Download Carbohydrate metabolism in cultured animal cells

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
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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).
The authors' work has been
s u p p o r t e d by t h e N u f f i e l d F o u n d a t i o n and the Medical Research
Council ( P r o g r a m m e Grant G977/997).
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