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Biotechnology Advances 30 (2012) 131141

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Systems biology of the metabolic network regulated by the Akt pathway


Ettore Mosca a,, Matteo Barcella a, Roberta Aleri a, Annamaria Bevilacqua b, c,
Gianfranco Canti c, Luciano Milanesi a,
a
b
c

Institute for Biomedical Technologies, CNR, via Fratelli Cervi 93, 20090 Segrate (Mi), Italy
Universit Telematica San Raffaele Roma, Via Val Cannuta 247, 00166 Rome, Italy
Department of Pharmacology, Universit degli Studi di Milano, Via Vanvitelli 32, 20129 Milan, Italy

a r t i c l e

i n f o

Available online 12 August 2011


Keywords:
PI3K/Akt pathway
Metabolic network
Systems biology
Kinetic models
Glycolysis
Cancer

a b s t r a c t
Cancer has been proposed as an example of systems biology disease or network disease. Accordingly, tumor
cells differ from their normal counterparts more in terms of intracellular network dynamics than single
markers. Here we shall focus on a recently recognized hallmark of cancer, the deregulation of cellular
energetics. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been
conrmed as an essential step toward cell transformation. We will consider how the effects of Akt activation
are connected with cell metabolism; more precisely, we will review existing metabolic models and discuss the
current knowledge available to construct a kinetic model of the most relevant metabolic processes regulated
by the PI3K/Akt pathway. The model will enable a systems biology approach to predict the metabolic targets
that may inhibit cell growth under hyper activation of Akt.
2011 Elsevier Inc. All rights reserved.

Contents
1.

2.
3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Glycolysis and Warburg effect . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Relevance of Akt in cancer . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.
Role of Akt in cancer cell metabolism . . . . . . . . . . . . . . . . . . . .
1.4.
Integrative systems biology approach to identify molecular targets in anticancer
Kinetic models of glucose/energy metabolism . . . . . . . . . . . . . . . . . . . .
A kinetic model to study the metabolic effects mediated by the PI3K/Akt pathway . .
3.1.
Integration of different kinetic models . . . . . . . . . . . . . . . . . . . .
3.2.
Modeling of the Akt-mediated metabolic effects . . . . . . . . . . . . . . .
Strategies for the analysis of the integrated model . . . . . . . . . . . . . . . . .

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132
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Abbreviations: ACC, acetyl-CoA carboxylase; ACCOA, acetyl-CoA; ACD, acyl-CoA dehydrogenase; ACL, ATP citrate lyase; ACS, acyl-CoA synthetase; ADP, adenosine diphosphate;
ALD, fructose 1,6 bisphosphate aldolase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; BPG, 1,3-bisphosphoglycerate; CAC, carnitine acyl-carnitine carrier; CIT,
citrate; COA, coenzyme A; CPT1A, carnitine palmitoyltransferase 1A; CPT II, carnitine palmitoyltransferase II; DE, differential equation; DHAP, dihydroxyacetone phosphate; ECH,
enoyl-CoA hydratase; EG, extended glycolysis, ENO, enolase; EP, phosphoribulose epimerase; E4P, erythrose-4-phosphate; FA, fatty acid; FASN, fatty acid synthase; F16BP, fructose1,6-bisphosphate; F26BP, fructose 2,6-bisphosphate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; GMM, glutamine mithocondrial metabolism; G6PD, glucose-6phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLC, glucose; GLUT, glucose transporter; GDH, glutamate dehydrogenase; GLS, glutaminase; G6P,
glucose-6-phosphate; HCD, 3-Hydroxyacyl CoA Dehydrogenases; HK, hexokinase; HPI, hexose-6-phosphate isomerase; KI, Ribose phosphate isomerase; LAC, lactic acid; LDH, lactate
dehydrogenase; MALCOA, malonyl-CoA; MCA, metabolic control analysis; MPM, mitochondrial pyruvate metabolism; NAD, nicotinamide adenine dinucleotides; NADH,
nicotinamide adenine dinucleotides; OCT, 3-oxoacyl-CoA thiolase; PDC, pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PFK-1, phosphofructokinase type 1; PFK-2,
phosphofructokinase type 2; 6PG, 6-phosphogluconate; 2PGA, 2-phosphoglycerate; 3PGA, 3-phosphoglycerate; PGAM, 3-phosphoglycerate mutase; 6PGD, phosphogluconate
dehydrogenase; PGK, phosphoglycerate kinase; Pi, inorganic phosphate; PI3K, phosphatidylinositol 3-kinase; PYC, pyruvate carrier; PYK, pyruvate kinase; PYR, pyruvate; PYRt,
pyruvate diffusion between cytosol and mitochondrial intermembrane space; PRPP, phosphoribosyl-pyrophosphate; PRPPS, phosphoribosyl-pyrophosphate synthetase; R5P,
ribose-5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; SA, sensitivity analysis; TA, transaldolase; TCA, tricarboxylic acid; TK1, transketolase 1; TK2,
transketolase 2; TPI, triosephosphate isomerase; X5P, xylulose-5-phosphate.
Corresponding authors.
E-mail addresses: ettore.mosca@itb.cnr.it (E. Mosca), matteo.barcella@itb.cnr.it (M. Barcella), roberta.aleri@itb.cnr.it (R. Aleri), annamaria.bevilacqua@unisanraffaele.gov.it
(A. Bevilacqua), gianfranco.canti@unimi.it (G. Canti), luciano.milanesi@itb.cnr.it (L. Milanesi).
0734-9750/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.08.004

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5.
Conclusions . . . . . .
Acknowledgments . . . . . .
Appendix A.
Supplementary
References . . . . . . . . .

E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

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1. Introduction
The intrinsic differences between cancer and normal cells are key
when trying to identify new targets for anticancer drugs and to
overcome chemo-resistance to anticancer therapy. Like drivers on busy
roads of big cities begin to turn around to reach their destination,
intracellular networks allow cancer cells to bypass the effect of a drug
using alternative pathways to exploit a critical function for their
survival: it is thus increasingly believed that a systems biology approach,
focused on the analysis of the structure and dynamics of these networks,
can lead to a better comprehension of cancer disease and could aid the
design of safer drugs and therapies (Wang, 2010). To summarize these
concepts, cancer has been designated as a systems biology disease
(Hornberg et al., 2006; Laubenbacher et al., 2009).
However, nowadays the development of systems biology in cancer
research is still limited, especially when more specic applications are
concerned. In this review, we will focus on a particular phenomenon
that has been recently recognized as a hallmark of cancer, the
deregulation of cellular energetics (Hanahan and Weinberg, 2011).
Quite a few tumor cells produce most of their ATP through the glycolytic
pathway, thereby producing more lactate than their untransformed
counterparts (Pedersen, 2007), and increase biosynthetic activities
required to create daughter cells (DeBerardinis et al., 2008). The
constitutive activation of the PI3K/Akt pathway has been conrmed as
an essential step toward cell transformation and contributes to both
metabolic changes in cell energetics and metabolic requirements to
support cell proliferation (Young and Anderson, 2008).
We will review existing models of central metabolism and we will
discuss the current knowledge available to construct a detailed kinetic
model of the most relevant metabolic effects of the PI3K/Akt pathway.
Such a kinetic model enables a systems biology approach to identify
potential metabolic targets that exploit the addiction of tumor cells to
increased glucose uptake and glycolysis.

1.1. Glycolysis and Warburg effect


Studies conducted in the early twentieth century demonstrated that,
unlike normal tissues, tumor cells are highly dependent on fermentation
reactions to survive. Starting from these studies Otto Warburg made one
of the rst hypotheses on the origins of cancer (Warburg, 1956). In
addition to the six recognized hallmarks of cancer (Hanahan and
Weinberg, 2000), aerobic glycolysis has been recently accepted as a
metabolic property of most tumors (Hanahan and Weinberg, 2011; Hsu
and Sabatini, 2008; Yeung et al., 2008). Overall energy metabolism is
greatly affected during cellular transformation (reviewed in Vander
Heiden et al., 2009). Primarily, cancer cells gain the ability to proliferate
even in the absence of growth signals. Furthermore, oncogenic
mutations often result in increased uptake of nutrients, particularly
glucose. Glucose is then metabolized into lactate regardless of oxygen
supply by the chain of reactions known as aerobic glycolysis. This
glycolytic switch was rst described by Warburg, who proposed that
this phenomenon was related to defects in mitochondria, but it has later
been shown that oxidative phosphorylation is not always impaired in
cancer cells (Moreno-Snchez et al., 2007).
Besides an enhanced glycolytic activity, cancer cells also display an
increased proliferation rate, sustained by the extra-cellular nutrients
uptake and conversion into biosynthetic precursors, such as nucleic
acids, proteins and lipids (Tong et al., 2009). Tumor cells can achieve

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139
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this goal through changes in the activation status of oncogenes, but, as


a consequence, they become oncogene-addicted (Weinstein, 2000).
1.2. Relevance of Akt in cancer
The constitutive activation of the PI3K/Akt pathway has been
conrmed by epidemiological and experimental studies as an essential
step toward the initiation and maintenance of human tumors
(Tokunaga et al., 2008). The PI3K/Akt pathway regulates several cellular
functions, including proliferation, growth, survival and mobility
(Carnero et al., 2008).
Notably, the PI3K/Akt pathway is related to both life and death
signaling, and it plays a major role not only in tumor growth but also
in the potential response of tumors to anticancer treatments (Huang
and Hung, 2009). Alterations of this signaling pathway are frequent in
human cancer and promote cancer cell resistance to anti-tumor drugs,
by overcoming the apoptotic pathway (Asnaghi et al., 2004; LoPiccolo
et al., 2007; Tokunaga et al., 2008).
In particular, the abnormal activation of the PI3K/Akt pathway in
tumor cells prevents the down-regulation of cell metabolism, protein
synthesis and cell growth when nutrients become limiting (Aki et al.,
2003; Bruno et al., 2007; Elstrom et al., 2004). Accordingly, the effects
of Akt activation on cell survival may be connected with its effects on
cell metabolism.
1.3. Role of Akt in cancer cell metabolism
The serine-threonine kinase Akt is a key molecule involved in the
signal transduction pathways of many extra-cellular inputs (Kandel and
Hay, 1999). One of the most important physiological functions of Akt is
to stimulate glucose uptake in response to insulin (Welsh et al., 2005).
The Akt transduction pathway is responsible for transmitting insulin
signal to the metabolic, transcription, and translation machinery of the
cell (Burgering and Coffer, 1995; Manning and Cantley, 2007). In
untransformed cells, the withdrawal of growth factors results in a
depletion of ATP and glucose-derived metabolites within the cell
(Rathmell et al., 2003). On the contrary, Akt constitutive activation
allows cells to continue to import glucose and amino acids (Edinger and
Thompson, 2002). Activated Akt has also been shown to increase the
glycolitic ux (Robey and Hay, 2009; Young and Anderson, 2008). Then,
where, precisely, does it act?
A series of proteins regulated by Akt are key players of the glucose
metabolism (Fig. 1). Upon insulin stimulation, Akt associates with
glucose transporter 4 (Glut4)-containing vesicles (Calera et al., 1998)
leading to Glut4 translocation to the plasma membrane. However,
constitutively active Akt, is able to induce glucose uptake by stimulating
translocation of Glut4 to the plasma membrane even in the absence of
insulin (Kohn et al., 1996). The constitutively active Akt also increases the
synthesis of Glut1, the main glucose transporter in most cell types (Kohn
et al., 1996). In particular, the activation of Akt enhances transcription
and translocation of Glut1 from the cytosol to the plasma membrane,
increasing glucose uptake (Barthel et al., 1999; Rathmell et al., 2003).
Akt activation can also alter glucose metabolism within cells. Glucose
conversion into glucose 6-phosphate represents the rst step of the
glycolytic pathway and it is accomplished by cellular HKs. The activity of
HK isoforms is nely regulated (reviewed by Pastorino and Hoek, 2008).
HK isoforms I and II bind to the mitochondrial outer membrane, where
high ATP concentrations favor enzymatic phosphorylation of glucose

E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

133

Fig. 1. Map of the glycolysis and pentose phosphate pathway. Detailed map of glycolysis and pentose phosphate shunt with the regulation exerted by the PI3K/Akt signaling pathway
(green dotted arrows where + indicates positive regulation). Black: metabolites; blue: proteins; red: co-factors.

(Majewski et al., 2004). Upon Akt activation, translocation of HKs to the


mitochondria is enhanced (Elstrom et al., 2004), although the
mechanism by which mitochondrial binding of HK is stimulated
remains elusive.
Akt has also effects on other regulatory elements of glycolysis; in fact it
has been shown that increasing Glut1 and HK expression does not
enhance the glycolytic ux to the levels observed with constitutive
activation of Akt (Rathmell et al., 2003). Glycolysis downstream targets of
Akt include PFK-2. Phosphorylation and activation of PFK-2 lead to
allosteric activation of PFK-1 (Deprez et al., 1997). These enzymes convert
F6P into F16BP, a key step in glucose metabolism.

Furthermore Akt can contribute to FAs oxidation and FAs synthesis


by regulating multiple steps of these pathways (Fig. 2). The TCA cycle
generates citrate that is next exported to the cytoplasm by the action of
citrate transport proteins. Cytosolic citrate is used for lipid and
cholesterol biosynthesis, it generates ACCOA by the ACL (ATP citrate
lyase), which is directly phosphorylated and activated by Akt (Berwick
et al., 2002). Activation of ACL supports increased ACCOA and MALCOA
production, inhibiting FAs oxidation and inducing lipid synthesis in Aktexpressing cells (Buzzai et al., 2005). Activated PI3K/Akt pathway
stimulates FA synthesis by a direct activation of ACL and inhibition of oxidation by down-regulating the expression of the -oxidation enzyme

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E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

Fig. 2. Map of FAs synthesis and -oxidation. Both the FAs synthesis and -oxidation are regulated by the PI3K/Akt signaling pathway (green dotted arrows where + and
indicate positive and negative regulation respectively). The processes of this module span the cytosol, the intermembrane space (IMS) and the mitochondrial matrix (MAT). The
Acyl-CoA is reported between parentheses to indicate that it is not always produced by OCT, as this depends on the acyl group of OCT substrate. Black: metabolites; blue: proteins;
red: co-factors.

CPT1A as described by DeBerardinis et al. (2006). It has also been


hypothesized that Akt might regulate other steps of lipid metabolism by
suppressing expression of proteins required for FA oxidation, but further
studies are needed to elucidate the mechanisms underlying additional
roles of Akt in cancer cell metabolism.

1.4. Integrative systems biology approach to identify molecular targets in


anticancer therapy
As introduced earlier, an integrative systems biology approach is
essential to study the complex network of pathways connecting gene

E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

regulation, signaling and cell metabolism, and the relevant alterations


occurring in malignant cells (Hornberg et al., 2006). A systems biology
approach combines empirical, mathematical and computational tools
to understand complex patho-physiological phenomena. As a consequence, a systems perspective on cancer prompts the application of
mathematical and computational models in order to deal with the
large amounts of data and the relationships within the datasets
(Anderson and Quaranta, 2008).
Computational models of cancer should capture the fundamental
biological processes that are the hallmarks of cancer. Progress has been
made in understanding the properties of cancer cells from a systems
biology point of view (Laubenbacher et al., 2009). It is now accepted that
oncogenic mutations affect cell behavior by changing the cellular
network to trigger malignancy (Bizzarri et al., 2008; Pawson and
Warner, 2007). Thus, network biology is useful to represent, compute
and model intracellular interactions and to get further insights into
cellular mechanisms (Kreeger and Lauffenburger, 2010).
In this review we outline recent studies of one of the most commonly
mutated signaling pathways in cancer: the PI3K/Akt pathway. Our focus
is on how Akt oncogenic activation changes the metabolic network and
how this information can be used to predict new targets and treatment
strategies in anticancer therapy. In the next sections, the available
knowledge for the construction of a detailed kinetic model to simulate
the PI3K/Akt pathway metabolic network will be discussed.
Such a model can be implemented linking different kinetic models to
each other. To reach this goal, a number of guidelines should be
addressed in order to ensure a uniform quality of the resulting model. The
members of the silicon cell project (Snoep, 2005), an international
consortium which aims at making computer replica of sub-cellular
systems, place the emphasis on using experimentally determined values
for model parameters, wherein the measurements should be made on an
isolated reaction step in order to ensure context independence.
Moreover, they underline the importance of linking models that have
been constructed under the same experimental conditions and of using
an experimentally measured law to represent the (dynamic) behavior of
boundary metabolites (Snoep et al., 2006). Another important property
that a metabolic model should have is the capability to reproduce steady
states, where metabolite concentrations and metabolic uxes are
constant. These states should represent stable behaviors of the metabolic
network, while transitions from one steady state to another represent the
responses of the cell to possible perturbations. Furthermore, the
availability of annotations, for example according to the MIRIAM
specications (Le Novre et al., 2005) and model codication using
standard languages, such as the SBML (Hucka et al., 2003) or CellML
(Cuellar et al., 2003), are also very useful for the understanding of model
denition and direct usability by means of several computational tools.
We will follow these guidelines to discuss the construction of a
detailed kinetic model for the metabolic processes regulated by the
PI3K/Akt pathway, whenever it is possible. In particular, the requirement of equal experimental conditions can be hardly met, since the
currently available metabolic models have been constructed relying on a
broad variety of experimental conditions.
The integrative model will capture the structure and dynamics of the
metabolic network regulated by the PI3K/Akt pathway. In this model the
effects of Akt activation will be reproduced by acting on the rate of the
biochemical processes regulated by PI3K/Akt pathway; more precisely,
this can be done introducing quantitative changes in the enzyme
kinetics values. This model might then be used to simulate normal and
aberrant behavior of the considered metabolic network, and to test
hypotheses about the mechanisms underlying the PI3K/Akt pathway
effects on tumor metabolism. The model will thus enable in silico
experiments. Eventually, the results derived from this systems biology
approach might be experimentally validated in cancer cells. Through
this approach, cancer systems biology will allow the integration of
computational and experimental data at various levels and has the
potential to hint possible anticancer therapeutic strategies.

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2. Kinetic models of glucose/energy metabolism


Compared to signal transduction and gene regulatory networks,
metabolic pathways are easier to study. The enzymes can be isolated and
characterized in vitro while reaction uxes can be quantied in vivo:
therefore, it is possible to collect data concerning the kinetics of each
biochemical reaction and the overall behavior of a metabolic pathway. As
a consequence, metabolic systems have been well characterized and
were among the rst to be reproduced by detailed kinetic models,
especially glycolysis. With modern genome-sequencing capabilities, the
size of metabolic models increased until the rst genome-scale metabolic
network was published (Edwards and Palsson, 1999). During the last
decade the eld of genome-scale metabolic network analysis has grown
rapidly and nowadays more than 50 genome-scale metabolic reconstructions are available and span several species of bacteria and
eukaryotes, including human. These models have already led to many
advances both at the theoretical and practical level (see Oberhardt et al.,
2009 for a review of the applications of genome scale metabolic
reconstructions), although these so-called structural models focus on
the reaction network structure and not on the kinetics. In fact, the
integration of detailed kinetics into these models and the determination
of an adequate amount of kinetics parameters (both steps enabling an
accurate study of the system dynamics) are standing challenges. Even if
some efforts are ongoing in this eld (Herrgard et al., 2006; Lee et al.,
2008), current detailed kinetics models include, compared to cellular
level models, a relatively limited number of biochemical processes.
The earliest models concerning glycolysis appeared in 1960s
(Chance et al., 1960, Garnkel and Hess, 1964), and nowadays tens of
models including the glycolytic reactions are publicly available in web
resources. Currently, the BioModels Database (Li et al., 2010), one of
the most important resources that allows users to store, search and
retrieve published mathematical models of biological interest, provides approximately 40 models concerning glycolysis, half of which
are classied as curated.
The differences among the available models regarding glycolysis
concern a number of factors leading to the inclusion of a different set of
biochemical processes or to a different mathematical formulation. There
are core metabolic models, where only the most important reactions
(e.g. important regulatory steps and branches) are included (e.g.
Galazzo and Bailey, 1990) and detailed models, where more or less
every biochemical reaction of the studied pathway is considered. An
important aspect is the approach used for the denition of the kinetic
parameters: while some models have been dened relying on extensive
tting of kinetics values on systemic datasets (e.g. Rizzi et al., 1997),
other exploit values experimentally determined by studying isolated
enzymes (e.g. Teusink et al., 2000). Even though each model can be
quantitatively different from another, it is possible to distinguish the
largest fraction of models in which glycolytic intermediates reach a
(stable) steady state, from models exhibiting oscillations of some
metabolites (e.g. Nielsen et al., 1998). Glycolysis models have been
constructed for several organisms and cell types; a large number of
models exist for yeast, while for the human species, detailed kinetic
models have been constructed mainly for erythrocytes (e.g. Mulquiney
and Kuchel, 1999), skeletal muscle (e.g. Lambeth and Kushmerick,
2002) and pancreatic beta-cells (e.g. Jiang et al., 2007).
Even if not ubiquitous in every glycolysis model, a number of (speciesspecic) glycolytic branches, such as those concerning disaccharides and
polysaccharides metabolism, and biochemical reactions concerning the
pyruvate destiny, such as those for lactate or ethanol fermentation and
Acetyl-CoA production, are usually considered (e.g. Conant and Wolfe,
2007), while the pentose phosphate pathway is detailed only in a few
models (e.g. Holzhtter, 2004). Due to the importance of the TCA cycle,
some models exist for it (e.g. Singh and Ghosh, 2006), even coupled with
the respiratory chain (e.g. Nazaret et al., 2009), with the FAs -oxidation
and the mitochondrial inner-membrane metabolite transport system
(e.g. Wu et al., 2007 and Yugi and Tomita, 2004).

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E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

3. A kinetic model to study the metabolic effects mediated by the


PI3K/Akt pathway
During the construction of a kinetic model to represent the metabolic
alterations regulated by the PI3K/Akt pathway, the optimal compromise
between two requirements has to be found. On the one hand, it is
important to consider as many metabolic reactions directly and
indirectly related to the PI3K/Akt pathway as possible, in order to ensure
an accurate reproduction of intracellular kinetics. On the other hand, as
the set of reactions considered increases, model construction and
analysis become harder due to a series of issues, such as the lack of
enzyme kinetics data or the number of parameters with uncertain values.
In this section we will describe a model that represents a possible
solution according to the metabolic pathways inuenced by Akt
activation and the available knowledge required for modeling such
metabolic processes.
It is possible to describe the model as composed of six modules:
(i) glycolysis and pentose phosphate pathway, (ii) TCA cycle,
(iii) respiratory chain, (iv) FAs synthesis and -oxidation, (v) the
glutamine mitochondrial metabolism and (vi) metabolite transporters,
as shown in Fig. 3 and Table 1.
The (i) glycolysis and pentose phosphate pathway (Fig. 1) include
the glucose transporter, the glycolytic pathway, the glycogen branch,
the pentose phosphate shunt and LDH reaction. The reactions and
kinetic laws for the glucose transporter, the ten glycolytic reactions, the
glycogen branch and LAC fermentation can be found in the model
recently proposed by Marn-Hernndez et al. (2011). The authors used
enzyme-specic kinetic laws and kinetic parameter values experimentally determined in cytosolic extracts of HeLa cells under the same
experimental conditions. Moreover, they simplied the glycogen branch
into two irreversible reactions representing the glycogen synthesis and
degradation rates which are, however, supported by experimental data

concerning the glycogen metabolism (e.g. glycogen content, synthesis


and degradation uxes). Their model also considers the pentose
phosphate shunt and the mitochondrial pyruvate destiny with
simplied reactions. As both these pathways play a relevant role in
relation to the PI3K/Akt pathway-mediated metabolic effects, it is useful
to replace these parts with detailed reactions from other models
available in the literature. Regarding the pentose phosphate shunt, it has
been proposed that one of the potential benets to a cancer cell of a high
glycolytic rate is the availability of glucose for the production of NADPH
by means of the oxidative arm of the pentose phosphate cycle, which
may be important in maintaining the redox state of a cell under
oxidative stress (Elstrom et al., 2004). To this end it is worthy to note the
pentose phosphate shunt model proposed by Holzhtter (2004), which
is in turn the integration of previous works where the kinetic
parameters were experimentally determined (Barman, 1969; Boyer,
1962; Lueck and Fromm, 1974; McIntyre et al., 1989).
It is important to consider the (ii) TCA and (iii) respiratory chain
modules due to their central role in metabolism and, as a
consequence, in cancer cells metabolism (Kroemer, 2006). These
modules can be entirely taken from the model proposed by Wu et al.
(2007). This model is particularly interesting since the authors
reconstructed the mitochondrial TCA cycle, the oxidative phosphorylation and the metabolite transports. The kinetic parameter values
used in this model were found in the literature or computationally
estimated in order to t a series of in vivo and in vitro data from
cardiac mitochondria and human skeletal muscle. Some mitochondrial reactions not available in the model of Wu et al. (2007) can be
found in the model of mitochondrial metabolism developed by Yugi
and Tomita (2004). The (iv) FAs synthesis and -oxidation module
encompasses the anabolic pathway of FAs synthesis and the catabolic
process that leads to the conversion of cytosolic FAs into mitochondrial ACCOA (Fig. 2). In this module, the PI3K/Akt pathway promotes

Fig. 3. Summary map of the metabolic network regulated by the PI3K/Akt signaling pathway. The metabolic network regulated by the PI3K/Akt pathway encompasses the
extracellular space, the cytosol and the mitochondrion. The biochemical processes composing the network can be divided into six modules: the glycolysis and the pentose phosphate
pathway, the tricarboxylic acid cycle, the respiratory chain, the fatty acids synthesis and -oxidation, the glutamine mitochondrial metabolism and the metabolite transporters. The
proteins positively and negatively regulated by the PI3K/Akt pathway are reported in green and red color respectively. The map is intended to provide an overall picture of the
metabolic network and, as a consequence, does not report all the molecular species and biochemical processes included in the network.

E. Mosca et al. / Biotechnology Advances 30 (2012) 131141


Table 1
Biochemical processes of the metabolic network regulated by the PI3K/AKT pathway
with references for the corresponding ux expressions.
Module name

List of processes

Flux expressions

Glycolysis and pentose


phosphate pathway

Glucose transport

Marn-Hernndez et
(2011)
Marn-Hernndez et
(2011)
Marn-Hernndez et
(2011)
Marn-Hernndez et
(2011)
Holzhtter (2004)

Glycolysis pathway
Glycogen production
LDH reaction

Tricarboxylic acid cycle

Respiratory chain
Fatty acid synthesis
and -oxidation

Pentose phosphate
pathway
PDH reaction
Tricarboxylic acid cycle
Pyruvate carboxylase
Respiratory chain
reactions
ACL reaction
ACC reaction
FASN reaction

ACS reaction
FA transport in
mitochondria
-Oxidation
Lipid metabolism
Glutamine mitochondrial Glutamine mitochondrial
metabolism
transport
GLS reaction
GDH reaction
Metabolite transporters
Malateaspartate
shuttle
Acetyl groups transporter
shuttle
Other transporters

al.
al.
al.
al.

Wu et al. (2007)
Wu et al. (2007)
Yugi and Tomita (2004)
Yugi and Tomita (2004)
Plowman and Cleland
(1967)
Kaushik et al. (2009)
Cox and Hammes
(1983)
Hall et al. (2003)
Yugi and Tomita (2004)
Yugi and Tomita (2004)
Mass-action irreversible
Steib et al. (1986)
Haser et al. (1985)
Bruggeman et al. (2005)
Yugi and Tomita. (2004);
Wu et al. (2007)
Yugi and Tomita (2004);
Swierczyski (1980)
Yugi and Tomita (2004);
Wu et al. (2007)

the FAs synthesis increasing the activity of ACL and inhibits the oxidation decreasing the concentration of CPT1A. FAs synthesis can
be simulated with four enzymatic reactions, respectively catalyzed
by ACL, ACC, FASN and an irreversible reaction representing the ux
toward lipid metabolism. The ACL, ACC and FASN kinetic laws
provided with experimentally determined kinetic parameters can be
found in different studies (respectively Plowman and Cleland, 1967;
Kaushik et al., 2009; Cox and Hammes, 1983), while a mass action
kinetics can be used for the irreversible reaction representing the ux
toward lipids, a boundary of the system. The reactions for FAs entry
into mitochondria and FAs -oxidation are also available in the model
by Yugi and Tomita (2004). This model lacks the pre-step of oxidation, catalyzed by the ACS; however, the kinetic law and related
parameters experimentally determined for puried murine ACS are
available (Hall et al., 2003).
Glutamine, which is highly transported into proliferating cells (Wise
et al., 2008), is a major source for energy and nitrogen for biosynthesis,
and a carbon substrate for anabolic processes in cancer cells (DeBerardinis
and Cheng, 2010). In cancer, glutamine can serve as an alternative
substrate for the TCA cycle in order to produce ATP and can become
critical for biosynthesis and survival. Hence, it can be used as an energy
substrate when glucose supply is limited (Yuneva, 2008), the so-called
glutamine addiction phenomenon (Wise and Thompson, 2010). Thus, it
is important to take into account a series of reactions needed for the
(v) glutamine metabolization in the TCA cycle. One solution consists in
representing this pathway with three biochemical processes. The rst one
is the transport of glutamine from the cytosol into mitochondria, for
which the kinetic law provided by Steib et al. (1986), with kinetic
parameters experimentally determined in rat brain, is suitable. The
second process is the conversion of glutamine into glutamate catalyzed by

137

the mitochondrial GLS; here the kinetic law and parameters experimentally determined in rat kidney (Haser et al., 1985) can be used. The ux
expression for the conversion of glutamate into oxoglutarate, operated by
the GDH, is available in the model of ammonium assimilation in E. coli
(Bruggeman et al., 2005).
Lastly, the (vi) metabolite transporters module, which includes the
malateaspartate shuttle, the acetyl groups transporter shuttle, and
transporters not included in the other modules, is required to
reproduce the metabolites trafc (or translocation) between cytosol
and mitochondria. These carriers can be found in the two models of
Wu et al. (2007) and Yugi and Tomita (2004) with the exception
of the malic enzyme (considered as a part of the acetyl groups
shuttle system), for which specic kinetic law and kinetic parameters
experimentally determined in rat skeletal muscle are described by
Swierczyski (1980).
3.1. Integration of different kinetic models
The linking of different kinetic models consists in the denition of a
unique model that combines the mathematics and possibly contains
some expressions not available in the starting models, but required for a
correct integration. Mathematically, these models are a set of
differential algebraic equations, in which each differential equation
(DE) describes the temporal variation of a metabolite concentration xi as
dxi
1 m
= si;k Jk x; p; t
Vi k = 1
dt
where the stoichiometric coefcient si,k species the amount of xi
produced (si,k 0) or consumed (si,k 0) by a reaction k, Jk is the ux of
reaction k, Vi is the volume of the compartment in which xi is
observed, and m is the total number of reactions. The uxes Jk are
functions of metabolite concentrations x and kinetic parameters p,
and are dened depending on the specic kinetic mechanism (e.g.
MichaelisMenten, Random Bi Bi or Multisite Ping Pong) of the
reaction that they describe. Algebraic equations are used to dene
conservation relations.
Since, as previously mentioned, it is common that the starting
models have been developed in different experimental conditions, a
preliminary step of model tting may be useful to obtain models that
are more compatible according to the known behaviors of the real
system that one wants to represent. For the modeling of the metabolic
network regulated by the PI3K/Akt pathway, the starting models may

Table 2
Preliminary tting of a glycolysis model. Comparison of steady state uxes ratios
computed using the glycolysis model by Marn-Hernndez et al. (2011) before and after
tting to uxes measured in HEK 293 cells by Henry et al. (2011) (Supplementary Fig. 1).
The expression J(X Y) indicates the ux from metabolite X to metabolite Y. The model
has been implemented for MATLAB (2007a, The MathWorks, Massachusetts, U.S.A.) and
simulated using parameters and initial conditions reported, respectively, in Supplementary Table 1 and Supplementary Table 2. Steady states were calculated using MATLAB
function fsolve. PYR_x indicates the mitochondrial concentration of PYR.
Fluxes ratios

J G6PF6P
J GLCG6P
J F6PG3P
J G6PF6P
J G3PPYR
JJ F6PG3P
J PYRPYR x
J PYRLAC
J G3PPYR
J PYRPYR x

Fluxes ratios value relative error


HEK 293
cells

Before preliminary
tting

After preliminary
tting

0.850

0.932 0.096

0.883 0.038

1.088

1.006 0.075

1.066 0.020

2.042

2.054 0.006

2.064 0.011

0.296

0.016 0.944

0.135 0.546

4.207

61.263 13.563

5.774 0.373

138

E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

undergo a preliminary tting in order to reproduce metabolic uxes


and concentrations of a given cell type. In Table 2 we report, as an
example, ratios between steady state uxes exhibited by the model of
Marn-Hernndez et al. (2011) using a different set of parameters and
initial conditions to reproduce the metabolic uxes experimentally
measured in HEK293 cells. In this case, the alternative tting is mainly
useful to reduce the rate of LAC production and increase the ux
toward the mitochondrial PYR metabolism.
Subsequently, it is useful to begin the actual integration process
identifying the list of the species and reactions in common among the
considered models. These points of connections should be managed to
obtain the mathematical formulation of the resulting model. The DE
for the species in common will have uxes originating from both
models, whenever the two models specify different reactions for the
same species. For reactions in common one ux expression must be
selected for use in the resulting model. To clarify this concept let us
consider two models already mentioned: the glycolysis model of
Marn-Hernndez et al. (2011), shortly G in the following, and the
mitochondrial model of Wu et al. (2007), shortly M. In these two
models the cytosolic PYR concentration is a common metabolite for
which G and M provide both different and common reactions. More
precisely, G contains four uxes concerning PYR and relative to PYK,
LDH and MPM, where MPM is a constant ux representing the
mitochondrial pyruvate metabolism; conversely, M considers only the
passive permeation through mitochondrial outer membrane, PYRt
(Fig. 4a). In this case, a possible solution to dene the DE for PYR is to
consider PYK and LDH from G and PYRt from M in place of MPM
(Fig. 4b). As previously said, the link between two models can demand
the addition of reactions not available in any of the two models. For
example, when linking G and M, the reactions required for transporting reducing equivalents from cytosolic NADH into the mitochondrial
matrix must be added for a correct coupling of the reactions in the two
compartments. Note that, in this case, the reactions that have to be
added are tissue specic: kidney and heart cells use the malate
aspartate shuttle while skeletal muscle and brain cells rely on the

glycerol 3-phosphate shuttle. After the mathematics representing the


resulting model has been dened, it is important to guarantee that all
the uxes are expressed in the same unit of measurement (e.g. mol/s),
since this is not often the case when considering the current models
available in the literature.
Once the integrated model is correctly implemented, a postintegration phase of tting is required to reproduce some other
known behaviors of the real system. Considering the metabolic network
regulated by Akt, experimental data concerning GLC consumption and
LAC production, oxygen and NAD(P)H consumption are useful for this
calibration step (Elstrom et al., 2004).
3.2. Modeling of the Akt-mediated metabolic effects
To correctly reproduce the metabolic effects of the PI3K/Akt signaling
pathway using the model as simulation tool, it is fundamental to take
into account how the PI3K/Akt pathway regulates the activity of its
targets: Glut, PFK-2, HK, ACL and CPT1A; see Fig. 3, where the global
regulation of the PI3K/AKT pathway on the different modules is shown.
In fact, the mechanistic details of the interactions can be simulated acting
on the kinetic parameters appearing in the rate equations of Akt targets.
The PI3K/Akt pathway-mediated regulation of Glut leads to an
increase of its concentration. Thus, this effect can be reproduced by
increasing the Vmax parameter (the maximum velocity) value
appearing in the monosubstrate MichaelisMenten equation used to
model the rate of glucose transport inside the cell. Similarly, but with
the opposite effect, the CPT1A concentration is decreased by the
activation of the PI3K/Akt pathway and thus one can reduce the
appropriate Vmax value in the kinetic law of this transporter. The
increased PFK-1 activity due to Akt is ultimately determined by a
higher concentration of one of the PFK-1 allosteric activators, F26BP,
which is in turn increased as a consequence of Akt regulation of PFK-2.
As this positive interaction is explicitly considered in the PFK-1 kinetic
law provided by Marn-Hernndez et al. (2011), it is possible to
increase the F26BP concentration value to reproduce the higher PFK-1
activity due to the PI3K/Akt pathway. Even if the mechanism by which
the PI3K/Akt pathway regulates the HK is not completely understood,
it is known that Akt activation increases the concentration of
mitochondrial bound HK, leading to a more efcient conversion of
GLC to G6P. Hence, it is possible to reproduce this effect increasing the
Vmax value of the bi-substrate MichaelisMenten kinetic law used for
this reaction. The activation of the PI3K/Akt pathway determines the
phosphorylation of ACL, which in turn increases the activity of the
enzyme. This event can be reproduced in silico introducing in the ACL
kinetic law the experimentally determined quantitative values of Vmax
and Km for phosphorylated ACL (Potapova et al., 2000).
To demonstrate this approach in practice, it is possible to consider
the kinetic model resulting from the integration of the two models G
and M (see the Supplementary materials). The integrated model was
simulated using four different parameterizations, where the kinetic
constants of Akt targets were modied in order to simulate the
metabolic effects downstream to Akt activity. As the strength of the
Akt activity is increased (from 10 to 100 times) glucose is consumed
faster while the production of LAC and ACCOA (a boundary metabolite
in the model) increases (Fig. 5).
4. Strategies for the analysis of the integrated model

Fig. 4. Denition of the differential equation for cytosolic PYR integrating ux


expressions from models G and M. a) Reactions involving cytosolic PYR included in
model G (blue) and model M (green); b) differential equation for PYR in model G, M and
in the resulting model (the subscript c refers to the cytosol). The red dotted cross
underlines the removal of MPM reaction during the integration of models G and M. JPYK,
JLDH, JPYRt: uxes of the processes controlled by PYK, LDH and PYRt respectively; JMPM:
ux for the mitochondrial PYR metabolism. Wc: volume of the cytosolic compartment.

Once a model reaches a satisfactory representation of some


behaviors of the real system, it can be used to improve the current
knowledge about the biological system.
A kinetic model can be perturbed by varying its kinetic constants
and species concentrations allowing in silico studies of a large number
of possible experimental conditions. Hence, complex and costly wet
experiments are reduced and limited to the most promising ones,
selected on the basis of model predictions.

E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

139

Fig. 5. Simulation of the metabolic effects of Akt activation. Simulation of a metabolic network resulting from the integration of the glycolysis model by Marn-Hernndez et al.
(2011) and mitochondrial model by Wu et al. (2007) (see Supplementary materials). Time evolution of a) GLC, b) LAC and c) cytosolic ACCOA computed with four different model
parameterizations that simulate the increasing strength of Akt regulation; x represents the varied parameters, i.e. Vmf, Vm, [F26BP] and Vm of respectively GLUT, HK, PFK1 and ACL ux
expressions (see Supplementary material). The initial conditions used are reported in Supplementary Table 3, while the ux expressions and the kinetic parameters are listed in
Supplementary Table 4. The simulations of the integrated model have been performed using the software MATLAB (2007a, The MathWorks, Massachusetts, U.S.A.): differential
equations were solved with the function ode15s.

One of the most relevant strategies of model analysis concerns the


identication of the model components mostly responsible for some
signicant properties. In fact, simple inspections are limited by the
complexity of biological models, and appropriate techniques, such as
sensitivity analysis (SA), must be used. SA can be done for different
purposes, such as model simplication, identication of model
parameters that must be known with the highest precision and study
of how the control is distributed inside the system. By comparing the
control coefcients dened in the framework of Metabolic Control
Analysis (MCA) (Heinrich and Schuster, 1998), a method of SA, between
normal and altered model parameterizations, it is possible to predict the
enzymes or transporters which show the highest control difference
between the two conditions. Considering the model of PI3K/Akt
pathway mediated metabolic effects that we described above, the
proteins with the highest control coefcient differences between
normal and altered condition will be candidates to be selective drug
targets. Bearing in mind that the oncologic clinical practice and MCA
have both demonstrated that the control of function is shared by
multiple steps (Moreno-Snchez et al., 2010), targeting the proteins
with the highest control coefcient differences might be a good strategy
to obtain one or a combination of desirable outcomes, such as the
inhibition of the aerobic glycolysis and glutamine metabolization
toward mitochondria, the re-activation of FAs -oxidation or the
inhibition of FAs synthesis. Importantly, due to the complex behavior
of the metabolic network, it is not granted that the optimal intervention
points are one or more of the metabolic targets of the PI3K/Akt pathway
that we discussed above (i.e. GLUT, HK, PFK-1, CPT and ACL).

Overall the in silico analyses will provide a combination of


targets that are more crucial (as they exert more control and are
more affected by perturbations) in cancer cells than in normal cells.
Then the question has to be asked whether these are the steps to
which the cancer cell is most sensitive.
As a fundamental facet of systems biology approach is to
experimentally validate in silico predictions, the proteins and relevant
reactions envisaged to be the best combination of specicity and
effectiveness may be selected for testing in cell-based assays. Molecular
compounds that specically inhibit key reactions, as well as small
interfering RNAs targeting critical enzymes or transporters, can be
administered to cells. Treated and control (untreated) cells may be
compared in order to measure which glycolytic steps the cells actually
regulate as a response to drug treatment. The cellular response, in terms
of growth rate, viability and cell death is useful to conrm the model
predictions. In conclusion, experimental data can then validate the
hypotheses or provide new ways to rene the model.
5. Conclusions
The constitutive activation of the PI3K/Akt pathway has been
conrmed as an essential step toward cell transformation. Cancer cells
use the PI3K/Akt pathway signaling to alter their metabolism in
several points in order to gain a number of selective advantages
compared to the other cells. Currently, several therapeutic strategies
that target the PI3K/Akt pathway for the treatment of cancer have
been proposed and are under clinical development (Engelman, 2009).

140

E. Mosca et al. / Biotechnology Advances 30 (2012) 131141

At the same time, it is becoming more and more evident that targeting
single gene products or pathways yields low rates of response and
should not be expected to cure cancer (Hayden, 2008). The PI3K/Akt
pathway regulates the activity of several proteins that, in turn, control
mass and energy ux distribution through a tightly interconnected
metabolic network. As a consequence, the altered metabolic network
shows different dynamics compared to the normal one. In this
context, an effective therapeutic approach should attack the altered
system in order to switch it off without affecting the normal system,
and having a model allows using SA methods to select robust and
sensitive parts of the system. With the aim of enabling a systems
biology approach for the metabolic network regulated by the PI3K/Akt
pathway, we reviewed and discussed the framework of knowledge
available for the modeling of the considered network, emphasizing
the use of enzyme-specic kinetic laws with experimentally derived
kinetic parameters.
The analysis of the current knowledge suggests that a series of detailed
rate equations are available for the most important biochemical processes
that link together in an integrated metabolic network all the processes
regulated by the PI3K/Akt pathway. The differences existing among the
experimental conditions used for the characterization of enzyme and
transporters kinetics demand a computational phase of parameter
estimation to obtain a coherent model which t the experimental
observations, before it can be exploited to gain in silico predictions.
Nevertheless, new data sets are continuously made available and
measuring everything is not mandatory (Alberghina and Westerhoff,
2005). The systems biology approach discussed here indeed allows
devising testable hypothesis based on a quantitative model that might
ultimately aid in understanding the complex metabolic network
mediated by the PI3K/Akt pathway.
Acknowledgments
This work has been supported by the MIUR FIRB ITALBIONET
(RBPR05ZK2Z), BIOPOPGEN and the Flagship INTEROMICS projects.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.biotechadv.2011.08.004.
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