tmpA40D TMP

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Current Chemical Biology, 2008, 2, 68-82
Emerging Roles for Metabolic Engineering - Understanding Primitive and

Complex Metabolic Models and Their Relevance to Healthy and Diseased
Kidney Podocytes
Mehmet M. Altintas1, Kutlu O. Ulgen2, Darryl Palmer-Toy3, Vivian E. Shih4, Dhinakar S. Kompala5
and Jochen Reiser*,1
1
Division of Nephrology and Program in Glomerular Disease, Department of Medicine, Massachusetts General
Hospital and Harvard Medical School, Boston, MA 02129-2020, USA
2
Department of Chemical Engineering, Bogazici University, Bebek 34342 Istanbul, Turkey
Regional Reference Laboratories, Southern California Permanente Medical Group, North Hollywood, California
91605, USA
4
Amino Acid Disorders Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, MA 021292020, USA
5
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309-0424, USA
Abstract: The central metabolism of a cell can determine its short- and long-term structure and function. When a disease
state arises, the metabolism (i.e., the transportation of nutrients into the cells, the overall substrate utilization and production, synthesis and accumulation of intracellular metabolites, etc.) is altered in a way that may permit organisms to survive
under the changing physiologic constraints. Although the response of cells to injury was studied thoroughly using molecular biology and structural morphology techniques, the knowledge regarding the metabolic signatures of the disease is limited. However, recent advances in analytical methods and mathematical tools have led to new approaches to those questions with the concept of computational biology which relies on the integration of experimentation, data processing and
modeling. The attempt to formulate current knowledge in mathematical terms has led to the development of several
mathematical modeling tools (i.e., metabolic flux analysis, metabolic control analysis, etc.) that helps us to understand an
entire biological system from basic structure to dynamic interactions. This review provides an overview and summarizes
the current status of applications of mathematical models for the quantification of fluxes. A specific example of kidney
podocyte cells illustrates how metabolic alterations, which occur during injury, can be used to aid in future therapeutic
development.
Keywords: Metabolic flux analysis, intracellular fluxes, podocyte metabolism, analysis of the health and disease.
INTRODUCTION
Biology in the past decades has been characterized by a
qualitative and descriptive approach designed to investigate
molecular behavior. Today, with computational technology
(i.e., modeling and simulation of complex processes), the
relationships between various parts of a biological system
(e.g., gene and protein networks involved in cell signaling,
metabolic pathways, organelles, cells, physiological systems,
organisms, etc.) are potentially understandable and predictive to some extent.
Together with the advances in biological science and
technology, an enormous number of new data types and formats are emerging daily. The success of mathematical models in biological and medicinal research requires an iterative
interaction between experimentation, modeling and simulation, and theory. These mathematical representations of
complex systems all have limitations, but they have the
*Address correspondence to this author at the Nephrology Division, Massachusetts General Hospital, Harvard Medical School, 149, 13th Street, Room
8214, Boston, MA 02129-2020, USA; Tel: 617-726-9363; Fax: 617-7265669; E-mail: jreiser@partners.org
1872-3136/08 $55.00+.00
potential to create a robust computational biology by the

collaborative work of biologists with mathematicians and
researchers from other disciplines [1].
Important progress has been made towards understanding
the complex systems in biological science from almost two
decades of research in metabolic engineering. The overall
goals of metabolic engineering are (i) to model and predict
the cellular metabolism in a quantitative manner and (ii) to
improve cellular properties by designing and implementing
rational genetic modifications [2-4]. In this sense, metabolic
engineering deals with the measurement of metabolic fluxes
and their control together with the evaluation of the impact
of genetic modifications on cellular physiology [5-8].
Based on this definition, metabolic engineering utilizes
various mathematical modeling tools (i.e., metabolic flux
analysis, metabolic control analysis, etc.) to identify fluxes
through critical metabolic pathways in the cell or tissue of
interest. Then, these quantitative methods allow precise control of the metabolic network throughout the pathway of interest and optimization of the metabolic flow to target metabolites or products. Besides providing a convenient framework for the integration of fluxes with the intracellular vari 2008 Bentham Science Publishers Ltd.
Emerging Roles for Metabolic Engineering
ables (metabolites, enzyme activities, proteins, etc.), metabolic engineering has a novel contribution with its capacity
to analyze more global networks of several enzymes and
reactions.
In this review, we describe the use of metabolic engineering techniques to analyze the physiology of normal and disease states in different model systems. We focus on the
pathway engineering approach towards novel therapies for
patients with injuries and/or chronic diseases, with an emphasis on disease processes occurring in podocytes, cells
essential to maintain kidney filtration.
MODELING METABOLIC NETWORKS
Mathematics and computation play critical roles in understanding the physiological behavior of cells and systematic integration of this information into a predictive model
that can be used for controlling the fate of the organism. In
this context, the essence of metabolic engineering is the capacity to engineer pathways that regulates the overall metabolism on the basis of a set of stoichiometric and/or kinetic
rules. In the following sections, mathematical tools of the
metabolic engineering will be discussed briefly before reviewing its applications to bacterial, yeast and mammalian
systems.
Construction of the Metabolic Networks
A metabolic pathway is defined as the series of feasible
and observable biochemical reactions connecting a specified
set of input and output metabolites [7]. A metabolic pathway
may be linear, cyclic, branched, tiered, directly reversible, or
indirectly reversible. Various metabolic pathways within a
cell (glycolysis, tricarboxylic acid cycle, pentose phosphate
pathway, gluconeogenesis, glyoxylate shunt, oxidative phosphorylation, etc.) form the cells metabolic network that is
generally a complex and nonlinear system of cellular (metabolites, nucleic acids, etc.) and extracellular constituents
(substrates, protons, etc.) and reactions. The metabolic networks including central carbon metabolism, regulation
mechanisms, energetic and transport reactions have a key
role in sustaining cellular functions by coordinating the activity of different metabolic pathways [9-11].
Since the metabolic pathways and fluxes are at the core
of metabolic modeling, mapping biochemical networks in
the cell or organ of interest is the priority for the application
of metabolic engineering. Those networks are currently organized into databases such as KEGG [12] and MetaCyc
[13]. These databases are based on information from experimental data and store valuable information about hundreds of pathways and cellular processes. In addition, the
complete metabolic network may not be fully described, i.e.,
pathway(s) within the network may consist of alternative
reaction(s) that produce the same set of metabolites from the
same set of precursor metabolites and cofactors [9]. Therefore, the network map is refined in an iterative fashion for
the most accurate reflection of the existing biochemical
knowledge (Fig. 1).
Measurement of Fluxes
Metabolic flux is the rate of material that flows along a
metabolic pathway or even through a single reaction connecting two or more metabolites. Measurement of metabolic
Current Chemical Biology, 2008, Vol. 2, No. 1
69
fluxes is an important quantitative approach in metabolic

engineering due to its capacity to characterize the flux distributions, reaction mechanisms and associated parameters.
It is possible to determine the intracellular fluxes by using isotope tracer methods. In isotopomer analysis, the biological system (of the microorganism) is fed with a specifically labeled substrate (usually 13C or 15N) during the stationary growth phase and the labeling patterns of the compounds in the central C or N metabolism (e.g., isotopic enrichment in the intracellular metabolite pools) are measured
after the labeling isotopic balances are reached. This technique is either based on nuclear magnetic resonance or mass
spectrometry [14-20]. The resulting data, together with the
metabolite balancing, provide a large amount of additional
information regarding pathway identification (i.e., identification of active pathways, analysis of cyclic, parallel and split
pathways, etc.), flux distribution and subcellular compartmentation.
Analysis of Fluxes by Mathematical Tools
Intracellular fluxes can also be determined from the
measured extracellular metabolite fluxes (e.g., the fluxes of
substrates into the cells and the fluxes of metabolites that are
secreted out of the cells) using mathematical tools such as
biochemical systems theory [21-25], metabolic control
analysis [26-29], metabolic flux analysis [30-35] and cybernetic modeling [36-42]. With the exception of metabolic flux
analysis (MFA), these approaches require information on the
kinetics of the individual reactions although the level of detail in the functional form of kinetics may be rather low.
The computational study of metabolic systems is a subject with a long history [43]. Mathematical methods for sensitivity analysis of metabolic regulation began in the late
1960s [44-46] and led to the biochemical systems theory
(BST). All processes are represented as products of powerlaw functions (either S-systems or the alternative variant of
generalized mass action systems, GMA) in BST to investigate metabolic and gene-regulatory systems. A major advantage of the BST approach is that it does not require a priori
structural information on the underlying pathway and models
are designed based solely on the identity of the reactants and
their reactional and regulatory interconnections.
The metabolic control analysis (MCA) shares the same
fundamental definition with BST. Both methods use the
same experimental information to study the responses of
metabolic systems to changes in their parameters that can be
exerted by genetic manipulations, enzymatic titrations or
enzymatic inhibitors. MCA (also known as metabolic control
theory) assumes that there is a definite amount of flux control that emerges as a parameter of critical importance in the
identification of feasible enzymatic modifications having
maximal impact on the network flux. In metabolic systems,
this control is normally spread quantitatively among several
enzymes in the system. Since its introduction three decades
ago, MCA has received much theoretical and experimental
attention since it allows us to understand how metabolic
fluxes are controlled by certain enzyme activities and metabolite concentrations [47].
Assuming that cellular metabolism is at a steady state,
MFA (also referred to as flux balance analysis, FBA) allows
us to calculate the unknown internal fluxes over each reac-
70 Current Chemical Biology, 2008, Vol. 2, No. 1
Altintas et al.
Fig. (1). The necessary steps to construct a metabolic model.
tion using data on external fluxes, metabolic stoichiometry

and mass balances around intracellular metabolites. Besides
quantification of pathway fluxes, MFA is useful for the identification of the rigidity (or flexibility) of key metabolic
branch points, the determination of non-measured extracellular fluxes, the calculation of maximum theoretical yields, the
identification of alternative pathways and the observation of
the function of metabolic pathways in vivo [6, 48, 49]. Although MFA has the ability to estimate the unmeasured internal fluxes, it does not incorporate any kind of regulation
mechanisms. On the other hand, MFA can incorporate additional information when it is available and is a powerful
technique when used in conjunction with novel physiological
experiments.
Another powerful methodology for describing the complex phenomena observed in biological cells is the cybernetic modeling framework which was developed by Ramkrishna and coworkers [50-52]. The cybernetic approach is
based on the idea of proposing an optimal mechanism such
that the cell regulates the synthesis and activity of the key
enzymes to promote perceived local or global cellular objective. The application of cybernetic framework to metabolic
engineering requires limited kinetic information combined
with cybernetic variables that modify the rates of enzyme
production and activation to modify the metabolic reaction
rates. The definition of the cybernetic variables varies depending on the nature of the metabolic pathway being examined [40].
In the presence of detailed kinetic information about the
specific cellular processes (e.g. enzyme catalyzed reactions,
protein-protein interactions, or protein-DNA binding), it is
possible to combine kinetics with the known stoichiometry

of the model to interpret and predict cellular behavior [25,
53, 54]. In particular, a kinetic model is constructed by (i)
defining all reactions with their stoichiometry, (ii) specifying
the kinetics of each reaction, and (iii) setting up the proper
mass balances for each metabolite. One major drawback of
kinetic modeling is that a realistic kinetic modeling of metabolic networks needs mechanistically-correct rate equations
that require detailed knowledge of all physiological effectors
influencing the activity of the catalyzing enzyme. Moreover,
our biochemical knowledge is limited to the enzymatic properties in vitro which are very different from the conditions
inside the microorganism, in vivo. Therefore, kinetic modeling is not suitable for developing metabolic engineering
strategies in the absence of well-characterized kinetic parameters [55, 56]. On the other hand, once kinetically judged
and experimentally validated, these models replace timeconsuming and expensive experiments for accurate prediction of the fluxes and form a valuable basis for viewing the
whole pathway, which adds to our understanding of the system of interest [57].
Beyond their intrinsic differences, all these modeling
approaches provide a mathematical basis for the representation of the existing relationship between metabolic network
components (metabolites, enzymes, etc.) and the cellular
systemic behavior exhibited by the network of interest. As
metabolite levels are governed by changes in fluxes and enzyme activities, understanding the changes on a metabolic
level will then help in understanding the regulatory mechanisms that lead to these changes in fluxes [58]. The metabolic model can thus form a solid biochemical basis on com-
puting the implications of the functions of the cell, such as

signaling and regulatory networks.
Despite the enormous potential and success of these approaches, the components of a system and their interactions
in the cellular network need to be studied further in order to
have a system-wide picture and system-level understanding
of biology [59, 60]. It is important as many properties of life
arise at the systems level only. More recently, systems biology has emerged as an integrative approach that investigates
pathways and networks by combining data about genes, proteins, enzymes and metabolites to generate a comprehensive
picture of the system (e.g., tissue, organ or organism) as a
whole [61-71].
Because of the biological complexity, system biology
enables a wide spectrum of mathematical modeling techniques such as the ones discussed above and bioinformatics
to visualize large networks of cellular components and generate hypotheses about the cell behavior. These hypotheses
can then be tested experimentally in systems biology framework by integrating the detailed information about the entire
genome of an organism (genomics), small molecules that
cells assimilate or synthesize (metabolomics), cellular proteins and their physical interactions (proteomics), the temporal and spatial distribution of all gene transcripts (transcriptomics) and the data from other accompanying highthroughput experiments.
METABOLIC MODELING OF HEALTHY AND DISEASED PODOCYTES
Podocytes, also known as the visceral glomerular epithelial cells, are terminally differentiated cells with a complex
cellular architecture contributing to many functions of the
71
normal kidney glomerulus [72]. They possess a highly

branched array of actin-rich foot processes (FP) that are essential to glomerular filtration on the kidney. The FPs of
neighboring podocytes regularly interdigitate, leaving between filtration slits that are bridged by an extracellular
structure, known as the slit diaphragm (SD). The FPs are
anchored on the outer aspect of the glomerular basement
membrane (GBM) (Fig. 2). Therefore, podocytes define the
final barrier to protein loss, which explains why podocyte
injury is typically associated with marked urinary protein
loss (proteinuria) which is a risk factor for morbidity and
mortality in these patients. Characteristically, podocyte FPs
are rearranged upon injury leading to loss of FP interdigitations and forming a flattened sheet of cytoplasm above the
GBM (referred to as FP effacement). If FP effacement is
reversed, coordinated kidney filtration is re-established.
However, in many cases, FP effacement progresses into
more severe and irreversible podocyte injury resulting in
progressive kidney failure. The metabolic signatures that are
involved to maintain normal and diseases podocyte structures need to be defined to better understand and treat proteinuric kidney disease.
Podocytes are terminally differentiated cells and their
metabolism is complicated and its understanding is just beginning. These complexities render the material balances for
podocyte cell cultivation a difficult problem to solve. Nevertheless, detailed material balance analysis in podocyte cell
culture systems would be desirable to provide insights toward a better understanding of podocyte cell metabolism.
One means for characterizing the intracellular metabolism of
cultured podocytes under normal and disease conditions is
the identification of the flux distributions by MFA as it
Fig. (2). Podocytes are highly specialized cells within the glomerulus that are essential for ultrafiltration. They form foot processes (FP) that
are highly dynamic cellular extentions. FPs, that rest on glomerular basement membrane (GBM), are interconnected by the slit diaphragms
(SD) to form the final component of the kidney permeability barrier.
Table 1.
Altintas et al.
Application of MFA to Different Microorganisms

Organism
Bacterium
Bacillus licheniformis
Bacillus subtilis
Clostridium acetobutylicum
Clostridium cellulolyticum
Corynebacterium glutamicum
Corynebacterium melassecola
Escherichia coli
Lactobacillus lactis
Streptomyces coelicolor
Streptomyces lividans
Zymomonas mobilis
Yeast
Aspergillus niger
Aspergillus oryzae
Candida milleri
Candida tropicalis
Penicillium chrysogenum
Saccharomyces cerevisiae
Saccharomyces kluyveri
Mammalian cells
BHK cells
CHO cells
HEK 293 cells
Hybridomas
Organ systems
Adipose tissue
Brain
Heart
Liver
Skeletal muscle
MFA Application
Ref.
Serine alkaline protease fermentation

Analysis of the energetic efficiency of growth
Glucose metabolism
Riboflavin production
Acetone fermentation
Fermentation characterization of the recombinant strains
Cellulose metabolism
Glutamate production
Lysine production from glucose
Lysine production from glucose and fructose
Lysine production from sucrose
Metabolism of mutant cells
Glucose and fructose metabolism
Acetate secretion under ATP maximization conditions
Byproduct secretion under various oxygenation rates
Effects of gene additions or deletions on metabolism
Effects of plasmid maintenance on metabolism
Growth and metabolism
Polyphosphate metabolism
Succinic acid/succinate production
Lactic acid production
Actinorhodin production
Calcium dependent antibiotic (CDA) production
Glucose, fructose and xylose metabolism
[134]
[135]
[136]
[137-140]
[141-143]
[144]
[145, 146]
[147-149]
[150-157]
[158]
[159]
[160, 161]
[162]
[163, 164]
[165]
[166-175]
[176]
[177-181]
[182]
[183-185]
[186]
[187]
[188]
[189, 190]
[191]
Glucose and glutamate metabolism

Nitrogen metabolism
Xylose metabolism
Dicarboxylic acid (DCA) production
Xylose metabolism
Penicillin production
Glucose metabolism and ethanol production
Growth on different carbon sources
Xylose metabolism and ethanol production
Nitrogen metabolism and ethanol production
Starch metabolism and ethanol production
Glycerol production
Heterologous protein production
Glucose metabolism
[192]
[193]
[194]
[195]
[196]
[197-200]
[201-206]
[207]
[205, 208-213]
[214]
[215]
[216]
[217]
[218]
Glucose and glutamine metabolism

Glycosylation
Adenoviral vector production
Effects of genetic engineering on metabolism
Glucose and glutamine metabolism
Energy metabolism
Glutamate production
Medium design and optimization
Response to changing medium composition
[219]
[220]
[111, 221, 222]
[223]
[113, 132]
[112]
[224]
[225]
[108-110, 226-228]
[229-233]
[107, 234]
Adipocyte formation
Fat synthesis
Glutamate metabolism
Neuron-astrocyte coupling
Characterization of the human mitochondrial metabolic network
Mitochondrial network properties in health and disease
Analysis of the hypermetabolic state
Collagen synthesis
Hepatocyte function in plasma
Interhepatic metabolism in liver failure
Postburn hepatic metabolism
Analysis of the postburn hypermetabolic state
[235]
[236]
[237]
[238]
[239]
[240]
[241-243]
[244]
[114, 245]
[115, 246]
[247-249]
[250]
offers the advantage of simplicity, i.e., it solely relies on the

known stoichiometry of a given biochemical reaction network. This approach has been employed successfully to analyze the metabolic physiology and behaviors of a variety of
species, from single-celled to multicellular organisms with
complex organ systems (Table 1).
Response of Podocytes to Injury
Podocytes are injured in many forms of human and experimental glomerular disease [73-76], including minimal
change disease (MCD), focal segmental glomerulosclerosis
(FSGS), diabetes mellitus (DM), membranous glomerulopathy (MG), crescentic (rapidly progressive) glomerulonephritis (CGN, RPGN) and lupus nephritis (LN). The early events
are characterized by alteration in SD and FP configuration,
resulting in FP effacement and loss of SD integrity. FP effacement is associated with the onset of proteinuria and is
accompanied by a reorganization of the actin cytoskeleton
into a dense network [77]. These early changes are fully reversible [78, 79].
In 1957, Farquhar et al. [80] were the first to describe
extensive FP effacement in biopsies of patients with nephrotic syndrome. Since then, FP effacement has been subject
of extensive investigation in humans and in animal models.
Based on recent insight into the molecular pathology of
podocyte injury, at least six major causes of FP effacement
and proteinuria can be identified: (i) interference with the
negative surface charge of podocytes [81-84] or slit membrane [85, 86]; (ii) interference with the activity of GLEPP1,
a membrane bound tyrosine phosphatase [87]; (iii) interference with the actin cytoskeleton and its associated protein
alpha-actinin-4 [88, 89]; (iv) interference with the GBM or
the podocyte-GBM interaction [90-97]; (v) interference with
the SD complex [98-100] and (vi) induction of danger signals in podocytes via upregulation of podocyte B7-1 [101].
Disease Models Under Investigation
In order to analyze the relationship of podocyte structure
and their central metabolism and the pathogenetic mechanisms of podocyte FP effacement, we utilize three in vitro
disease models: (i) podocytes with alpha3 integrin deletion
(genetic model [102]), (ii) podocytes treated with lipopolysaccharides (LPS, immunological model [103]), (iii) podocytes treated with puromycin aminonucleoside (PAN, toxic
model [104]). The common denominator of all these cell
models is the rearrangement of the actin cytoskeleton.
The deletion of alpha3 integrin in mice leads to severe
podocyte FP effacement and perinatal death of mutant mice
from kidney and lung failure [91]. We have developed conditionally immortalized mouse podocyte cell lines from these
mice. These cells have functional and structural deficits and
serve as a genetic in vitro model of podocyte damage [102].
In a recent study, we have established that these cells have
an induced expression of the protease cathepsin L. Moreover, these cells exhibit different cytoskeletal dynamics, e.g.
increased cellular motility when compared to wildtype podocytes.
Similar structural changes as seen in alpha3 integrin deficient podocytes can be achieved by treating wildtype podocytes with PAN. PAN treatment leads to the reorganization
of the actin cytoskeleton, focal contacts, and SD proteins in
73
cultured podocytes [104]. We also observed that application

of PAN to wildtype podocytes for 48 hours caused a dosedependent increase in autologous B7-1 expression, a transmembrane protein previously known as a B-cell costimulatory molecule. B7-1 in podocytes is capable to induce FP
effacement and disruption of the SD complex, thereby modifying glomerular permselectivity [103]. In another work, we
reported that PAN treatment strongly increased the migration
of wildtype podocytes [102].
The application of LPS to cultured podocytes represents
a more recent model. Podocytes sense LPS by expressing
toll-like receptor-4 (TLR-4) and its coreceptor CD14, which
in turn triggers B7-1 expression and the reorganization of the
podocyte actin cytoskeleton. Most significantly, LPS injection caused podocyte FP effacement and severe proteinuria
within 24 hours when injected into mice. The LPS-induced
proteinuria is transient and returns to base-line levels after 72
hours [103]. In summary, the above models have similar
structural deficits in common but the underlying pathways
are diverse. These models are therefore well suited to dissect
different metabolic profiles for a similar appearing structural
disease condition.
Podocyte Metabolic Network
Traditional MFA starts with a predetermined metabolic
network. Although the central metabolic pathways are well
known, the network models used in the literature differ due
to the simplifications made in the case of non-identifiable
fluxes. Those simplifications are made by either lumping the
reactions in a pathway that can not be determined independently or assuming unidirectionality (e.g., zero exchange flux)
in some fluxes, generally the ones belonging to product formations [105]. Also, there are cases where a pathway or reaction may assumed to be inactive or not expressed [106].
The metabolic network model for podocytes (Fig. 3), is a
moderately detailed model that we use as a basis for quantification of the fluxes for the cultured podocyte cells. It is an
adaptation of a network previously used for hybridomas
[107-110], CHO cells [111], HEK 293 cells [112, 113] and
hepatocytes [114]. In contrast to CHO cells, HEK 293 cells
and hepatocytes, podocytes represent highly differentiated
cells. This means that the metabolism of podocytes is tailored to maintain such a differentiated phenotype and may
quantitatively differ from proliferative cells such as HEK
293 cells. On the other hand, the operative metabolic enzyme
composition is likely similar between various mammalian
cell types, so that we decided to use available information of
mammalian cells to construct our podocyte metabolic network. This network-adaptation approach was employed successfully to analyze the podocyte behavior by manually adding or removing the corresponding pathways in the metabolic map.
In the model, there are 40 intracellular fluxes and 24
fluxes for transport rates and biosynthesis rates that can be
measured. The transport fluxes are formally defined for each
measured metabolite (all 20 amino acids, glucose, lactate,
ornithine and citrulline), and each rate is defined with a positive sign for production. Each extracellular metabolite is
linked to its intracellular counterpart metabolite pool. Sixtythree metabolites constitute the nodes for pseudo steady-state
mass balances. Intracellular fluxes are overall biochemical
Altintas et al.
Fig. (3). The metabolic network model for podocyte cultures. Arrows indicate the direction of reaction.
reactions representing kidney-specific functions and pathways: glycolysis (flux nos. 1 to 5), reduction of pyruvate to
lactate (no. 6), Krebs cycle (nos. 7 to 14), urea cycle (nos. 15
to 17), amino acid degradation (nos. 18 to 37), pentose phosphate pathway (no. 38), oxygen uptake and electron transport
(nos. 39 and 40) (Fig. 3). All pathways were verified as feasible for Mus musculus by online bioinformatic databases
[12, 13].
Formulation of the Model
MFA starts with setting up a stoichiometric matrix for a
network of reactions occuring in the cell. Then, the mass
balance constraints around intracellular metabolites are
specified. These constraints identify a series of linear equations of individual reaction fluxes that must be fulfilled to
enable steady state criterion. Mathematically, the reaction
network and the mass balance constraints can be summarized

in the following matrix notation:
S11 S12
S r = :
:
Sm1 Sm2
S1n
r1

:
: : = 0
Smn mxn rn nx1
(Eq. 1)
S is the mxn stoichiometry matrix, with m as the number

of metabolites, and n as the number of reactions. The vector
r represents all the individual fluxes of intracellular and extracellular compounds (Fig. 4). In this equation, S11 denotes
the stoichiometric coefficient of the first metabolite in the
first reaction while S12 denotes for the stoichiometric coefficient of the first metabolite in the second reaction, etc. For
example, if the third metabolite participates only in its formation in reaction 3 and its disappearance in reaction 7 with
75
Fig. (4). Principles of metabolic flux analysis (MFA). MFA technique is based on relatively simple linear algebra. If the stoichiometry of the
relevant intracellular reactions and the cellular composition are known, and the uptake and secretion rates of the relevant metabolites (rates
denoted by e in the figure) have been measured, the reaction rates (rates denoted by r in the figure) can be determined using the appropriate mass balance equations.
the stoichiometric coefficient of 1, then S33 = -S37 = 1, while

all other coefficients in that row are zero. The right hand side
of this equation is made equal to zero assuming the cultured
podocytes are in metabolic steady state where the intracellular levels of metabolites are constant [48].
By separating r into measured and unknown components,
and rcalc, respectively, and partitioning matrix S into
r
meas
and Scalc, where they contain the stoichiometric coeffiS
cients of measured and unknown reactions (i.e., internal and
transport fluxes), respectively, we obtained:
meas
Scalc x rcalc + Smeas x rmeas = 0
(Eq. 2a)
and
Scalc x rcalc = Smeas x rmeas
(Eq. 2b)
calc
is not a square matrix, i.e., the number of

Because S
rows is greater than the number of columns, Eq. (2b) cannot
be solved by simple inversion. One approach was to use the
Moore-Penrose pseudo-inverse method [48], in which each
side of Eq. (2b) is multiplied by the transpose of Scalc:
(Scalc)T x Scalc x rcalc = (Scalc)T x Smeas x rmeas
calc
The matrix multiplier of r (i.e., (S

invertible, and Eq. (3) is easily solved:
calc T
) xS
(Eq. 3)
calc
) is now
rcalc = ((Scalc)T x Scalc)-1 x (Scalc)T x Smeas x rmeas
(Eq. 4)
Since the system of linear equations is overdetermined

(more equations than unknown fluxes), fluxes are determined by linear regression [48, 115].
Proteome Analysis to Complement MFA
Proteomics is one among various omics fields that allows the analysis of complex mixtures of proteins, for example from cell lysates, subcellular fractions or immunoprecipitated protein complexes [116-118]. The general approach to
cellular proteomics is characterized by (i) protein extraction
from a crude homogenate, (ii) separation of the intact proteins (e.g. by 2D-PAGE) or peptide fragments of specific
proteolytic digestion (e.g. by trypsin digestion followed by
liquid chromatography) and (iii) identification of these proteins by mass spectrometry and bioinformatics [119]. Quantitative comparisons of two or more proteomes are possible
if the protein samples are distinctly labeled, stoichiometrically combined, and the mixture is then separated and analyzed [120, 121]. While 2D-PAGE has been the central technology in proteomics for over 30 years, there have been tremendous developments in liquid chromatography-mass spectrometry (LC-MS) to combine separation and analysis into a
single on-line process capable of the rapid identification of

hundreds or thousands of proteins from cells [122].
Ransom [123] attempted to construct a whole cellular
proteome map for podocyte cells for the first time. He and
his coworkers performed a differential analysis using 2DPAGE of changes in protein expression in cultured murine
podocytes in response to glucocortioids [124]. A total of 106
proteins representing 88 unique proteins were identified in
their podocyte proteome map.
To elucidate the molecular basis of biological processes,
the analysis of dynamic changes of the subcellular distribution of proteins is necessary [125]. In order to describe the
subcellular proteome of the podocyte and to identify podocyte proteins whose expression is altered by toxic challenges, cytoplasmic andmitochondrial proteome analysis of
wildtype and disease models of cultured podocytes were
performed by LC-MS. The results are summarized in Fig. 5.
Organellar proteomics is a valuable tool to complement and
substantiate MFA studies in cultured cells, i.e. podocytes.
Comparison of organellar proteomes in healthy and diseased
podocyte cells will complement metabolic studies and provide further insights into disease pathogenesis. Such a comparison will also lay ground for additional functional studies
to prove or adapt results obtained by MFA. In particular,
quantitative changes in proteome permit us understand
measureable changes in metabolite fluxes at the enzyme
level.
Evaluation of the Model
In batch cultures of diseased podocytes, we observed an
increased use of both glucose and glutamine when compared
to the healthy cells. An increase in the oxidation of glucose
and glutamine in diseased cells promoted higher utilization
of other amino acids as well. MFA showed that essential
amino acids contributed to about 80-90% of the total amino
acid consumption and glutamine is needed to supply the essential amino acids in diseased cells. On the other hand, the
nitrogen-balance calculations revealed that the amount of
nitrogen incorporated by podocytes (mainly from glutamine
uptake) exceeded significantly the amount of nitrogen removed as glutamate, alanine and aspartate in disease models.
The ammonia measurements in disease podocytes correlated
very well with the nitrogen-balance calculations suggesting
that a considerable part of nitrogen is released in the form of
ammonia. This ammonium buildup as a result of high glutamine consumption in diseased podocytes makes sense
since glutamine metabolism and glutamine chemical decomposition are the primary sources of ammonium buildup in the
cell cytoplasm and the culture media [126] and specific production rate of ammonium is closely correlated to glutamine
[127]. Schneider et al. [128] summarized a large number of
articles dealing with numerous different effects caused by
elevated ammonia and ammonium concentrations on cell
cultures. The challenge here is to generate or employ a metabolic engineering tool to help diseased cells alleviate ammonia stress.
High glucose and glutamine uptake and the large amount
of lactate release by diseased podocyte cultures prompted us
to think about the possibility that these cells consume
amounts of glucose and glutamine higher than that required
to satisfy their energy and biosynthetic needs which may
Altintas et al.
result in an accumulation of lactate and ammonia in the medium. If the metabolism of glucose or glutamine can be improved, then it is possible to obtain higher consumption rates
of these nutrients and lower lactate and ammonia production.
It is also known that there is a close interrelation of the consumption of these two nutrients by the cells to satisfy their
carbon and nitrogen demands [129-132]. One way to direct
carbon sources effectively through pathways other than the
lactate production pathway is to promote the conversion of
pyruvate to acetyl-CoA and CO2 via pyruvate dehydrogenase
(Fig. 3). This branching enzyme from glycolysis into the
TCA cycle is not active in mammalian cell lines [133] as
validated by our proteome analysis. The stable expression of
pyruvate dehydrogenase in podocyte cells would improve
the utilization of glucose and limit the production of lactate
and ammonia, which are deleterious to the cell.
In mammalian cells, the removal of amino nitrogen from
the glutamate family of amino acids (arginine, ornithine,
proline, histidine and glutamine) is achieved through a welldescribed transdeamination system involving aspartate
transaminase (catalyzing the transfer of an amino group from
glutamate to oxaloacetate, forming alpha-ketoglutarate and
aspartate) and glutamate dehydrogenase (catalyzing the reversible oxidative deamination of glutamate to alphaketoglutarate and ammonia). Proteomic analysis revealed
that these two enzymes were down-regulated in PAN-treated
cells indicating the possibility of ammonia accumulation in
these cells which is also pointed out by the comparative
MFA on untreated (control model) and PAN-treated (toxic
model) cells (Fig. 5).
CONCLUSION
The glomerular podocyte is increasingly recognized as a
primary determinant of many glomerular diseases (e.g., focal
segmental glomerular sclerosis, glomerulonephritis, membranous nephropathy, glomerular hypertension, etc.) leading
to chronic kidney disease. Recent discoveries have highlighted the importance of podocyte proteins in maintaining
the glomerular filtration barrier in both health and disease.
Although the response of podocytes to injury was studied
thoroughly using molecular biology and structural morphology research tools, the metabolic signatures of podocyte FP
effacement are not known. Metabolic analysis with a clear
correspondence between disease and the related metabolic
changes by employing metabolic flux analysis (MFA) and
proteomic analyses help to define important metabolic pathways in healthy and diseased podocytes and can also be employed for other eukaryotic disease models.
Although MFA has been applied extensively to study the
cellular function (i.e., to increase the production of the primary or secondary metabolites, to produce desired chemicals
from less expensive feedstocks, to generate alternative pathways for the production of specialty chemicals, etc.) from
bacteria, yeasts, fungi, and mammalian cells, its application
to problems in physiology and medicine is less well recognized [6]. Here, we developed a thorough stoichiometric
model and used MFA to obtain a quantitative estimate of
stationary metabolic flux rates without knowledge of the
detailed kinetics of individual reactions, which are usually
accompanied with problems concerning availability and in
vivo/in vitro discrepancies. The proposed metabolic model-
77
Fig. (5). Relative abundances of some key enzymes in the podocyte metabolic network in health and disease. Subcellular proteome data were
obtained from wildtype and disease (toxic) models of cultured podocytes, respectively.
ing has the capacity to determine the metabolic capabilities

of podocytes under normal and disease conditions by employing the framework of metabolite balancing in combination with linear programming methods. The changes observed in the profiles of glucose, glutamine and ammonia
give credence to the indication that not only the structure
(e.g., morphology) but also the metabolism of podocyte cells
is changed in disease.
Reiser). J. Reiser was also supported by the KMD foundation. M. M. Altintas was supported by an NIH training grant
and T32DK007540.
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68

Current Chemical Biology, 2008, 2, 68-82

Emerging Roles for Metabolic Engineering - Understanding Primitive and

Department of Chemical Engineering, Bogazici University, Bebek 34342 Istanbul, Turkey

potential to create a robust computational biology by the

Emerging Roles for Metabolic Engineering

Current Chemical Biology, 2008, Vol. 2, No. 1

fluxes is an important quantitative approach in metabolic

70 Current Chemical Biology, 2008, Vol. 2, No. 1

Fig. (1). The necessary steps to construct a metabolic model.

tion using data on external fluxes, metabolic stoichiometry

possible to combine kinetics with the known stoichiometry

Emerging Roles for Metabolic Engineering

puting the implications of the functions of the cell, such as

Current Chemical Biology, 2008, Vol. 2, No. 1

normal kidney glomerulus [72]. They possess a highly

72 Current Chemical Biology, 2008, Vol. 2, No. 1

Application of MFA to Different Microorganisms

Serine alkaline protease fermentation

Glucose and glutamate metabolism

Glucose and glutamine metabolism

Emerging Roles for Metabolic Engineering

offers the advantage of simplicity, i.e., it solely relies on the

Current Chemical Biology, 2008, Vol. 2, No. 1

cultured podocytes [104]. We also observed that application

74 Current Chemical Biology, 2008, Vol. 2, No. 1

network and the mass balance constraints can be summarized

S is the mxn stoichiometry matrix, with m as the number

Emerging Roles for Metabolic Engineering

Current Chemical Biology, 2008, Vol. 2, No. 1

the stoichiometric coefficient of 1, then S33 = -S37 = 1, while

Scalc x rcalc + Smeas x rmeas = 0

is not a square matrix, i.e., the number of

The matrix multiplier of r (i.e., (S

rcalc = ((Scalc)T x Scalc)-1 x (Scalc)T x Smeas x rmeas

Since the system of linear equations is overdetermined

76 Current Chemical Biology, 2008, Vol. 2, No. 1

single on-line process capable of the rapid identification of

Emerging Roles for Metabolic Engineering

Current Chemical Biology, 2008, Vol. 2, No. 1

ing has the capacity to determine the metabolic capabilities

We also performed a differential proteomic analysis of

78 Current Chemical Biology, 2008, Vol. 2, No. 1

Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The

Varner J, Ramkrishna D. Metabolic engineering from a cybernetic

Emerging Roles for Metabolic Engineering

Liu ET. Systems biology, integrative biology, predictive biology.

Current Chemical Biology, 2008, Vol. 2, No. 1

focal segmental glomerulosclerosis. J Am Soc Nephrol 2000; 11:

80 Current Chemical Biology, 2008, Vol. 2, No. 1

Palmer-Toy DE, Kuzdzal S, Chan DW. Proteomic approaches to

Desai RP, Nielsen LK, Papoutsakis ET. Stoichiometric modeling of

Emerging Roles for Metabolic Engineering

thase in batch and continuous cultures. Biotechnol Bioeng 1999;

Current Chemical Biology, 2008, Vol. 2, No. 1

Kim HB, Smith CP, Micklefield J, Mavituna F. Metabolic flux

82 Current Chemical Biology, 2008, Vol. 2, No. 1

Received: December 7, 2006

Xie L, Wang D. Stoichiometric analysis of animal cell growth and

Revised: February 1, 2007

Accepted: March 12, 2007

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