygl ucose-6-phosphat e (2-DOG-6-P), and 3-O-MeG in mouse 3T3 cells, in DNA- and RNAtransformed 3T3 ceils, and in a flat r e v e r t a n t of transformed 3T3 (.'ells. The concentration of the unphosphorylated species was the same in each cell type, attaining an intracellular concentration equivalent to t h a t of t h e i n c u b a t i o n m e d i u m ; d i f f e r e n c e s in uptake could be accounted for by differences in the rat e and ext ent of phosphorylation. T h e y c o n c l u d e d that changes in hexose uptake following malignant transformation r e f l e c t e d changes in the rat e of hexose metabolism r a t h e r than t r a n s p o r t per se (see below). Kletzien and Perdue ( 1 9 7 4 a , b , c ) s t u d i e d t h e uptake of 2-DOG and 3-O-MeG in chick embryo fibroblasts growing at di fferent rates and in chick embryo f i b r o b l a s t s t r a n s f o m e d by a t e m p e r a t u r e I s e n s i t i v e mutant of Rous s a r c o m a vi r us . T h e r e was no change in the Kin, but the Vma x increased substantially (Table 2). The change in Vma x was shown to be dependent on both post-translational and post-transcriptional events (Kletzien & Perdue, 1975a,b). The increase in 2-DOG uptake was shown to be due to an increased rat e of phosphorylation which could be o b s e r v e d in whole ceils, but not in cell e x t r a c t s (Kletzien & Perdue, 1974a). Indeed the r a t e of phosphorylation of 2-DOG was not only similar in e x t r a c t s of all cell types, but was t~_ to 6-fold higher t h a n t h e r a t e observed in whole cells. It was concluded that a reaction prior to phosphorylation and part of the transport process is responsible for the altered r at e of sugar transport. Phosphorylation was shown to be dependent on the rat e of entry of 2-DOG, i.e. transport was the rate-limiting step. Kletzien and Perdue (197tta) pointed out that this was not necessarily true in the work reported by Romano and Colby (1973). However, it is well known that hexokinase is p o w e r f u l l y i n h i b i t e d by glucose-6-phosphate (G-6-P) (Colowick, 1973) and by 2-DOG-6-P (Barban, 1962). This inhibition would go some way towards explaining the di fferent rates of phosphorylation in vivo and in vitro, observed by Kletzien and Perdue, since 2-DOG-6-P is rapidly c o n c e n t r a t e d in whole cells (up to l0 raM) whereas such concentrations would only be achieved in homogenates a f t e r very long incubation periods. An inhibition of 2-DOG uptake, due to an inhibition of hexokinase by G-6-P, has been demonstrated in glucosephosphate-isomerase(GPI)deficient Chinese-hamster ceils (Morgan & Faik, 1980; Pouyssegur et al., 1980). These mutants accumulate G-6-P while growing on glucose (up to i00 nmol/mg of protein) and have a greatly reduced uptake of 2-DOG compared to the wild-type ceils. When the GPI mutants are starved for glucose, the levels of G-6-P fall and 2-DOG transport increases to that found in the wild-type cells (Pouyssegur et al., 1980; F;aik & Morgan, unpublished). F u r th er evidence that transport is regulated at least in chick ceils comes from studies with the oncostatic methylase inhibitor, 5'-deoxy5'-S-isobutyl-thioadenosine (SIBA) (Pierr~ & Robert-G6ro, 1981). SIBA was shown to p r e v e n t t h e i n c r e a s e in 2-DOG uptake, following transformation, without af f ect i ng phosphorylation in vivo or in vitro. While the case for regulation of hexose transport per se may still 676 MORGAN & FAIK Table 3. Variation in the activities of some glyeolytic enzymes Specific activity (nmol/min/mg Enzyme Wild-type (I) Glycolytic mutant (1) Normal Transformed protein) Control Stimulated Ilexokinase 25.7 31.1 170 (2) 48 (3) 317 (2) 86 (3) 61.8 (4) 58.3 (4) Phosphofructokinase 37.4 71.3 50 (2) 188 (3) 171 (2) 319 (3) 31.6 (5) 108.4 (5) 6.9 Phosphog]ycerate kinase 3464 4470 (2) 12750 (2) Pyruvate kinase 841 555 1340 (3) 2550 (3) Lactate dehydrogenase 2951 3505 2280 (3) 4480 (3) Glucose-6-P dehydrogenase i00 6-phosphogluconate dehydrogenase - 106.5 45 (3) 128 (2) 76 (3) II0 (2) 78 (2) 25 (3) 91 (2) 24 (3) (i) Morgan & Faik, 1980; (2) Gregory & Bose, 1977; (4) Kletzien & Perdue, 1974e; (5) Diamond et al., 1978. (3) Singh et al., 1974b; not be proven (Romano, 1976), it is fairly convincing. However, reported rates of glucose utilization are often in excess of hexose transport (Table 2) and it is still uncertain that transport is always (if ever) the rate-limiting step in glycolysis. Glycolytic enzymes The changes in glucose transport discussed above have frequently been invoked to explain changes in growth rates and glucose utilization f o l l o w i n g t r a n s f o r m a t i o n or o t h e r s t i m u l i . There is convincing evidence that changes in the activities of specific glycolytic enzymes are also effected by such stimuli (Table 3), Romano & Colby (1973) concluded that changes in 2-DOG uptake were due to changes in the a c t i v i t y of hexokinase. An increase in mitochondrial hexokinase acitivity has been observed in rat hepatoma cells compared to normal liver and this hexokinase appears to be less sensitive to inhibition by G-6-P ( B u s t a m a n t e & Pedersen~ 1977). Similarly 9 Wohlhueter and Plagemann (1981) have shown that pH-dependent changes in 2-DOG uptake are due to changes in phosphorylation rather than transport. Fodge and Rubin (1973) attributed an increase in glycolysis following infection of chick embryo ceils with Rous sarcoma virus to an increase in the activity of phosphofructokinase. A similar increase in the activity o f phosphofructokinase has been observed in extracts of 3T3 ceils following serum stimulation (Schneider et al.~ 1978) and following stimulation of lymphocytes with concanavalin A (Wang et a l . 1980). Activation of phosphofructokinase is an a t t r a c t i v e proposition since this e n z y m e has long been thought to play an important role in the regulation of glycolysis in animal tissues. A similar activation of phosphofructokinase9 through the agency of fructose-2~6-bisphosphate~ CARBOHYDRATE METABOLISM IN ANIMAL CELLS 677 has been proposed to explain the glucose-induced stimulation of glycolysis in pancreatic islet cells (Malaisse et al., 1981). Gregory & Bose (1977) were unable to show an activation of phosphofructokinase (or other glycolytic enzymes) on transformation of normal rat kidney c e l l s with K i r s t e n s a r c o m a virus and a t t r i b u t e d the changes in g l y c o l y t i c a c t i v i t y to changes in hexose transport. Singh et al. (197/ta,b) in an extensive study analysed normal and Rous-sarcomav i r u s - i n f e c t e d chick e m b r y o cells for the activities of glycolytic enzymes and intermediates. They determined crossover points from the increases and decreases in the levels of glycolytic intermediates and concluded that the increase in glucose flux was due to increases in the a c t i v i t i e s of hexokinas% phosphofructokinas% and pyruvate kinase (Table 3; and Singh et al., 197Lta). Further analysis confirmed that these enzymes were present at elevated levels in extracts of the transformed cells (Singh et al., 1974b). Thus although it is clear that g l y c o l y s i s is regulated~ the basis is obscur% as is any connection between growth regulation and glycolytic activity (see below). The use of mutants with alterations in the activities of the glycolytic pathway might well aid the elucidation of the controls. Pentose phosphate pathway The two major products of this pathway are NADPH and ribose5-phosphat% the former of major importance for reductive synthesis and t h e latter for nucleic acid synthesis. In liver the generation of NADPH may be of greatest importance and the activity of the pentose pathway may be regulated in accord with lipogenesis (Nepokroeff et al., 1974). How is the pentose pathway regulated in cultured cells? The supply of ribose-5-phosphate for nucleic acid synthesis may be of more importance than the supply of NADPH for actively growing cells. Smith and Buchanan (1979) have shown that one of the earliest measurable responses in serum-stimulated fibroblasts is an increase in the synthesis of phosphoribosylpyrophosphate following from an increase in the flux from glucose to ribose-5-phosphate. Thus the regulation of the pentose phosphate pathway may be an important factor in growth control and deserves further investigation. Reitzer et al. (1980) have shown that at least one-third of the ribose-5-phosphate synthesized in HeLa ceils growing on glucose and almost all of the ribose-5-phosphate g e n e r a t e d during g r o w t h on fructose is required for nucleic acid synthesis. They further suggest that the supply of ribose-5-phosphate is growth-limiting in fructose-grown cells and that the only essential f u n c t i o n of sugar metabolism is to provide carbon in the pentose cycle. This is rather an extreme view which appears to ignore the role of sugar metabolism in providing amino sugars and nucleotide sugar products. Nevertheless, these data support the concept that the essential role of carbohydrates is to provide carbon for biosynthesis and not for energy (see below). Williams (1980) has claimed that the reaction sequence as usually represented in textbooks requires a number of modifications. These i n c l u d e d i f f e r e n t i n t e r m e d i a t e s , and a d d i t i o n a l e n z y m a t i c steps catalysed by a new epimeras% a new phosphotransferase, and aldolase; there is no role for transaldolase. There is a cruciat difference in the role of GPh in the classic scheme the formation of G-6-P from the 678 MORGAN & FAIK rearrangement reactions is entirely dependent on GPI, but in Williams's modification G-6-P can also be formed by a reaction catalysed by transketolase. Mutants of Chinese-hamster cells lacking GPI activity (Morgan & Faik, 1980; Pouyssegur et al., 19g0) have been used to i n v e s t i g a t e the reaction sequence in these cells. If the Williams pathway operates, then extracts of GPI-deficient cells should still be able to synthesize G-6-P from added ribose-5-phosphate. This has been shown not to be the case: such extracts convert ribose-5p h o s p h a t e only to fructose-6-phosphate and it has therefore been concluded that the modified pentose pathway does not operate in these ceils (Morgan, 1981). Tricarboxylic acid cycle and respiration There have been few investigations of the control of this pathway in ceils in culture (see below), but recent reports of the isolation of a succinic-acid-dehydrogenase-deficient mutant (Soderberg et al., 1977) and a N A D H - c o e n z y m e - Q - r e d u c t a s e mutant (De Francesco et al., 1976) d e m o n s t r a t e that the biochemical genetic analysis of these mitochondrial functions is a viable proposition. Genetic analysis of a number of these respiration-deficient cells has so far revealed seven complementation groups (Soderberg et al., 1979). Complementation groups I, II, and VII have all been shown to have defects in complex I of the e l e c t r o n - t r a n s p o r t chain (Breen & Scheffler, 1979). The mutant in group IV is defective in succinate dehydrogenase (Soderberg et al., 1977) and the mutant in group V has a defect in mitochondrial protein synthesis (Ditta et al., 1977). The analysis of the remaining complementational groups is yet to be reported. Complex I of the e l e c t r o n t r a n s p o r t chain ( N A D H / c o e n z y m e Q reductase) contains around 16 d i f f e r e n t polypeptide chains. Biochemical and genetic analysis of these and other respiration-deficient ceils should provide insight into mammalian mitochondrial functions and biogenesis. The Provision of Energy It has generally been accepted that glucose is the major energy source in ceil-culture media, and that energy is derived from the conversion of glucose to pyruvate (glycolysis) and the oxidation of pyruvate to carbon dioxide by the operation of the TCA cycle (Paul, 1965). Many cells in culture, however, appear to convert glucose almost exclusively to lactic acid (aerobic glycolysis) with only a small p e r c e n t a g e being oxidized (Warburg, 1926; Aisenberg, 1961). This apparent lack of respiratory a c t i v i t y has been extensively investigated, often in the expectation that an understanding of the phenomenon would go some way to explaining the basis of malignancy (Eigenbrodt & Glossmann, 1980). Recent results (Donnelly & Scheffler, 1976; Zielke et al., 1976, 1978; Reitzer et al., 1979) suggest a different i n t e r p r e t a t i o n of the phenomenon, demonstrating that the cell in culture is metabolically adept since it can use a variety of energy s o u r c e s and t h a t r e s p i r a t o r y a c t i v i t y is not suppressed. A r e s p i r a t i o n - d e f i c i e n t mutant of Chinese-hamster cells has been described (Scheffler, 197#; Donnelly & Scheffler, 1976) which obtains its energy solely by glycolysis and excretes large amounts of lactic CARBOHYDRATE METABOLISM IN ANIMAL CELLS 679 acid. The mutant oxidizes [1-t#C]glucose to t#CO2, but is unable to oxidize [6-t#C]glucose to I#CO 2, Even the wild-type ceils oxidize [6-t#C]glucose very slowly and it was shown that the oxidation of glucose via the TCA cycle contributed only a tiny amount to the energy needs of the cell. However, it was then shown that other oyidizable substances present in the medium (principally glutamine) were utilized even in the presence of glucose (Donnelly & Scheffler, 1976) and contributed as much as #0% of the energy needs of the cells. R e s p i r a t o r y inhibitors such as rotenone completely inhibit respiration in wild-type cells, but do not kill them (within 2# h), provided that the glucose supply is adequate. Under these conditions t h e g l y c o l y t i c r a t e increases to provide the extra ATP normally derived from the oxidation of glutamine. Thus in the short term glycolysis can provide all the energy needs of the ceil. The a n a l y s i s of glycolysis-deficient mutants (Pouyssegur et al., 1980; Morgan & Faik, 1980) has shown th&t cells can obtain their energy needs entirely from respiration. One mutant (Pouyssegur et al.~ 1980) is deficient in GPI and the other (Morgan & Faik, 1980) is d e f i c i e n t in both GPI and phosphoglycerate kinase (PGK). Both mutants grow on glucose almost as well as the wild-type cells, but n e i t h e r p r o d u c e s lactic acid. The oxidation of [1-t#C]glucose is normal whereas the oxidation o~ [6-1#C]glucose is negligible. Both mutants are rapidly killed by the application of respiratory inhibitors such as cyanide and oligomycin at concentrations which do not kill the wi[ld-type cells~ and both oxidize glutamine extensively (Pouyssegur, personal communication; Morgan, unpublished results). Thus both these ceils rely on respiration for their energy needs and appear to be able to grow as readily as their glycolysis-competent parents. One of the m u t a n t s ( P o u y s s e g u r et al., i980) has been shown to be just as capable of forming tumours in experimental animals as the wild-type cells, implying that there is no necessary connection between the rate of glycolysis and malignancy. Further evidence that glutamine is a major energy source of cells in culture comes from studies on glutamine utilization in HeLa cells growing on glucose, galactose, or fructose (Reitzer et al., 1979) and human diploid cells growing in high and low concentrations of glucose (Zielke et al., 1976, 1978). The utilization rates of glucose and glutamine appear to have a reciprocal relationship, glutamine utiliz a t i o n being lower in the presence of high (5 raM) glucose and greater in the presence of low (50 pM) glucose, although growth rates were essentially indentical. Glutamine was shown to contribute up to 30% of the energy requirements of the cell (in good accord with the data of Donnelly & Scheffler, 1976). In the presence of fructose or g a l a c t o s e , s u b s t r a t e s which are only slowly metabolized via the glycolytic route, HeLa ceils derive more than 9896 of their energy needs via glutamine oxidation, and even on glucose almost 5096 of the energy requirements are met by the oxidation of glutamine (Reitzer et alo, 1979). A further factor to be considered is the operation of the m a l a t e a s p a r t a t e s h u t t l e (or other NADH shuttle systems) which enables cytosolic NADH formed by glyceraldehyde-3-phosphate dehydrogenase to be oxidized by the mitochondrial respiratory chain. The operation of the m a l a t e - a s p a r t a t e shuttle in ascites cells has been shown to 680 MORGAN & FAIK contribute 13-48% of the total ATP yield from glucose metabolism (Greenhouse & Lehninger, 1976). For Chinese-hamster, human diploid, and HeLa cells, and probably for the majority of cells in culture, it may be concluded that either glycolysis or respiration can satisfy the energy requirements and that m usual cell-culture media a combination of the two is used. It is clear, then, that many ceils do not oxidize glucose completely (the r e a s o n f o r this is still obscure) and that t here is no necessary relationship between the rate of glycolysis and growth control. There may be a relationship between energy provision, from whatever source, and growth control, but this remains to be investigated. It should be e m p h a s i z e d t h a t n e i t h e r glucose (or another suitable hexose) nor glutamine can be eliminated completely from culture media; both are essential precursors in biosynthetic pathways. The distinction must be kept very clearly in mind if the essential role of these compounds, which may alter following malignant change, is not to be obscured by t h e i r i n t e r c h a n g e a b l e role as energy sources. Thus, although the glycolytic mutants (Morgan & Faik, 1980; Pouyssegur et al., 1980) utilize glutamine for their energy needs, they cannot grow in the absence of a s u i t a b l e precursor of sugar phosphates. The place of glutamine as a respiratory fuel has not yet been extensively investigated. Other compounds including glutamate, o x a l o a c e t a t e , pyruvate, and atanine can be oxidized (Morgan & Bowness, unpublished data). Thus a number of oxidizable substrates may be able to substitute for glutamine in cell-culture media. Carbohydrate Metabolism and D i f f e r e n t i a t i o n Differentiated cells It has been appreciated for some time that the composition of culture medium a f f e c t s the expression of d i f f e r e n t i a t e d functions and indeed the ability of d i f f e r e n t i a t e d cells to grow in vitro (Rizzino et al., 1979). In many instances this has been shown to be due to serum factors, especially hormones and growth factors. The carbohydrate source can also have a selective infiuence: some tissues are charact e r i z e d by their metabolic versatility; the liver, for example, can m e t a b o l i z e l a c t a t e and o x a l o a c e t a t e since it carries out gluconeogenesis. Medium c o n t a i n i n g such substrates would, t h e r e f o r e , be selective for liver-like cells. It should also be possible by altering the car b o h y d r at e source to select cell lines from d i f f e r e n t i a t e d tissue and tumours which are abie to metabolize these carbohydrates. Bert ol ot t i ( 1 9 7 7 a ) has isolated variants of d i f f e r e n t i a t e d rat hepatoma ceils which p r o l i f e r a t e in medium in which oxaloacetic acid is substituted for glucose. The variants express the gluconeogenic enzymes ( f r u c t o s e diphosphatase and phosphoenolpyruvate carboxykinase) essential for the synthesis of phosphorylated sugars which are themselves essential for growth in the absence of exogenous hexose. In addition, they also e x p r e s s some liver-specific functions. D i f f e r e n t i a t e d variants have been isolated from d e d i f f e r e n t i a t e d rat hepatoma cells by selection in glucose-free medium supplemented with oxaloacetic acid ( D e s c h a t r e t t e e t al., 1 980) . T h e y h a v e been shown to contain a number of liver-specific functions including gluconeogenic enzymes, liver aldolase, CARBOHYDRATE METABOLISM IN ANIMAL CELLS 681 and serum albumin. The pleiotropic nature of this change suggests t h a t a m o d i f i c a t i o n has occurred in some function that regulates several liver genes. S o m a t i c cel l hybrids of d i f f e r e n t i a t e d rat h ep ato ma cells and mouse lymphoblastoma cells (Bertolotti, 1977b) were unable to grow in glucose-free medium when initially isolated, i.e. the d i f f e r e n t i a t e d functions were extinguished. However, it was possible to isolate d i f f e r e n t i a t e d segregants a f t e r selection in glucosef r e e medium. Those selected in oxaloacetic acid medium expressed f r u c t o s e diphosphatase and phosphoenolpyruvate carboxykinase, whereas t h o s e s e l e c t e d in d i h y d r o x y a c e t o n e m e d i u m c o n t a i n e d f r u c t o s e diphosphatase and triokinase. These results show that re-expression of these enzymes is not necessarily under coordinate control, in agreement with the finding in Chinese-hamster ceils (Faik & Morgan, 1980; see below). Such ceils should provide a useful model system for studying the regulation of gluconeogenesis. Teratocarcinomas Avner et al. (1977) have shown that the differentiation of the PCC3/A/1 t e r a t o c a r c i n o m a into cartilage, keratin, lipid, muscle, nerve cells, etc. is dependent on the presence of either glucose or mannose in the culture medium. In the absence of glucose, mainly endoderm is formed. A number of differentiation variant cell lines were obtained by c u l t u r i n g t h e t e r a t o c a r c i n o m a ceils in medium in which other c a r b o h y d r a t e s were substituted for glucose. Thus, by varying the carbon source it may be possible to isolate cell lines which correspond to t h o s e occurring at di f f er e nt stages of the normal in-vitro diff e r e n t i a t i o n sequence. Cell lines Cell lines should contain the same genetic information (even if it has been subjected to r e a r r a n g e m e n t s ) as the animal from which they were derived. Assuming that inactive genes can be r e - a c t i v a t e d , it should be possible to isolate variants that re-express enzymes necessary for growth on what are usually considered non-utilizable carbohydrates. For example, ceils lining the small intestine synthesize a lactase (usually only in young animals) and are able to metabolize lactose. Although cell lines contain lysosomal acid-B-galactosidase, they are unable to grow on lactose. Since t here is no permeability barrier to lactose (Faik & Morgan, 1977a), the cell lines, presumably, are unable to hydrolyse the disaccharide. Medium containing lactose would be selective for lactose-utilizing variants which might express a gut-like lactase. Variants of Chinese-hamster ovary cells have been described which grow on lactose in addition to glucose (Faik & Morgan, 1977b). The b i o c h e m i c a l analysis of these variants is incomplete, but there is preliminary evidence that the lactose + ceils synthesize a lactase that is similar in properties to the lactase found in Chinese-hamster gut (Faik, unpublished da t a ) . If this is confirmed it will provide a system in which two cell lines differ only in the ability to express a single gene: analysis of such a system should yield important information about the regulation of gene expression. Variants able to grow on 682 MORGAN & FAIK o t h e r d i s a c c h a r i d e s such as sucrose (Faik & Morgan, t977b) and maltose (Scannell & Morgan, 19g0) have been isolated, but not fully characterized. The isolation of pentose-utilizing variants has been reported by two laboratories (Faik & Morgan, I976; Hoffee et al., 1977). The ribose + ceils isolated by Faik and Morgan (t977b) grow on xylose but not on arabinose and can be placed into two classes. One class differs from its wild-type parent not only in its ability to utilize ribose and xylose, but also in its inability to excrete lactic acid while growing on glucose. This variant is deficient in both GPI and PGK (Morgan & Faik, 1980) and has been described earlier. The second class of ribose + variant differs from the wild-type cell only in its ability to grow on ribose and xylose. The wild-type and both types of ribose+ cells are able to accumulate ribose from the medium and contain the enzymes of the classical pentose phosphate pathway and a ribokinase. The activities of the pentose-phosphate-pathway enzymes are elevated about 2-fold in the ribose + variant% but this does not seem sufficient to explain the ribose + phenotype (Faik & Morga% unpublished data). Indeed it may be more pertinent to examine why the parental cells are unable to grow on ribose (ribose toxicity has been reported: Demetrakopoulos & Amos, 1976). One further property of the ribose + GPI-PGK- mutant is its inability to grow on mannose; indeed mannose is toxic during growth on either glucose, fructose, galactose~ or ribose (Faik & Morgan, 1977b). Fructose diphosphate accumulates in these ceils (up to l0 mM) within one hour of transfer to mannose and may itself be the toxic agent since mannose+ revertants do not accumulate fructose diphosphate (Morgan, in preparation). Hybrids of wild-type and ribose + ceils retain the ribose + phenotype (Morgan et a l , t9g0), and thus in these cells the ability to grow on ribose is a dominant characteristic. The ribose + ceils have a different morphology when transferred from glucose to ribose and vice versa. Indeed, all the carbohydrate variants of Chinese-hamster ceils that have been isolated (Faik & Morgan, I977b, t9g0; Scannell & Morgan~ i980) exhibit morphological c h a n g e s on different carbohydrates. Similar morphological changes have been observed when wild-type cells are transferred from glucose to o t h e r carbohydrates (Cox & Gesner, 1965; Amos et a l , 1976; 3ohnson & Schwartz, t976) and in other pentose + cells (3argiell% 1980). The biochemical basis of these changes is not established, but it seems likely that the cause is an alteration in the glycoproteins and/or glycolipids of the cell membrane resulting from changes in 'the pool of sugar-derivative precursors (Amos et a l , 1976). The ability to reversibly, but stably, alter cell morphology may provide a useful s y s t e m for s t u d y i n g the regulation of glycoprotein and glycolipid biosynthesis. P e n t o s e - u t i l i z i n g variants of Novikoff hepatoma ceils have been isolated in medium in which glucose was replaced by either ribose, xylose, arabinose, or deoxyribose (Hoffee et a l , 1977; 3argiell% 1978). Subsequent analysis showed a significant degree of cross-utilization, although non-cross-utilizing ribose + variants have also been isolated (3argieil% 1978). An increase in ribokinase activity may be associated with the ribose + phenotype (3argiell% 1980), and the location of ribokinase within the cells (either membrane-bound or free) may also CARBOHYDRATE METABOLISM IN ANIMAL CELLS 683 be important. In hybrids of these ribose + cells and the parental cells, the ribose + phenotype is first extinguished and then re-expressed. In this instance, then, the ribose phenotype is recessive (Silnutzer & 3argiello, 1981). The ability to grow on l a c t a t e should depend on the ability to perform gluconeogenesis (see earlier). L act at e-ut i l i zi ng variants of C h i n e s e - h a m s t e r o v a r y c e l l s have been isolated (Faik & Morgan, 1977b) and shown to incorporate l a c t a t e into cellular material (Faik & Morgan, 1980). The wild type is able to accumulate l a c t a t e , but does not appear to be able to incorporate it. The l a c t a t e cells have a higher PEP carboxykinase activity than the wild type, but the activity is unchanged on glucose or l a c t a t e (Faik, unpublished data) while the level of glucose-6-phosphatase appears to increase when the cells are shifted from glucose to l a c t a t e . Thus, in these cells as in hepatoma cells (Bertolotti, 1977b) t her e appears to be no coordinate expression of these gluconeogenic enzymes. Media containing carbohydrates other than glucose are, t h e r e f o r e , selective for cells able to express the enzymes essential for the metabolism of t h e s e s u b s t r a t e s . D i f f e r e n t i a t e d cells can be selected for and maintained, and cell lines can be isolated in which previously unexpressed functions are activated. The analysis of these cells should provide an insight into the control of differentiation. Concluding Remarks The study of carbohydrate metabolism in cultured cells is providing a fresh insight into its regulation in vivo and its relationship to growth control. The a b i l i t y to i s o l a t e w e l l - c h a r a c t e r i z e d mutants is a p o w e r f u l t o o l , which has already yielded rich dividends with the promise of more to come. The analogy with the progress of prok a r y o t i c b i o c h e m i c a l g e n e t i c s , with its emphasis on carbohydrat e metabolism, is striking. It is perhaps not folly to predict that the b i o c h e m i c a l genetic analysis of carbohydrate mutants will have an i m p o r t a n t i m p a c t on our understanding of the regulation of both c a r b o h y d r a t e metabolism and gene expression in mammals. Acknowledgements This review was written during the tenure of a Nuffield Foundation Science Research Fellowship (MJM). 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