Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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Cloning and expression of heterologous proteins in animal cells 73

Cell metabolism and its

contr ol in culture

Paola Amable and Michael Butler

4.1 Introduction

Metabolism, the sum of all chemical transformations taking place in a cell,

is a highly coordinated network of activities in which many multi-enzyme

systems and pathways are involved. The purposes of this network can be

summarized as follows:

(i) to obtain chemical energy by capturing solar energy or degrading

energy-rich nutrients from the environment;

(ii) to convert nutrient precursor molecules into the characteristic macro-

molecules of the cell;

(iii) to polymerize monomers into macromolecules (proteins, nucleic

acids, and polysaccharides);

(iv) to synthesize and degrade biomolecules required for specialized

cellular functions, such as membrane lipids, intracellular messengers,

and pigments.

The network operates through a series of enzyme-catalyzed reactions

that constitute the metabolism. Each of the consecutive steps in a metabolic

pathway brings about a speciﬁc chemical change, usually the removal,

transfer, or addition of a particular atom or functional group. The pre-

cursor is converted into a product through a series of metabolic intermedi-

ates called metabolites. The term intermediary metabolism is often applied

to the combined activities of all the metabolic pathways that interconvert

precursors, metabolites, and products of low molecular weight.

Some metabolic pathways are linear, some are cyclic, and some are

branched, yielding multiple end products from a single precursor or

converting several precursors into a single product.

Metabolic pathways are regulated at several levels within a cell. The

most immediate regulation is by the availability of substrate. A second

type of rapid control is allosteric regulation by a metabolic intermediate or

coenzyme (an amino acid or ATP, for example) that signals the cell’s

internal metabolic state. In multicellular organisms the metabolic activities

of different tissues are also regulated and integrated by growth factors and

hormones that act from outside the cell (Nelson and Cox, 2004).

Cells, removed from animal tissues or whole animals, will continue to

grow if supplied with nutrients and growth factors, allowing single cells to

act as independent units that are able to divide by mitosis. Cell lines can

live in vitro for a ﬁnite time (ﬁnite cell lines) or indeﬁnitely (permanent

cell lines). Permanent cell lines may continue growth in a culture vessel

until limited by some parameter associated with the mode of culture. The

limitation for growth in a culture may arise from nutrient depletion,

accumulation of metabolic byproducts or lack of surface for anchorage-

dependent cells.

Multiple culture passages can be established by transfer of cells from a

high density growth-limited culture into fresh medium. In this way,

growth of cells can be maintained for prolonged periods in culture.

Normal animal cells have an intrinsic ﬁnite growth capacity but some cells

acquire a capacity for inﬁnite growth and such a population can be called

an established or continuous cell line. This requires the cell to go through

a process called transformation, which causes the cells to lose their

sensitivity to the stimuli associated with growth control. Transformed

cells may lose their anchorage dependence and often show some chromo-

some fragmentation. This genetic state is referred to as aneuploidy, which

means that there is slight alteration from the normal diploid state. The

transformed cells have a high capacity for growth in relatively simple

growth media and without the need for growth factors (Butler, 2004).

4.2 Energy sources

In contrast to plant cells, which normally get their cellular energy from

photosynthesis, animal cells need a carbohydrate source, usually glucose,

and the amino acid glutamine. The catabolism of these substrates allows

the production of two coenzymes (ATP and NADH – nicotinamide

adenine dinucleotide), which are essential for maintaining the viability of

the cells. These coenzymes can be used for the maintenance, metabolism

and/or for the synthesis of particular desired products (Wagner, 1997).

The nature and concentration of carbon and nitrogen substrates in the

culture medium affect both the energy status and metabolism of the cells.

Medium generally contains both a carbohydrate (normally glucose) and a

range of amino acids to satisfy the cellular growth requirements (Butler,

2004). Lipids are also important, not only as an energy source, but also as

precursors for the synthesis of the cellular membrane components.

As a consequence of transformation, cells in culture assume a modiﬁed

metabolism when compared with the same cell in the organism of origin.

For example, the consumption rates of glucose and glutamine are very

high initially but decline throughout a normal batch culture, as does the

speciﬁc growth rate. These high uptake rates exceed the cellular need for

both precursors and energy, and result in increased ﬂuxes in glycolysis and

glutaminolysis, which in turn leads to overﬂow metabolism and formation

of byproducts such as lactate, ammonium, and alanine (Doverskog et al.,

1997).

4.2.1 Glucose

Glucose is a monosaccharide sugar that functions as an important fuel for

energy metabolism in all mammalian cells. It is also a remarkably versatile

76 Animal Cell Technology

precursor, capable of supplying a huge array of metabolic intermediates

for biosynthetic reactions. In animals and vascular plants, glucose may be

stored (as a polysaccharide such as glycogen or starch), oxidized to a

three-carbon compound (pyruvate) via glycolysis to provide ATP and

metabolic intermediates, or oxidized via the pentose phosphate cycle

(PPC) to yield ribose 5-phosphate for nucleic acid synthesis and reduced

nicotinamide adenine dinucleotide phosphate (NADPH) generation for

reductive biosynthetic processes.

The cytoplasmic membrane of mammalian cells is impermeable to

glucose, and therefore the uptake is by means of transport proteins located

in the plasma membrane. In the majority of mammalian cells the glucose

transport mechanism is facilitated diffusion. This is saturable, bidirec-

tional, and driven by the concentration gradient across the cell membrane.

Once glucose enters the cell cytoplasm, it is rapidly phosphorylated by

hexokinase to glucose-6-phosphate. This enzymatic reaction leaves low

amounts of free glucose in the cytoplasm, stimulating the transport of

more glucose in the uptake direction. Six different isoforms of glucose

transporters have been isolated in different tissues and designated GLUT1,

GLUT2, GLUT3, GLUT4, GLUT5, and GLUT7 (Craik and Elliot, 1979;

Kayano et al., 1988; Carruthers, 1990; Gould et al., 1991; Burant et al.,

1992; Kahn, 1992; Pessin and Bell, 1992; Waddell et al., 1992).

In the cytosol, glucose-6-phosphate is converted by two main pathways:

glycolysis and the PPC.

Glycolysis occurs completely in the cytosol and does not require O

.It

produces a small amount of ATP, NADH, and the three-carbon com-

pound pyruvate. The sequence of reactions is shown in Figure 4.1

Four molecules of ATP are formed per glucose molecule during

glycolysis, catalyzed by phosphoglycerate kinase and pyruvate kinase.

However, early in the glycolytic pathway, two ATP molecules are

consumed: one by the addition of a phosphate residue to glucose in the

reaction catalyzed by hexokinase and another by the addition of a second

phosphate to fructose 6-phosphate in the reaction catalyzed by phospho-

fructokinase-1. Thus glycolysis yields a net of only two ATP molecules

per glucose molecule.

The overall chemical equation for this ﬁrst stage of glucose metabolism

is:

þ 2NAD

þ 2ADP

þ 2P

! 2C

þ 2NADH þ 2ATP

The PPC allows the generation of NADPH reduction equivalents

required for cell anabolism, and ribose 5-phosphate molecules for the

synthesis of nucleic acids. Alternatively, ribose 5-phosphate can also be

generated or transformed into fructose 6-phosphate or glyceraldehyde

3-phosphate, providing metabolic ﬂexibility to the cell, in order to balance

the ﬂuxes through these pathways. The ﬂux through the PPC is related to

the nucleic acid requirements for DNA duplication or RNA transcription,

and could probably be controlled by the cell cycle (Wagner, 1997).

In the presence of oxygen, however, pyruvate formed in glycolysis is

transported into mitochondria, where it is oxidized by O

to CO

and

O in a series of oxidation reactions, collectively called the tricarboxylic

Cell metabolism and its control in culture 77

acid (TCA) cycle, citric acid cycle, or Kreb’s cycle (Figure 4.2). These

reactions generate 8 NADH, 2 GTP (guanosine 59-triphosphate; inter-

changeable with ATP), and 2 FADH

(1,5-dihydro-ﬂavin adenine di-

nucleotide). When NADH and FADH

are further metabolized through

Glucose

Glucose 6-phosphate

Fructose 6-phosphate

HEXOKINASE

Fructose 1,6-biphosphate

ATP

ADP

PHOSPHOGLUCOSE

ISOMERASE

PHOSPHOFRUCTOKINASE 1

TRIOSE

PHOSPHATE

ISOMERASE

GLYCERALDEHYDE

3-PHOSPHATE

DEHYDROGENASE

ATP

ADP

Glyceraldehyde 3-phosphate

(2 molecules)

ALDOLASE

1,3-Bisphosphoglycerate

(2 molecules)

2 ADP

2 ATP

PHOSPHOGLYCERATE

KINASE

3-Phosphoglycerate

(2 molecules)

PHOSPHOGLYCEROMUTASE

2-Phosphoglycerate

(2 molecules)

ENOLASE

Phosphoenolpyruvate

(2 molecules)

2 ADP

2 ATP

PYRUVATE KINASE

LACTATE DEHYDROGENASE

Pyruvate

(2 molecules)

ALDOLASE

2 NAD 2 P

⫹

2 NAD

⫹

2 NADH 2 H⫹

⫹

2 NADH

2 H⫹

⫹

Lactate

(2 molecules)

Figure 4.1

The glycolytic pathway by which glucose is degraded to pyruvate.

78 Animal Cell Technology

the oxidative phosphorylation and GTP is converted into ATP, the TCA

cycle renders a ﬁnal amount of 25 ATP molecules per glucose molecule,

far outstripping the ATP yield from glycolysis. ATP is the most important

molecule for capturing and transferring free energy in biological systems.

Lactate

Alanine

Serine

Cysteine

Pyruvate

NAD

⫹

NADH H⫹

⫹

Acetyl CoA

Fatty acids

Leucine

Isoleucine

LACTATE

DEHYDROGENASE

PYRUVATE

DEHYDROGENASE

FADH

FAD

NAD

⫹

NADH

CoA

Citrate

ACONITASE

Aconitate

Isocitrate

ISOCITRATE

DEHYDROGENASE

GLUTAMATE

DEHYDROGENASE

ACONITASE

H2O

NADP

⫹

NADPH

Glutamate

Histidine

Proline

Arginine

NADH

NAD

⫹

α-KETOGLUTARATE

DEHYDROGENASE

FADH

FAD

Succinyl-CoA

Oxaloacetate

FUMARASE

Malate

MALATE

DEHYDROGENASE

NAD H⫹

⫹

NADH

Fumarate

SUCCINATE

DEHYDROGENASE

FADH

FAD CoQH

CoQ

Succinate

CoA

SUCCINYL

CoA LIGASE

GTP

GDP

P⫹

Acetoacetyl-CoA

Acetoacetate

3-OXOACID

CoA

TRANSFERASE

NAD

⫹

NADH

CITRATE

SYNTHETASE

Aspartate

Tyrosine

Phenylalanine

ACETYL CoA

TRANSFERASE

CoA

β-HYDROXYBUTARATE

DEHYDROGENASE

2⫻

α-ketoglutarate

β-hydroxylbutyrate

Figure 4.2

The tricarboxylic acid (TCA) cycle, in which acetyl groups transferred from acetyl CoA are oxidized to

carbon dioxide.

Cell metabolism and its control in culture 79

The released energy of almost all the metabolic pathways is converted to

the chemical energy of phosphoanhydride bonds in ATP. Cells use the

energy released during hydrolysis of the ATP bonds to power energeti-

cally unfavorable processes.

The TCA cycle takes place inside the mitochondria. It is not only the

metabolic pathway that accounts for the complete combustion of the

product of glycolysis, but also the pathway that accounts for the complete

combustion of carbohydrates, fatty acids, and amino acids. The main func-

tions of the cycle are to provide different compounds that are precursors for

the cell anabolism and to generate most of the metabolic energy.

The connection between TCA and glycolysis is made through pyruvate.

The pyruvate produced in glycolysis is transported from the cytosol across

the mitochondrial membranes to the inner mitochondrial matrix. There, it

reacts with coenzyme A (CoA), forming CO

and the intermediate acetyl-

CoA. This reaction, catalyzed by pyruvate dehydrogenase, is highly

exergonic and essentially irreversible.

Although the TCA cycle directly generates only one ATP per turn, there

are four oxidation steps in the cycle that provide NADH and FADH

These compounds generate a large ﬂow of electrons into the respiratory

chain and thus lead to formation of a large number of ATP molecules during

oxidative phosphorylation. In this pathway, passage of two electrons from

NADH or FADH

to O

drives the formation of ATP. The number of

ATP molecules that may be formed through oxidative phosphorylation

may vary depending on the cell line. It is considered that the metabolism of

a NADH molecule generates 2.5 ATP molecules, and a FADH

molecule

generates 1.5 ATP molecules (Elliott and Elliott, 2001). These values are

known as P/O ratios and represent the relationship between ATP synthesis

and oxygen consumption. Then, the overall yield of ATP from the complete

oxidation of glucose is 32 ATP molecules per glucose molecule when both

pyruvate molecules are completely oxidized.

Unlike mammalian cells in vivo, established mammalian cell lines are

highly deregulated. They show a high glycolytic ﬂux (Donelly and

Schefﬂer, 1976) and are unable to completely oxidize glucose, generating

high amounts of lactate (a maximum of 2 moles of lactate per mole of

glucose) as an end product, even under fully aerobic conditions. Further-

more, in the presence of non-limiting glucose concentrations they con-

sume glucose at a much higher rate than would be strictly required to

maintain cell growth. This indicates that a low percentage of pyruvate is

incorporated into the TCA cycle for a complete oxidation, a pathway

much more efﬁcient regarding the energetic yield (32 ATP instead of

2 ATP when lactate is the end product).

There are several hypotheses related to lactate generation in mammalian

cells in culture (Ha

ggstro

m, 2000):

(i) the cells convert pyruvate to lactate as a way to re-equilibrate their

redox potential and as a means of NADH regeneration in the cytosol;

(ii) lactate dehydrogenase competes with pyruvate dehydrogenase for

conversion of pyruvate to lactate in the cytosol;

(iii) too low concentrations of aspartate in the cytosol may limit the

malate–aspartate shuttle activity;

80 Animal Cell Technology

(iv) respiration may be suppressed by low availability of ADP (adenosine

diphosphate), which would be consumed due to rapid ﬂux of the

glycolysis pathway;

(v) malate may accumulate inside the mitochondria due to glutamine

metabolism, which would make more difﬁcult the inﬂux of malate

into the mitochondria, as required by the malate–aspartate shuttle;

(vi) the respiratory system may be saturated by NADH generated by the

metabolism of glutamine inside the mitochondria;

(vii) there may be a deﬁciency in a key enzymatic activity linking the

glycolysis pathway with the TCA cycle, such as pyruvate dehydro-

genase complex, phosphoenolpyruvate carboxykinase, or pyruvate

carboxylase. In this situation, the metabolism of pyruvate through the

lactate dehydrogenase enzyme would be favored (Fitzpatrick et al.,

1993; Petch and Butler 1994; Neerman and Wagner, 1996).

From all the hypotheses mentioned above, the low ﬂux of pyruvate into

the TCA cycle is better explained by a decreased activity of several

enzymes: pyruvate carboxylase, pyruvate phosphoenol carboxykinase,

pyruvate dehydrogenase, and malic enzyme II (Figure 4.3). Then, the

pyruvate accumulated is converted to lactate by the enhanced catalytic

action of the lactate dehydrogenase, as an alternative pathway to generate

energy for cellular processes.

There are many studies supporting this hypothesis. Gstraunthaler et al.

(1999) have demonstrated that when renal proximal tubular cells are

brought into culture, they revert from oxidative metabolism and gluco-

neogenesis to high rates of glycolysis. They investigated the metabolism of

continuous renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum

kidney) and found that glucose was quantitatively converted to lactate,

which accumulated in a 2:1 molar ratio (lactate:glucose). In LLC-PK(1)

cells, increased glucose supply caused increases in hexokinase, phospho-

fructokinase, pyruvate kinase, and lactate dehydrogenase activities. This

higher lactate dehydrogenase activity is responsible for the increased

lactate generation and the reduced TCA cycle activity. Neerman and

Wagner (1996) compared glucose metabolism in a mammalian (BHK) and

an insect (Sf9) cell line. They found no activity of the key enzymes

connecting glycolysis with the TCA cycle, such as pyruvate dehydrogen-

ase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, indi-

cating that glucose is mainly metabolized via glycolysis and lactate

formation.

Glutamine is normally another important energy source in cultured cell

lines. However, in the absence of glutamine, the cells may change their

metabolism, to increase the energy yield from glucose. Chen and Harcum

(2005) demonstrated that, when Chinook salmon embryo (CHSE) cells

were cultured in a glutamine-free culture, hexokinase activity remained

constant and lactate dehydrogenase activity decreased. In this way, the

lack of glutamine reduced lactate production to make the energy metabo-

lism more efﬁcient.

Acetyl-CoA enters the TCA cycle by reacting with oxaloacetate to form

citrate. At this point, there is an important ﬂux of citrate leaving the TCA

cycle and being exported to the cytosol, where it contributes to lipid

Cell metabolism and its control in culture 81