BBA - General Subjects 1864 (2020) 129687

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BBA - General Subjects

journal homepage: www.elsevier.com/locate/bbagen

Kinetic modeling of glucose central metabolism in hepatocytes and T

hepatoma cells
Álvaro Marín-Hernándeza, , Juan Carlos Gallardo-Péreza, Marco Antonio Reyes-Garcíaa,
⁎

Marcela Sosa-Garrochob, Marina Macías-Silvab, Sara Rodríguez-Enríqueza,

Rafael Moreno-Sáncheza, Emma Saavedraa,
⁎

a
Departamento de Bioquímica, Instituto Nacional de Cardiología, Mexico City 14080, Mexico
b
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico

ARTICLE INFO ABSTRACT

Keywords: Background: Kinetic modeling and control analysis of a metabolic pathway may identify the steps with the
Glycogen synthase highest control in tumor cells, and low control in normal cells, which can be proposed as the best therapeutic
Glycogen phosphorylase targets.
Phosphoglucomutase Methods: Enzyme kinetic characterization, pathway kinetic modeling and control analysis of the glucose central
Hexose-6- phosphate isomerase
metabolism were carried out in rat (hepatoma AS-30D) and human (cervix HeLa) cancer cells and normal rat
Metabolic Control Analysis
hepatocytes.
Oxamate
Results: The glycogen metabolism enzymes in AS-30D, HeLa cells and hepatocytes showed similar kinetic
properties, except for higher AS-30D glycogen phosphorylase (GP) sensitivity to AMP. Pathway modeling in-
dicated that fluxes of glycogen degradation and PPP were mainly controlled by GP and NADPH consumption,
respectively, in both hepatocytes and cancer cells. Likewise, hexose-6-phosphate isomerase (HPI) and phos-
phoglucomutase (PGM) exerted significant control on glycolysis and glycogen synthesis fluxes in cancer cells but
not in hepatocytes. Modeling also indicated that glycolytic and glycogen synthesis fluxes could be strongly
decreased when HPI and PGM were simultaneously inhibited in AS-30D cells but not in hepatocytes.
Experimental assessment of these predictions showed that both the glycolytic and glycogen synthesis fluxes of
AS-30D cells, but not of hepatocytes, were inhibited by oxamate, by inducing increased Fru1,6BP levels, a
competitive inhibitor of HPI and PGM.
Conclusion: HPI and PGM seem suitable targets for decreasing glycolytic and glycogen synthesis fluxes in AS-30D
cells but not in hepatocytes.
General significance: The present study identified new therapeutic targets within glucose central metabolism in
the analyzed cancer cells, with no effects on non-cancer cells.

1. Introduction mechanisms in cancer [1–6]. Glycogen allows cells to contend with

One of the typical characteristics that distinguish cancer cells from
normal cells is an accelerated glycolysis. However, other pathways of
the glucose central catabolism such as glycogen metabolism also show
changes in the expression of enzyme isoforms and regulatory
nutritional stress and also acts as an osmotically silent polymer for
glucose storage. In diverse human tumors (kidney, colon, rectum) [3,4]
and cancer cell lines [7], high levels of glycogen in comparison to
normal cells and tissues have been reported. The increase in glycogen
content correlates with a high activity of glycogen synthase (GS) in

Abbreviations: 2-DG, 2-deoxy-glucose; DHAP, dihydroxyacetone phosphate; Ery4P, erythrose-4-phosphate; Fru6P, fructose-6-phosphate; Fru1,6BP, fructose-1,6-
bisphosphate; Fru2,6BP, fructose-2,6- bisphosphate; Glc, glucose; GLUT, glucose transporter; Glc1P, glucose-1-phosphate; Glc6P, glucose-6-phosphate; Glc6PDH,
glucose-6-phosphate dehydrogenase; 6PG, 6-phosphogluconate; 6PGDH, 6-phosphogluconate dehydrogenase; G3P, glyceraldehyde-3-phosphate; Glc16BP, glucose-
1,6-bisphosphate; GS, glycogen synthase; GP, glycogen phosphorylase; GR, glutathione reductase; GPx, glutathione peroxidase; HPI, hexose phosphate isomerase;
HK, hexokinase; LDH, lactate dehydrogenase; NDK, nucleoside diphosphate kinase; PPP, pentose phosphate pathway; PEP, phosphoenolpyruvate; PGM, phos-
phoglucomutase; PYK, pyruvate kinase; Pyr, pyruvate; UGP, UDP-glucose pyrophosphorylase; UDP-Glc, UDP-glucose; Xu5P, xylulose-5-phosphate
⁎
Corresponding authors at: Instituto Nacional de Cardiología Ignacio Chávez, Departamento de Bioquímica, Juan Badiano No. 1, Col. Sección XVI, Tlalpan, Mexico
City 14080, Mexico.
E-mail addresses: marinhernndez@yahoo.com.mx (Á. Marín-Hernández), emma_saavedra2002@yahoo.com (E. Saavedra).

https://doi.org/10.1016/j.bbagen.2020.129687
Received 8 January 2020; Received in revised form 28 June 2020; Accepted 20 July 2020
Available online 23 July 2020
0304-4165/ © 2020 Elsevier B.V. All rights reserved.
Á. Marín-Hernández, et al.
human renal clear cell carcinomas [3]. In addition, some cancer cells
(Ehrlich ascites, HT29) are not sensitive to glucagon and epinephrine,
the main hormones that stimulate glycogen degradation [2] or to in-
sulin that induces glycogen synthesis [8].
A high glycogen level: (i) improves cancer cell survival under stress
conditions such as hypoxia [9,10]; (ii) correlates with radio-resistance
and metastasis [11,12]; and (iii) may exert significant control on the
glycolytic flux through its degradation [13,14]. For these reasons, tar-
geting of glycogen metabolism could provide a novel strategy for an-
ticancer treatment.
The changes in glycogen metabolism of cancer cells can be attrib-
uted to activation of some oncogenes (Rab25) and/or transcriptional
factors (HIF-1α). Rab25 oncogene induces glycogen storage in ovarian
cancer cells to maintain ATP levels during starvation [15], whereas
HIF-1α is a positive transcriptional regulator of the genes encoding
enzymes of glycogen synthesis such as phosphoglucomutase (PGM),
UDP-glucose pyrophosphorylase (UGP), glycogen branching enzyme
and GS [9,10]. Furthermore, expression of the GS1 (muscle) isoform has
been described in rat hepatomas [1]. In turn, prolonged hypoxia in-
duces glycogen phosphorylase (GP) expression by a HIF-1α in-
dependent mechanism [9]. It has been reported the dominant expres-
sion of the brain-type GP isoform (GPB) in human colorectal
carcinomas and gastric cancer [5]. GPB is also predominantly expressed
in 59 human cell lines of diverse origin, where low or null levels of liver
(GPL) and muscle (GPM) isoforms are observed [6]. The GPB isoform is
poorly activated by phosphorylation and is more sensitive to direct
activation by AMP than the liver and muscle isoforms [16]. In con-
sequence, glycogen degradation becomes stimulated in cancer cells by
nutritional stress conditions such as hypoglycemia and hypoxia. Hence,
the different kinetic properties of the GP and GS isoforms expressed in
cancer cells suggest changes and adjustments in the mechanisms of
control and regulation of the glycogen synthesis and degradation
pathways.
To understand how the glycogen metabolism in cancer cells is
controlled, kinetic metabolic modeling and the fundamentals of
Metabolic Control Analysis (MCA) can be applied. Kinetic modeling of a
metabolic pathway integrates detailed kinetic properties of individual
pathway enzymes and transporters into a computational model able to
simulate the metabolite concentrations and pathway fluxes found in a
cell under different physiological conditions.
Several kinetic models of glucose central metabolism in liver and
heart have been reported [17–22]. Some of these models are restricted
to a single direct pathway such as the pentose phosphate pathway (PPP)
[19]. In addition, these models have been built with kinetic parameters
collected from the literature and hence determined under different
conditions of pH, temperature and assay medium, or by parameter
optimization to fit metabolite and flux data [17–22]. Furthermore, in
the kinetic models where all relevant pathways of glucose central me-
tabolism were incorporated [17,18,20–22], analysis of the steps that
exert control of the pathway fluxes has not been generally undertaken.
The simultaneous interplay among the different components of a
pathway (i.e., all enzymes, transporters and metabolites) can reveal the
intrinsic properties and mechanisms that control both the pathway flux
and intermediary metabolite concentrations, and this can be carried out
by MCA [13,23,24]. Thus, experimentally validated kinetic metabolic
models that perform MCA allow elucidation of the mechanisms by
which a metabolic pathway is controlled and identify which enzymes
and transporters exert, and which others do not have, leading roles in
the pathway function. With this approach, the enzymes and transpor-
ters with the highest control in cancer cells, but low control in normal
cells, are identified and they can be proposed as the best and most
adequate therapeutic targets.
The main controlling steps of glycolysis in human (HeLa) and rat
(AS-30D) cancer cells were previously identified through kinetic mod-
eling and MCA [13,14]. Although the flux control coefficient values of
the main controlling steps might vary, depending on the environmental
2
BBA - General Subjects 1864 (2020) 129687
conditions (normoxia/hypoxia; hyper- normo- or hypo-glycemia) and
among different cancer cell lines, previous results have indicated that
the same steps preserve the main flux control of cancer glycolysis, but
with variable high values [13,14].
Then, the aims of the present study were (i) to produce a detailed
biochemical characterization of the glycogen metabolism in malignant
human (HeLa) and rat (AS-30D) cancer cells and (ii) to build an ex-
perimental- and mechanism-based kinetic model platform of glucose
central metabolism in cancer cells that includes, in addition to glyco-
lysis, the glycogen metabolism and the initial reactions of PPP. As some
of the enzymes of glucose metabolism in cancer cells have changed
their mechanisms of regulation (e.g. decreased sensitivity to physiolo-
gical inhibitors, lower affinity for products), due to expression of iso-
forms that differ from those found in normal cells [25], the experi-
mental analyses were also performed in fasted and fed hepatocytes, as
control.
Furthermore, the multi-pathway modeling analysis indicated that
HPI and PGM exert significant control on glycolytic and glycogen
synthesis fluxes, respectively in cancer cells (AS-30D) but not in normal
cells (rat fed hepatocytes). This observation suggested that both en-
zymes could be suitable therapeutic targets to specifically inhibit the
glucose metabolism in cancer cells.
2. Materials and methods
2.1. Chemicals
ATP, ADP, AMP, alanine, CaCl2, EGTA, Ery4P, Fru6P, Fru1,6BP,
Glc, Glc1P, Glc6P, Glc16BP, glycogen, Hepes, IGEPAL NP40, KCl, KOH,
L-lactate, Mops, NaCl, NADP+, NADH, Na2SO4, MgCl2, oxamate, PEP,
phenylalanine, SDS, sodium deoxycholate, Tris-HCl, UDP-Glc, 6PG, Pi
and PPi were purchased from Sigma Chemical (St. Louis, MO, USA).
Glc6P, PGM, HK, Glc6PDH, PYK and LDH were purchased from Roche
(Mannheim, Germany).
2.2. Isolation of cancer and liver cells
AS-30D hepatoma cells were isolated as described elsewhere
[13,26]. Hepatocytes were isolated by perfusion of isolated liver with
collagenase IV (Worthington; Lakewood, NJ, USA) from ad libitum fed
or 24 h-fasted male Wistar rats of 200–250 g weight [27]. HeLa cells
were cultured as described previously [14]. The cellular viability as-
sayed by trypan blue exclusion was ≥90% (AS-30D and HeLa cells) and
80–85% (hepatocytes). Animal manipulation was carried out in ac-
cordance with the recommendations stated by the Mexican Official
Standard NOM-062-ZOO-1999 norm.
2.3. Western blot assays
Cytosol-enriched fractions of cancer and normal cells (7–14 mg of
cytosolic protein) were suspended in RIPA lysis buffer (1× PBS pH 7.2,
1% IGEPAL NP40, 0.1% SDS and 0.05% sodium deoxycholate) plus
2.5 mM phenyl methanesulfonyl fluoride and 1 tablet of complete
protease inhibitors cocktail (Roche; Mannheim, Germany;
11697498001). Fifty μg protein of detergent-dissolved cytosol-enriched
fractions were further re-suspended in loading buffer (50 mM Tris-HCl
pH 6.8, 2% SDS, 10% glycerol, 0.002% bromophenol blue plus 5% β-
mercaptoethanol) and loaded onto a 10 or 12.5% polyacrylamide gel
under denaturing conditions. After SDS-PAGE separation, the proteins
were blotted onto PVDF membranes (Merck Millipore; Darmstadt,
Germany; IPVH00010) followed by immunoblotting at 4 °C overnight.
The following primary antibodies (1:500 or 1:1000 dilution) were used:
anti-PGM (Abcam; Cambridge, MA, USA; ab173588), anti-UGP (Abcam,
ab57686), anti-GS (Abcam, ab40867), anti-GP (Abcam, ab88078) and
anti-α-tubulin (Santa Cruz Biotechnology; Santa Cruz, CA, USA; sc-
5286). Hybridization bands were revealed with the corresponding
Á. Marín-Hernández, et al.
secondary antibodies conjugated with peroxidase (Santa Cruz
Biotechnology; sc-2005 and sc-2317). The signal was detected by
chemi-luminiscence using the ECL-Plus detection system (Amersham;
Little Chalfont, Buckinghamshire, UK; RPN2232). Densitometric ana-
lyses were performed with the Scion Image Software (Scion; Bethesda,
MD, USA).
To evaluate the phosphorylation status of GP, proteins were im-
muno-precipitated by incubating the cytosol-enriched fractions of AS-
30D, HeLa, fasted and fed hepatocytes with the anti-GP antibody (1 μg)
plus protein A (Sigma-Aldrich; P3391) for 1 h. Then, phosphorylation
was detected with anti-P-Ser antibody (Q5Phospho-Ser 1:100) (Qiagen;
Venlo, Netherlands; 37430) following the manufacturer instructions.
Densitometry analysis was performed using the Scion Image Software.
2.4. Enzyme activities
To evaluate enzyme activities in whole cell extracts of AS-30D,
hepatocytes and HeLa, the cells were disrupted by freezing in liquid N2
and thawing at 37 °C by three times. The enzyme activities were de-
termined as described below by using 0.01–0.2 mg of cell protein.
Cytosol-enriched fractions of cancer and liver cells were obtained ac-
cording to Marín-Hernández et al. [27]. In all enzyme determinations,
control reactions were undertaken to assess assay specificity by omit-
ting one of the enzyme's specific substrates; this spurious activity, if
any, was subtracted from the activity attained with the complete re-
action mix. The reaction was determined under conditions of initial
velocity (no product accumulation) and excess of coupling enzymes,
which prevented product transformation by other enzymes present in
the cell extract. Furthermore, it was ensured linearity of the activities
with respect to cell protein added and that the reaction was linear for
several minutes.
Enzyme activities were determined in KME buffer (120 mM KCl,
20 mM Mops, 0.5 mM EGTA, pH 7.0) at 37 °C by following the NAD(P)+
reduction or NAD(P)H oxidation at 340 nm in a spectrophotometer. In
our previous studies, a mean pH value of 7.0 was chosen because the
intracellular pH varies from 7.2 to 6.8 when cells are actively con-
suming glucose [13]. The PGM assay contained 0.5 mM NADP+, 2 mM
MgCl2, 1 U Glc6PDH and 0.01–0.05 mg protein of cytosol-enriched
fraction; the reaction was started by adding Glc1P (0.25–10 mM). The
0.1–5 μM Glc16BP activation and 0.25–3 mM Fru1,6BP inhibition on
PGM was evaluated at different Glc1P concentrations. The UGP activity
was assayed in the direction of Glc1P formation using 0.5 mM NADP+,
2 mM PPi, 2 mM MgCl2, 1 U PGM, 1 U Glc6PDH, 2 μM Glc1,6BP and
0.01–0.03 mg of cytosol-enriched fraction; the reaction was started by
adding UDP-Glc (0–0.6 mM); when PPi was varied (0.3–2 mM) UDP-Glc
was 0.5–0.6 mM. The GP activity was assayed using glycogen
(0.2–40 mM glucose equivalents), 2 mM MgCl2, 2 μM Glc1,6BP, 0.1 mM
AMP, 0.5 mM NADP+, 1 U PGM, 1 U Glc6PDH and 0.1–0.3 mg of cy-
tosol-enriched fraction; the reaction was started by adding Pi
(0.5–100 mM). The AMP (0.1–1.5 mM) activation on GP was evaluated
at different glycogen concentrations.
When the effect of alanine (10–30 mM) and phenylalanine
(1–2.5 mM) on PYK activity was determined, the assay contained 1 U
LDH, 5 mM MgCl2, 0.15 mM NADH, 1–3 μg of cytosol-enriched fraction
and 0.05–2 mM PEP. The reaction was started by adding 3 mM ADP.
When the effect of lactate on PYK activity was evaluated, the assay
contained 2 U Glc6PDH, 2 U HK, 0.5 mM NADP+, 5 mM MgCl2, 3 mM
ADP and 1–3 μg of cytosol-enriched fraction. After a baseline was at-
tained, the reaction was started by adding PEP (0.05–1.5 mM).
GS activity was determined by spectrofluorometry. The assay con-
tained 10 U PYK, 10 U LDH, 0.25 mM PEP, 15 μM NADH, glycogen
(10 mM glucose equivalents), 5 mM MgCl2 and 0.15–0.3 mg of cytosol-
enriched fraction. After a baseline was attained, the reaction was
started by adding UDP-Glc (0.05–5.9 mM). Glc6P (2–7.7 mM) activa-
tion was evaluated at different UDP-Glc concentrations.
To determine the kinetic parameters, one substrate was varied at
3
BBA - General Subjects 1864 (2020) 129687
different fixed-variable concentrations of the second substrate and vice
versa. In order to obtain the Ki and Ka values, inhibitors, activators and
substrates were also varied at different concentrations. To calculate the
kinetic parameters (Vmax, Km, Ka, α, β, Ki), the experimental data were
fitted by non-linear regression analysis to either the Michaelis-Menten,
non-essential mixed type activation, essential mixed type activation, or
simple competitive inhibition equations [28], using the Microcal Origin
v. 5 software.
2.5. Fluxes and metabolite concentrations
Suspensions of AS-30D cells (15 mg cell protein/mL) were incubated
in Krebs-Ringer (125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.4 mM CaCl2,
1 mM KH2PO4, 25 mM HEPES, pH 7.4) medium under orbital shaking at
150 rpm and 37 °C. After 1 min, a cellular sample was withdrawn
(t = 0), the remaining cell suspension was divided in two and one of
them was supplemented with glucose (1–30 mM) whereas the other
served as control (no glucose added). Samples were collected after 7.5,
15, 30 and 60 min and immediately mixed with ice-cold perchloric acid
(3% v/v), vortexed and centrifuged at 1800xg for 1 min at 4 °C. The
supernatants were neutralized with 3 M KOH/0.1 M Tris and stored at
−72 °C until use for determination of ATP, AMP, DHAP, Glc1P, Glc6P,
Glc1,6BP, G3P, Fru6P, Fru1,6BP, PEP, Pyr, UDP-Glc, PPi and L-lactate
contents as described by Bergmeyer [29]. In AS-30D cells, total lactate
production did not require correction provided by 2-deoxyglucose in-
hibition (lactate produced by glycolysis) because lactate production by
endogenous substrates is negligible in the absence of added glucose
[27]. For determination of Glc1P and UDP-Glc content, it was required
to use a spectrofluorometer instead of a spectrophotometer. For gly-
cogen determination, cellular samples (500 μL), withdrawn at the same
times indicated before, were centrifuged at 20,800 xg for 20 s. The cell
pellets were frozen in liquid nitrogen and kept at 70 °C until use. Gly-
cogen content was determined according to Marín-Hernández et al.
[26]. The Glc1,6BP content was determined by the fluorometric method
described by Passonneau et al. [30]. This last method is based on the
stimulation of PGM activity by Glc1,6BP. Glc1,6BP was determined at
37 °C in 50 mM imidazole-HCl, pH 7.0, containing 1 mM MgCl2, 0.1 mM
EDTA, 0.01% bovine serum albumin, 0.05 mM NADP+, 10 mU PGM
and 50–100 μL of neutralized supernatants previously stored at −72 °C.
The reaction was started by adding 15 μM G1P. The Glc1,6BP content
was calculated by interpolation from a standard curve of Glc1,6BP
(4.9–490 picomoles).
To assess the effect of oxamate on glycolysis and glycogen meta-
bolism, AS-30D cells and hepatocytes (15 mg cell protein /mL) were
incubated in the absence or presence of 10 mM oxamate in Krebs-Ringer
buffer without glucose for 15 min under orbital shaking and 37 °C.
Thereafter, an aliquot was withdrawn (t = 0) and 5 mM glucose was
added to the remaining cell suspension; 15 min later another aliquot
was removed and frozen in liquid N2. The aliquots were immediately
processed as described above for determination of glycogen, Fru1,6BP,
ATP and L-lactate contents.
In hepatocytes, the glycogen synthesis flux was determined by using
a radio-isotopic method because the enzymatic method was unable to
detect changes in the content of glycogen [26]. Hepatocytes (15 mg cell
protein /mL) were incubated in Krebs-Ringer buffer without glucose in
the absence or presence of 10 mM oxamate for 15 min under orbital
shaking and 37 °C. Thereafter, an aliquot was withdrawn and 5 mM D-
[2-3H-glucose] (1.3 Bq/nmol, American Radiolabeled Chemicals, St
Louis, MO, USA) was added to the cell suspension; 15 and 30 min later
other cell aliquots were removed. The cellular samples were centrifuged
at 20,800 xg for 20 s and the supernatant was discarded. The cell pellet
was frozen in liquid nitrogen and kept at −70 °C until use. The cell
samples were thawed at room temperature, re-suspended in 30% KOH
and heated for 60 min at 90 °C. Afterwards, they were mixed with 6%
Na2SO4 and 100% ethanol and centrifuged at 20,800 xg for 5 min. The
pellet was washed once with 1 mL of 80% ethanol and incubated at
Á. Marín-Hernández, et al. BBA - General Subjects 1864 (2020) 129687

room temperature. Finally, the dried samples (glycogen) were re-sus- [AMP ]
1+
[AMP ]

pended in water and their radioactivity determined with a Packard Vmax

1+
KAMP
[AMP ]
KAMP
[AMP ] ( [A][B] [P ]
Keq )
Scintillation Counter (TRI-CARB 2100TR, Meriden, CT, USA). KAMP Ka 1 +
KAMP
Kb
v=
[AMP ] [AMP ]
[A] 1 + [A] 1 + [B]
KAMP [B ] KAMP [P ]
1+ + + +
2.6. Kinetic modeling [AMP] Kb [AMP] Kp
(4)
Ka 1 + Ka 1 + Kb
KAMP KAMP

Using as scaffold the previously published kinetic model of glyco- [AMP ]

[AMP ]
lysis in AS-30D and HeLa cells [14,23], detailed kinetic equations for 1+

( [A][B] )
1+
KAMP KAMP [P ]
Vmax
the enzymes of glycogen metabolism (PGM, UGP, GS and GP), PPP 1+
[AMP ]
Ka 1 +
[AMP ]
Kb
Keq
KAMP KAMP
(Glc6PH and 6PGDH) and cytosolic NADPH consumption (GR and GPx) v=
[AMP ] [AMP ]
were now included, keeping the other rate equations unaltered or with [A] 1 +
KAMP [B ]
[A] 1 +
KAMP
[B]
[P ]
1+ + + +
some modifications. The PYK equation now included competitive in- [AMP] Kb [AMP ] Kp
(5)
Ka 1 + Ka 1 + Kb
hibition by alanine, phenylalanine and lactate; and ENO, PYK and LDH
KAMP KAMP

equations incorporated inhibition by oxamate (Ki). The rate equations for Glc6PDH and 6PGDH (Eq. (6)) were ordered
To compare the flux control distribution of the glucose central bi-bi reversible Michaelis-Menten equations for non-interacting sub-
metabolism pathways in hepatocytes and AS-30D cells, a kinetic model strates (α = 1 and β = 1). CO2 was fixed according to Moreno-Sánchez
for rat fed hepatocytes was also constructed using as template the AS- et al. [24]. In this equation A, B, P, Q correspond to NADP+, specific
30D kinetic model but with the kinetic parameters determined in he- substrate (Glc6P or 6PG), specific product (6PG or ribulose-5-phos-
patocytes. The reactions included in the models are described in Table phate) and NADPH, respectively. Ka, Kb, Kp and Kq are the Km values
S1 and the kinetic parameters used are shown in Table S2. for the corresponding substrates and products; and Keq is the equili-
Concentrations for alanine, phenylalanine, lactate, external glucose brium constant of the reaction.
(Glc out), Glc1,6BP, glycogen, Ery4P, Fru2,6BP, Pi, PPi, citrate, H2O2,
xylulose-5-phosphate (Xu5P) and ribulose-5-phosphate were fixed in
the models at the values shown in Table S3, which were within their v=
Vmf
KaKb ( [A][B] [P ][Q]
Keq )
[A] [A][B] [P ][A] [Q][B ] [Q][P ] [Q]
respective physiological ranges. The model simulations were carried 1+ Ka
+ KaKb
+ KpKa
+ KqKb
+ KqKp
+ Kq (6)
out with COPASI software [31] which can perform MCA and can cal-
culate the flux control coefficients from the elasticity coefficients. The GR kinetics (Eq. (7)) was described by an ordered bi-ter Michaelis-
rate equations used for the glycogen metabolism, pentose phosphate Menten equation [24], where A = NADPH, B = GSSG, P=NADP+,
pathway, NADPH consumption reactions, nucleotide diphosphate ki- Q = GSH and R = GSH.
nase (NDK), ENO, PYK and LDH are as follow.
The PGM rate equation (Eq. (1)) was a mono-substrate reversible
v=
Vmf
KaKb ( [A][B] [P ][Q][R]
Keq )
Michaelis-Menten equation with competitive inhibition by Fru1,6BP 1+
[A]
+
[A][B]
+
[P ][Q][R]
+
[Q][R]
+
[Q]
+
[R]
and mixed-type activation by Glc1,6BP [A] [28]: Ka KaKb KpKqKr KqKr Kq Kr (7)

The rate equation for GPx (Eq. (8)) was represented by a simplified
Vmf
1+
[A]
Ka ( ) Vmr 1 + [Glc1P](1 + )
[Glc 6P ] 1 +
[A]
Ka
[A]
Ka
[A]
Ka ordered ter-uni system in which the product H2O was not considered in

v=
1+
[A]
Ka
K (1 + )
Glc 6P
[A]
Ka
1+ K (1 + )
[A]
Ka Glc1P
[A]
Ka the reaction [24]. A = H2O2, B = GSH, C = GSH and P = GSSG.
[Glc 6P ] (1 + ) + [Glc1P] (1 + ) + [Fru1, 6BP]
[A] [A]

1+
K Glc6P
Ka

(1 + ) K (1 + ) Ki
[A]
Ka
Glc1P
Ka
[A]
Ka
Fru1,6BP
(1) v=
Vmf
KaKbKc ( [A][B][C] [P ]
Keq )
[A] [A][B] [A][B][C ] [P ]
1+ Ka
+ KaKb
+ KaKbKc
+ Kp (8)
The UGP rate equation (Eq. (2)) was defined by a reversible bi-
substrate ordered Michaelis-Menten equation. A, B, P and Q are UTP, The ENO equation (Eq. (9)) was a monoreactant reversible equation
Glc1P, UDP-Glc and PPi, respectively; Ka, Kb, Kp and Kq are the Km with competitive inhibition:
values for the corresponding substrates and products. [S] [P ]
Vmf Ks
Vmr Kp
[A][B ] [P ][Q] v=
Vmf Vmr 1+
[S ] [P ] [I ]
+ Kp + Ki
v=
K a Kb Kp K q
Ks (9)
[A] [A][B] [P ][Q] [Q]
1+ + + +
Ka K a Kb Kp K q Kq (2) Where S = [2PG], P = [PEP] and I = [oxamate] with their re-
spective affinity constants (Ks, Kp or Ki).
The rate equation for GS (Eq. (3)) was a mono-substrate irreversible
The PYK rate equation (Eq. (10)) was a random BieBi system with
Michaelis-Menten equation with non-essential mixed-type activation by
competitive inhibition:
Glc6P [28].

Vmax 1 +
[Glc6P ]
KGlc6P v=
Vmf
KaKb ( [A][B] [P ][Q]
Keq )
[UDPGlc] [I1 ] [I2 ] [I3 ] [I4 ] [A] [B ] [A][B ] [P ] [Q]
1+
[Glc 6P ] 1+ Ki1
+ Ki2
+ Ki3
+ Ki 4
+ Ka
+ Kb
+ KaKb
+ Kp
+ Kq
KGlc6P
v= [P ][Q] [A][Q] [P ][B ]
1+
[Glc 6P ] + KpKq
+ KaKq
+ KpKb (10)
KGlc 6P
Km + [UDPGlc]
[Glc 6P ]
1+
KGlc6P (3) Where A = [PEP], B = [ADP], P = [Pyr], Q = [ATP] with their
respective affinity constants (Ka, Kb, Kp and Kq) and I1 = [Alanine],
The GP rate equation was represented by an ordered bi-uni I2 = [Phenylalanine], I3 = [Lactate] and I4 = [oxamate] with their re-
Michaelis-Menten equation with allosteric modulation by AMP, which spective inhibition constants (Ki1, Ki2, Ki3 and Ki4). In the hepatocytes
was an essential mixed-type activation (Eq. (4)) in the AS30D model, or model, the PYK equation only included inhibition by oxamate.
non-essential mixed-type activation (Eq. (5)) [28] in the rat fed hepa- The rate equation for LDH (Eq. (11)) was a random BieBi system for
tocytes model. A, B and P are glycogen, Pi and Glc1P, respectively. Ka, non-interacting substrates (α1 = 1; β = 1) with mixed type inhibition
Kb and Kp are the Km values for the corresponding substrates. by oxamate:

4
Á. Marín-Hernández, et al.
Vmf
[A][B]
Vmr
[P][Q]
1 KaKb KpKq
v= [A] [ B] [A][B] [P][Q] [P ] [Q] [I ]
1+ + + + + Kp + + Ki
Ka Kb 1 KaKb KpKq Kq
where A = [NADH], B = [Pyr], P = [NAD+], Q = [Lactate] with their
respective affinity constants (Ka, Kb, Kp and Kq). α2 is the factor by
which Ka and Kb change when oxamate is bound to the enzyme.
I = [oxamate] and Ki is the inhibition constant for oxamate.
In the kinetic model, NDK (ATP + UDP → ADP + UTP) and NADPH
consumption (representing anabolic reactions) were incorporated.
These reactions were included as reversible mass-action and irrever-
sible constant flux equations, respectively; their rate constants and flux
were parametrized to allow the model to make simulations.
2.7. Statistical analysis
Statistical differences were analyzed by Student's t-test for non-
paired samples or by one-way ANOVA with Scheffe comparison test
considering P < .05 as criterion of significance.
3. Results
3.1. Content of glycogen metabolism enzymes
UGP and GS showed decreased protein levels (70 and 61%, re-
spectively) in AS-30D hepatoma cells as compared to rat fed hepato-
cytes, whereas the GP and PGM contents were similar (Fig. 1A). On the
other hand, AS-30D cells showed increased levels of both GS (9.2-fold)
and GP (2.1-fold) with respect to fasted-hepatocytes (Fig. 1A). In ad-
dition, the protein contents of UGP, GS and GP were significantly lower
(51%, 96% and 44%, respectively) in fasted-cells in comparison to fed
cells (Fig. 1A). In the immunoblot against GP, two distinct bands were
observed, but only the inferior one was used for densitometric analysis;
the identity of the upper band is unknown. When GP was further im-
munoprecipitated, the upper band was not observed (Fig. 1B).
3.2. Enzyme activities of glycogen metabolism
To assess changes in the expression of enzyme isoforms of glycogen
metabolism, thorough kinetic characterization of all the glycogen en-
zymes in cytosol-enriched fractions of AS-30D cells and hepatocytes
was carried out. Effects of other competing enzymes on the determi-
nation of the targeted enzyme's kinetic parameters in cytosol-enriched
protein fractions can be ruled out, since the activity assays were under
conditions of initial velocity and it was ensured to be highly specific as
described in Methods section. Moreover, the affinity constant values
were similar to the values reported for purified enzymes (Table 1). An
advantage of performing kinetic studies in sub-cellular fractions is that
the actual expression of different isoforms with their covalent mod-
ifications unaltered can be determined; these features are lost during
enzyme purification.
Activity of GP was detected in the absence of added AMP (GP ac-
tivator) in fed- and fasted-hepatocytes cytosolic fractions (Table 1),
being in the latter cells 19-fold higher in comparison to the first ones.
This difference in activity may be attributed to predominance of the GP
inactive form in fed hepatocytes and active form in fasted hepatocytes,
since these GP activities did not correlate with their similar protein
contents in both hepatocyte types (Fig. 1A). In contrast, GP activity in
AS-30D hepatoma cells was only detected in the presence of AMP
(0.1 mM), indicating that AMP was an essential activator for this en-
zyme in these cancer cells. Similar activity dependence on AMP has
been reported for GP from Novikoff hepatoma cells [1]. Thus, it seemed
that AS-30D cells expressed the muscle or brain GP isoform that are
highly sensitive to AMP, but not the low AMP-sensitive liver isoform
[1,16].
BBA - General Subjects 1864 (2020) 129687
Notwithstanding the differences in the content of active enzyme
[B][I ] [A][B][I ] (i.e., Vmax = kcat x [enzyme]total) in the cells, GP from fasted-hepato-
+ +
2 KbKi 1 KaKb 2 Ki cytes and AS-30D cells showed similar kinetic parameters for its ligands
(11) (Table 1). AMP was a mixed type essential activator with respect to
glycogen in AS-30D hepatoma cells and it was a non-essential mixed
type activator in fasted-hepatocytes (Fig. S1), suggesting the expression
of different GP isoforms. A mixed type activator increases both sub-
strate affinity and Vmax values and it is essential when its presence is
mandatory for enzyme activity; in contrast, a non-essential activator is
not strictly required for enzyme activity. ATP and Glc inhibited GP
activity but their effect was observed at high non-physiological con-
centrations (KiATP = 8–12 mM, KiGlc = 10–27 mM).
In addition to AMP, GP activity is also regulated by phosphorylation
at serine 14 that leads to enzyme activation [16]. Indeed, greater serine
GP phosphorylation was observed in AS-30D (5.5-fold) and fasted he-
patocytes (3.8-fold) in comparison to fed hepatocytes (Fig. 1B). The
level of GP phosphorylation correlated with the low and high activity
(without AMP) in fed and fasted hepatocytes, respectively. In AS-30D
cells, the high levels of phosphorylation did not correspond with the
null GP activity in the absence of AMP. This observation suggested that
AS-30D cells expressed the brain GP isoform, which is poorly activated
by phosphorylation [16].
GS activity was detected in the absence of added glycogen in AS-30
cytosol- enriched fraction, suggesting the presence of glycogen traces in
the fraction. Maximal activity was determined with the addition of
glycogen (10 mM glucose equivalents). The Km UDP-Glc value de-
termined (Table 1) was similar to values reported for cancer and non-
cancer enzymes [32,38]. Glc6P (a physiological modulator) was a non-
essential mixed-type activator versus UDP-Glc, mainly modifying the
substrate affinity and with low effect on Vmax (Table 1, Fig. S2). In
contrast, GS activity was not possible to be determined in cytosol en-
riched fractions from rat fed liver because its activity was extremely
low.
PGM and UGP activities were determined in the direction of Glc6P
formation. Both enzymes showed similar kinetic parameters (Table 1)
in AS-30D and fasted-hepatocytes. The PGM Vmax was 2.8-fold higher
in rat fed hepatocytes than in AS-30D cells. The effect of the two PGM
modulators, Glc1,6BP and Fru1,6BP, was also evaluated. The former
was a non-essential activator with respect to Glc1P, modifying substrate
affinity but not Vmax. The Ka values were similar for the enzymes from
AS-30D and fasted-hepatocytes (Table 1). Fru1,6BP behaved as a
competitive inhibitor with respect to Glc1P, with similar Ki values for
the enzymes from AS-30D cells and fasted-hepatocytes (Table 1). Other
metabolites such as erythrose-4-phosphate (50 μM) and 6-phosphoglu-
conate (1 mM) did not modify PGM activity (data not shown).
3.3. Fluxes of synthesis and degradation of glycogen and glycolysis in AS-
30D hepatoma cells
In AS-30D, glycogen synthesis and glycolysis were stimulated by
exogenous glucose (Fig. 2), despite the variable initial glycogen content
observed among cell batches. At 1 mM glucose, the glycogen synthesis
flux in these cells (Table 2) was similar to that reported for astrocytoma
cells, of 3.1 nmol/min*mg protein [40]. Maximal fluxes of glycogen
synthesis and glycolysis were achieved with 2.5 mM glucose; higher
glucose concentrations (5–30 mM) did not further stimulate glycogen
synthesis or glycolysis fluxes (Fig. 2 and Table 2).
On the other hand, glycogen degradation was only evident with no
glucose added; under this condition, lactate production was not ob-
served, but conversely, lactate consumption became apparent (Fig. 2
and Table 2). Thus, it seemed likely that pyruvate produced by gly-
cogen degradation was oxidized by mitochondria.
The low glycogen degradation flux (−0.5 ± 0.2 nmol Glc equiva-
lents/min*mg protein; Table 2) could be attributed to the low glycogen
content in AS-30D cells (128 ± 59 nmol Glc equivalents/mg protein;
n = 9). To test this hypothesis, cells were incubated with 30 mM
5
Á. Marín-Hernández, et al. BBA - General Subjects 1864 (2020) 129687

Hepatocytes
A)

% of band intensity vs. α-tubulin

PGM 125 AS-30D
62 KDa
Fasted hepatocytes
100 Fed hepatocytes **
UGP
57 KDa

GS
80 KDa 40
GP **
97 KDa

20 **
-tubulin
55 KDa
**
0
PGM UGP GS GP

B) 140
Hepatocytes AS-30D
Fasted Hepatocytes
% of band intensity vs. GP
120 Fed Hepatocytes
HeLa

*
100
GP
WB 80
P-Ser
60

*
40

*
20

0
P-Ser
Fig. 1. Glycogen metabolism protein levels (A) and GP phosphorylation status (B) in AS-30D cancer cells and hepatocytes from fed or fasted rat. In both figures
representative Western blot are shown. Densitometric analysis represents the mean ± SD of three independent preparations. Statistical analysis was performed using
one-way ANOVA with Scheffe comparison test. *P < .05, **P < .01 versus AS-30D cancer cells. See the Materials and Methods section for more experimental
details.

glucose for 1 h. As expected, the glycogen content indeed increased by 3.4. Modeling glucose central metabolism in AS-30D hepatoma cells
2.5-times (321 ± 81 nmol Glc equivalents/mg protein; n = 3); how-
ever, when these last cells were incubated with no glucose, the glycogen In the previous kinetic model of glycolysis of AS-30D cells [23], the
degradation flux was still unchanged (−0.6 ± 0.6 nmol/min*mg; pathways of glycogen synthesis, glycogen degradation and oxidative
n = 4). This observation suggested that glycogen content did not limit and non-oxidative (only transketolase reaction) branches of PPP were
GP activity. represented as constant fluxes. In the updated models here reported the
An alternative way to increase the glycogen degradation flux would reactions of PGM, UGP, GS, GP, Glc6PDH and 6PGDH were now ex-
be to increase the AMP levels, the GP activator, by subjecting AS-30D plicitly included with the appropriate rate-equations (Fig. 3), using the
cells to an energy metabolism stress. To this end, cells were incubated kinetic parameters experimentally here determined (Table 1) or pre-
for 60 min with 0.5 μM oligomycin (a potent and specific OxPhos in- viously reported by our group [24]. To maintain freely variable the
hibitor) in the absence of glucose. The glycogen degradation flux in- NADPH/NADP+ concentrations, a NADPH demand reaction was in-
deed increased to 1.4 ± 0.6 nmol/min*mg protein (n = 4); 5 μM oli- cluded, that represents pathways that consume NADPH such as ana-
gomycin did not further increase the flux (1.3 ± 0.5 nmol/min*mg; bolic reactions for amino acids, nucleotides, cholesterol and fatty acid
n = 4). Increased AMP levels activating muscle or brain GP isoform syntheses. Two successive reactions (GR and GPx), associated to oxi-
may explain this last observation; however, the total AMP levels in dative stress and NADPH consumption (ROS metabolism), were also
oligomycin treated cells (2.2 ± 0.3 mM; n = 3) were similar to those included using the kinetic parameters previously reported by our group
determined in non-treated cells (2.3 ± 0.6 mM, n = 3), indicating that [24]. Also, to maintain variable the UDP and UTP concentrations for
GP activity was not further stimulated by AMP. An alternative ex- the UGP reaction and to maintain a link with the ADP and ATP levels,
planation is that GP may be activated by an increase in cytosolic Pi (GP the NDK reaction was included.
substrate), as induced by OxPhos inhibition. In this regard, increased The gluconeogenesis pathway was not considered in the kinetic
cytosolic Pi concentrations have been reported when OxPhos was in- models because, in our experimental conditions, external glucose is
hibited in perfused rat liver; these changes in Pi levels were associated always present at different concentrations (1–25 mM) and hence de
with an increase in glycogenolysis rate [41]. novo glucose synthesis most likely becomes negligible. External glucose
represents the glucose present in circulation. Although several reports

6
Á. Marín-Hernández, et al. BBA - General Subjects 1864 (2020) 129687

Table 1
Kinetic parameters of glycogen metabolism enzymes from hepatoma AS-30D, hepatocytes and HeLa cells.
Enzyme AS-30D Hepatocytes HeLa Km values reported (mM)

Fed Fasted

1
GP Vmax 12 ± 3 (4) 1 ± 1 (3) 19 ± 11 (3) 3.2 ± 2 (3)
Km Pi 4.4 ± 0.9 (3) ND 2.6 ± 1.7 (3) 3.9 (1) 1–55,7
Km Glycogen 1.5 ± 0.3 (4) ND 1.7 ± 0.6 (3) 1.6 ± 0.4 (3) 1.2–1.46
Ka AMP 0.7 ± 0.1 (3) ND 1.2 ± 0.16 (3) 0.4 ± 0.25 (3)
α 0.1 ± 0.05 (3) ND 0.5 ± 0.16 (3) 0.6 ± 0.35 (3)
β 1.2 ± 0.18 (3) 2.4 ± 1 (3)
PGM Vmaxr 205 ± 97 (10) 574 ± 105 (3)* 345 ± 173 (4) 468 ± 74 (3)
3,9
Km Glc1P 1.0 ± 0.3 (10) 1.2 (2) 0.9 ± 0.1 (4) 1.1 ± 0.2 (3) 1.3–1.6
Ka Glc16BP 0.006 ± 0.003 (4) ND 0.01 (1) 0.015 (1)
α 0.03 ± 0.02 (4) ND 0.02 (1) 0.01 (1)
β 0.98 ± 0.03 (4) ND 1.1 (1) 0.96 (1)
KiFru1,6BP 0.12 ± 0.07 (4) ND 0.14 (2) 0.13 (1)
UGP Vmax 340 ± 23 (3) 341 ± 105 (3) 321 (1) 150 (1)
Km UDPGlc 0.06 ± 0.01 (3) ND 0.07 (1) 0.07 (1) 0.05–0.0664
K0.5 PPi 0.7 ± 0.1 (3) ND 0.5 (1) 2.1 (1) 0.08–0.214
GS Vmax 6.2 ± 2.6 (5) ND ND ND
2,8
Km UDPGlc 0.6 ± 0.3 (5) ND ND ND 0.5–1
Ka Glc6P 3.8 ± 1.8 (3) ND ND ND
α 0.1 ± 0.05 (3) ND ND ND
β 1.1 ± 0.1 (3) ND ND ND

Vmax values in nmol/min*mg cell soluble protein; Km, Ki, Ka, K0.5 values in mM. 1GP activity was determined in the presence of 0.1 mM AMP. *P < .0001 vs. AS-
30D. ND, not determined. In fed-hepatocytes, only the Vmax of GP, PGM and UGP are reported, because the low GP activity did not allow for kinetic parameters (Km
Glycogen, Km Pi and Ka AMP values) determination. For PGM and UGP, it was assumed that kinetic parameters (Km Glc1P, Ka Glc1,6BP, Ki Fru1,6BP) were similar to those of
the fasted-hepatocytes enzymes. The data shown represent the mean ± SD, with the number of independent cell preparations assayed in parentheses. Values
reported in 2 [32], 3 [33], 4 [34], 5 [35], 6 [36], 7 [37], 8 [38], 9 [39].

400 Table 2
A) Metabolic fluxes in AS-30D cells exposed to several external glucose con-
centrations.
(nmol glc equivalents)

Glucose Fluxes of glycogen synthesis/ Glycolytic flux (nmol lactate/

300
Glycogen

(mM) degradation (nmol Glc equivalents/ min*mg cellular protein)

min*mg cellular protein)

0 −0.5 ± 0.2 (7)* −0.28 ± 0.35 (7)*

200 1 1.4 ± 0.7 (6) 5.2 ± 1.3(5)
2.5 5.9 ± 1.4 (4) 8.3 ± 1.6 (4)
5 6.8 ± 2.2 (7) 9.2 ± 2.1 (6)
10 6.8 ± 0.7 (4) 10.5 ± 3.8 (4)
30 7.0 ± 2.9 (5) 9.3 ± 2.5 (6)
100
0 10 20 30 40 50 60 *The negative value indicates glycogen degradation or lactate consumption.
Values shown represent the mean ± SD. The number of preparations assayed is
B) shown in parentheses.
300
indicate that cancer cells may synthesize ribose-5-phosphate, glycerol-
225 3P, and serine from lactate and amino acids, this only occurs during
Lactate
(nmol/mg)

strict glucose deprivation [42].

150 Furthermore, since the Vmax values corresponded to the cytosol-
enriched fractions (Table 1), these were also experimentally determined
in whole cell extracts (Table S4) and used in the model. As the pH effect
75
on Vmax was not included in the rate equations of HK and HPI, their
Vmax values were also determined at pH = 6.8, the cytosolic pH when
0 glycolysis is active in AS-30D cells [13]. At pH = 6.8 the Vmax values of
0 10 20 30 40 50 60 both enzymes lowered by about 20% vs. pH 7.0 (pH condition used
Time (min) initially to determine all activities). In the case of PYK, the effect of
alanine, phenylalanine, proline, threonine, valine, isoleucine, trypto-
Fig. 2. Glycogen synthesis/degradation (A) and lactate production (B) in AS-
30D cells. AS-30D cells were incubated without (■) or with 5 (●) or 30 (Δ) mM
phan, and lactate were evaluated. Only alanine, phenylalanine, and
glucose. The initial contents (time = 0) of glycogen were 131 ± 43 (without lactate inhibited PYK activity at physiological concentrations; they
glucose, n = 8), 158 ± 70 (+ 5 mM external glucose, n = 10) and 128 ± 59 were competitive inhibitors with respect to PEP, with Ki values of
(+ 30 mM external glucose, n = 8) nmol Glc equivalents/mg protein. 4.5 ± 1.2 (n = 3), 0.67 ± 0.3 (n = 3) and 14.9 (n = 2) mM, respec-
tively (Fig. S3). In consequence, the inhibitory effect of these

7
Á. Marín-Hernández, et al. BBA - General Subjects 1864 (2020) 129687

Glc out

GLUT
(ROS) H2O2 Glycolysis Glycogen synthesis
GPx 2 GSH Glc in
ATP Glc6P
GSSG GR (- ) HK
Fru1,6BP Glc1,6BP (+)
ADP Glc1P UDP-Glc Glycogen
+ (- ) (+)
NADPH NADP
+ NADPH NADP UGP GS
PGM
Ribulose 5P 6PG Glc6P Glc1P UTP PPi UDP
6PGDH Glc6PDH ( -)
(-)
Fru1,6BP HPI Fru1,6BP Glc1P Glycogen
6PG GP
Ery4P Fru6P
DHAP (+) Pi
ATP (-) ATP
PFK-1 Citrate
Ery4P (+) AMP
ADP Fru2,6BP
TK Fru1,6BP Glycogen degradation
Xu5P
ALDO ( -)
DHAP
Pentose Phosphate Pathway TPI
G3P ATP + UDP ADP + UTP
NDK
NAD+ + Pi
NADH NAD+ GAPDH
DHases NADH ATP + AMP 2 ADP
AK
1,3BPG
ADP
PGK
ATP
3PG

NADP+ NADPH PGAM

NADPH Consumption 2PG
ENO
Lactate
PEP
ADP (- ) Alanine
PYK Phenylalanine
ADP + Pi ATP
ATPases
Pyr + 12.5 ADP + 12.5 Pi 12.5 ATP
MPM
NADH
LDH
NAD+

Lactate

Fig. 3. Pathway reactions included in the kinetic model of glucose metabolism in AS-30D and hepatocytes. AK, adenylate kinase; ALDO, fructose-1,6-bisphosphate
aldolase; ATPases, ATP-consuming processes; DHAP, dihydroxyacetone phosphate; DHases, NADH-consuming reactions; ENO, enolase; Ery4P, erythrose-4- phos-
phate; Fru1,6BP, fructose-1,6-bisphosphate; Fru6P, fructose-6-phosphate; Fru2,6BP, fructose-2,6- bisphosphate; GAPDH, glyceraldehyde-3-phosphate dehy-
drogenase; G3P, glyceraldehyde-3-phosphate; Glc out, external glucose; Glc in, intracellular glucose; Glc1P, glucose-1-phosphate; Glc6P, glucose-6-phosphate;
Glc6PDH, glucose-6-phosphate dehydrogenase; Glc16BP, glucose-1,6-bisphosphate; GS, glycogen synthase; GP, glycogen phosphorylase; GR, glutathione reductase;
GPx, glutathione peroxidase; LDH, lactate dehydrogenase; MPM, mitochondrial pyruvate metabolism; NDK, nucleoside diphosphate kinase; PEP, phosphoenolpyr-
uvate; PGAM, 3-phosphoglycerate mutase; PGK, 3-phosphoglycerate kinase; PGM, phosphoglucomutase; Pyr, pyruvate; PYK, pyruvate kinase; TPI, triosephosphate
isomerase; TK, transketolase; UGP, UDP-glucose pyrophosphorylase, UDP-glc, UDP-glucose; Xu5P, xylulose-5-phosphate; 2PG, 2-phosphoglycerate; 3PG, 3-phos-
phoglycerate; 1,3BPG, 1,3 bisphosphoglycerate; 6PG, 6- phosphogluconate; 6PGDH, 6-phosphogluconate dehydrogenase.

metabolites was included in the PYK rate equation, which allowed the
model to better predict the steady-state PEP concentrations.
The PGM rate equation included the regulatory effects of Glc1,6BP
(activator) and Fru1,6BP (competitive inhibitor). However, the high
levels of Fru1,6BP found in AS-30D cells in comparison to normal cells
(Table 3), and the low levels of Glc1,6BP (8.8 ± 1.4 μM; n = 4) and
Glc1P relative to the Ka and Km values (Table 1), drastically decreased
the PGM rate and the kinetic model did not reach a steady-state. Thus,
the parameters involved in activation (Ka) and inhibition (Ki) induced
by Glc1,6BP and Fru1,6BP, respectively, required to be parameterized.
Although the values obtained by parameterization (KaGlc1,6BP = 2 mM
and Ki Fru1,6BP = 18 mM) were higher than the experimentally-de-
termined values (Table 1), the simulation could reach stable steady-
states. Then, it is clear that the PGM kinetics still requires further ex-
perimental analysis.
For GP, AMP activation was the only regulatory mechanism in-
cluded in its rate equation, since the effect of other modulators (glucose
and ATP) was negligible. Glc6P is a GP inhibitor and GS activator; thus,
the GS rate equation included a Glc6P activation term. The Glc6P effect
on GP activity was not here experimentally determined due to technical
restriction by the coupled enzymatic assay used, and hence this
inhibition was not included in the respective rate equation. Therefore,
the Vmax value of GP used in the model required to be parameterized
and be decreased to 41% of the activity at 1.2 mM AMP (intracellular
concentration, Table 3); such decrease was estimated by using the
equation with essential mixed-type activation and the activity de-
termined in whole cell extracts with high (2 mM) AMP (Table S4). This
adjustment enabled the model to better simulate the net fluxes of gly-
cogen metabolism in the presence and absence of glucose (Table 2).
These AMP-dependent changes in GP activity may have a physiological
basis as AMP may be as low as 0.1 to 0.5 mM, when considering that
24% of total AMP can be found in the cytosol [43]. This lower AMP
concentration, together with Glc6P that behaves as inhibitor, should
maintain a low GP activity, when the cells are consuming glucose.
With the modifications described above, the model was able to
closely simulate (i) the glycogen metabolism at 0, 1, 2.5, 10 and 30 mM
external glucose (Fig. 4); and (ii) the metabolite concentrations and
fluxes at 5 mM external glucose, except for Fru6P, NADP+ and NADPH
concentrations (Table 3). The model could simulate the glycolytic
fluxes at glucose concentrations < 5 mM; however, the glycolytic flux
was not well simulated when external glucose was more than 10 mM.
Lactate accumulation can induce intracellular acidification (pH = 6.8)

8
Á. Marín-Hernández, et al. BBA - General Subjects 1864 (2020) 129687

Table 3 10
Metabolic fluxes and intermediary concentrations determined In vivo and si- A)

synthesis/degradation
mulated by in silico modeling for AS-30D cells and hepatocytes.

flux (nmol/min*mg)
8
Metabolite / flux AS-30D Rat fed hepatocytes
6

Glycogen
In vivo Model Reported* Model
4
Glcin 6.24 1.9 N.D. 3.3
Glc6P 3.1 ± 1.3 (7) 2.4 0.12–0.96 1.28
Fru6P 1.2 ± 0.4 (3) 0.07 0.05–0.4 0.7
2
Fru1,6BP 3.9 ± 2.6 (4) 1.9 0.014–0.1 0.002
DHAP 7.8 ± 2 (3) 4.0 0.017–0.6 0.03 0
G3P 0.7 ± 0.4 (3) 0.12 0.005–0.1 0.002
PEP 0.1 ± 0.06 (3) 0.011 0.07–0.15 0.33 -2
Pyr 1.3 ± 0.4 (3) 1.3 0.08–1.6 0.29 0 5 10 15 20 25 30
ATP 5 ± 2.7 (4) 5.1 2.0–3.6 3.4
ADP 0.6 ± 0.6 (3) 0.37 0.68–1.6 1.6
20
AMP 1.2 ± 0.7 (5) 1.3 0.16–0.22 0.36
B)
Glycolytic flux 9.2 ± 2.1 (6) 11.1 0.84–2.4 2.9

(nmol/min*mg)
15

Glycolytic flux
Glc1P 0.1 ± 0.03 (3) 0.05 0.012–0.022 0.07
UDPGlc 0.4 ± 0.2 (7) 0.1 0.38 0.03
UTP 0.26–1.13 0.48 0.26–0.553 0.58
UDP 0.1–0.3 3 0.7 0.05–0.263 0.27 10
Glycogen synthesis flux N.D. 7.44 N.D. 0.55
Glycogen degradation flux N.D. −1.97 N.D. −0.35
Glycogen net flux 6.8 ± 2.2 (7) 5.47 0.01–0.38 0.2 5
6PG 0.22–0.481 0.14 0.016–0.18 0.012
NADP+ 0.322 0.02 0.32 0.07
NADPH 0.22 0.49 0.242 0.47 0
PPP flux 0.06–0.1261 0.29 0.43 0.44
GSH 3.42 3.4 2.22 2.3 0 5 10 15 20 25 30
GSSG 0.012 0.01 0.0712 0.014
Glucose (mM)
Metabolite concentrations in mM, flux in nmol/min*mg cell protein. Values
Fig. 4. Experimental and modeled fluxes obtained in AS-30D cells at various
represent the mean ± SD. The number of different cell preparations assayed is
external glucose concentrations. (A) Glycogen synthesis/degradation and (B)
shown in parentheses. *Values taken from [18,27,46–50]. Values reported in
glycolysis. The solid lines are the predicted fluxes by kinetic modeling whereas
nmol/ g tissue wet weight were converted to concentrations considering a vo-
the symbols represent experimental data (Table 2). For model simulations,
lume of 0.8 mL/1 g tissue wet weight [51]. Values taken from 1 [13], 2 [24], 3
ATPase k values was modified (0.0017 min-1) to avoid ATP exhaustion. The
[51] and 4 [27]. Simulations were performed at 5 mM glucose. Glycogen net
dotted line represents the simulation with the pH effect on HPI activity (Vmf
flux is estimated from glycogen synthesis flux minus glycogen degradation flux.
and Vmr were decreased 40%).
N.D., Not determined.

3.5. Modeling glucose metabolism in rat fed hepatocytes

in AS-30D [13]. This change in pH can cause glycolysis inhibition [44],
and HPI is one of the enzymes that decrease its activity under this
condition [45]. When HPI inhibition by acidification is simulated, the
model prediction of pathway fluxes improves (Fig. 4). To be noted, the
glycolytic flux was more sensitive than glycogen metabolism to pH
changes, as previously reported [44].
The concentrations of metabolites determined in intact AS-30D cells
(with 5 mM glucose) after 15 min of incubation were used for model
validation (Table 3) because at this time the fluxes of glycogen and
glycolysis were linear (Fig. 2).
The model robustness was also assessed. The flux control coeffi-
cients and metabolite concentrations were not significantly altered by
decreasing by half or increasing by 2 times the kinetic parameters of
most steps, except for GLUT Vmax value and HK, HPI, PGM, UGP and
GS Vmax and Km values, all which were controlling steps (see below
Fig. 5 and Table S5). Changes in the latter parameters altered by more
than 50% their flux control coefficients, with the consequent impact in
most of pathway variables. Metabolite concentration variations by
more than 50% were attained when changing either HPI, PGM and UGP
Vmax and Km values (Fru1,6BP and DHAP); TPI, PGK, PGAM, ENO and
PYK Vmax and Km values (DHAP); or Glc6PDH and 6PGDH Vmax and
Km values (6PG). The PPP flux varied by 50–100% when GPx Vmax and
Km values were changed or when NADPH consumption flux was varied.
The model was also sensitive to changes in the ATPase k value. Not-
withstanding the enlisted exceptions, the predicted pathway behavior
showed satisfactory robustness which was within the physiological
determined values at different glucose concentrations (Fig. 4; Table 3)
and different cell types (Table 3).
To determine the main controlling steps of glucose central meta-
bolism in rat fed hepatocytes, a kinetic model was also built, for which
the kinetic parameters previously reported by our group and others
were used (Table 3 and Table S2). The Vmax values and some kinetic
parameters were experimentally determined in whole cell extracts
(Table S4) and cytosoli-enriched fractions (Table 1). Metabolite con-
centrations and pathway fluxes reported previously for hepatocytes of
fed rats were used to validate the model simulations (Table 3).
Glycolytic metabolites (Glc6P, Fru6P, Fru1,6BP, DHAP) and Glc1P
concentrations reported or estimated for hepatocytes were lower (7–78-
fold) than those observed in hepatoma cells (Table 3). The published
glycogen synthesis and glycolytic fluxes were also lower in rat hepa-
tocytes (4–18-fold) in comparison with the fluxes determined in AS-30D
cells (Table 3).
In the hepatocytes kinetic model, GS and GP Vmax needed to be
adjusted to simulate the fluxes of glycogen metabolism found in the
cells (Table 3, Table S2). With these adjustments, the model closely
predicted the concentrations of metabolites and fluxes, except for
Fru1,6BP and UDPGlc concentrations (Table 3).
The robustness of the hepatocytes kinetic model was equally eval-
uated by decreasing by half or increasing by 2 times the kinetic para-
meters of the pathway enzymes. Variations in Vmax (for GLUT and HK)
and/or Km (for GLUT, UGP and GS) values led to a greater than 50%
variation in the flux control coefficients. Variations in the Vmax (for
PFK-I, PGM, UGP, GS, Glc6PDH, 6PGDH) and/or Km (for PGM, UGP,
GS, Glc6PDH, 6PGDH) values resulted in significant changes of the
AMP or 6PG concentrations. In addition, the PPP flux showed

9
Á. Marín-Hernández, et al. BBA - General Subjects 1864 (2020) 129687

Fig. 5. Flux control coefficients of enzymes/pro-

A) Glycolysis C) Glycogen Degradation
cesses of the glucose central metabolism pathways
0.8 1.0 obtained by kinetic modeling. The flux control dis-
0.8 tribution obtained in rat fed hepatocytes and AS-
0.4
0.4 30D cells for glycolysis (A), glycogen synthesis (B),
AS-30D
CJEi

CJEi
0.2 Hepatocytes glycogen degradation (C) and pentose phosphate
0.2 pathway (D). The CJEi shown were those determined
0.0 for the fluxes through LDH (glycolysis), GS (gly-
0.0 cogen synthesis), GP (glycogen degradation) and
6PGDH (PPP) (Table S5).
-0.2

NADPH consumption
-0.2

NADPH consumption
ATPases
ATPases

Glc6PDH-6PGDH
Glc6PDH-6PGDH
ALD-LDH

PFK-1
PFK-1

GLUT
GLUT

MPM

MPM
ALD-LDH

PGM
PGM

UGP

UGP
GPx

GPx
HPI

HPI

GR
HK
GR
GS

GP
HK

GP

GS
1.2 0.9
0.8
0.4 0.6
0.2
CJEi

0.0 CJEi
-0.2 0.2
-0.4
-0.6
0.0
B) Glycogen Synthesis D) Pentose Phosphate Pathway

significant changes when Vmax and Km for GPx varied. Like the AS-30D 3.7. Experimental and modeled effect of oxamate on glucose central
model, the fed hepatocyte model exhibited high sensitivity to variation metabolism in AS-30D cells
in the ATPase k value. Thus, the hepatocyte model displayed adequate
robustness within the physiological intervals determined in vivo. We previously demonstrated that oxamate is a glycolysis flux in-
hibitor, by inhibiting ENO, PYK and LDH activities and inducing in-
3.6. Flux control distribution of glucose central metabolism in hepatoma creased levels of Fru1,6BP; the latter in turn is a competitive inhibitor
cells and rat fed hepatocytes of HPI [23,55] and PGM. Hence, oxamate inhibition on LDH, PYK, and
ENO rate equations were included. The inhibition of glycolytic and
At 5 mM external glucose, the main controlling steps of glycolysis glycogen synthesis fluxes was well simulated when the concentration of
flux were HPI > GLUT = HK in AS-30D cells whereas for rat fed he- oxamate was varied, which correlated with an increase in the levels of
patocytes were HK > > PFK > GLUT (Fig. 5 and Table S5). A con- Fru1,6BP. Interestingly, this oxamate effect was only observed in the
trasting flux control distribution for glycolysis, when this pathway is AS-30D model but not in the rat fed hepatocytes model (Fig. S4).
modeled alone, has also been reported for AS-30D cells [13] as com- The model prediction that HPI and PGM exert significant control of
pared to that of hepatocytes or other non-cancer cells [52,53]. glycolysis and glycogen synthesis in hepatoma cells but not in rat fed
For the glycogen synthesis flux, the main controlling steps were hepatocytes was experimentally assessed using oxamate. The glycolytic
PGM > GLUT = HK in AS-30D cells, and HK > > GS > GP and glycogen synthesis fluxes and ATP concentration decreased by 50%
> MPM = GLUT in rat fed hepatocytes (Fig. 5 and Table S5). It has in comparison with non-treated cells after incubation of AS-30D cells
been also reported that GLUT, HK and GS exert control on glycogen with 10 mM oxamate. This effect correlated with an increased (2.8-fold)
synthesis in rat muscle [54]. level in Fru1,6BP (Table 4). In contrast, at the same oxamate con-
For glycogen degradation flux, the main controlling step was GP in centration, glycolysis and glycogen synthesis fluxes as well as ATP le-
both AS-30D cells and rat fed hepatocytes, exerting high control derived vels were unaffected in rat fed hepatocytes, which correlated with a
from its very low activity despite of AMP activation. Regardless of the decreased Fru1,6BP level (Table 4). These experimental results sup-
type of rate equation used to describe GP kinetics (reversible or irre- ported the modeling prediction that HPI and PGM activities in AS-30D
versible), the predicted metabolite concentrations, fluxes and control cells exert more flux control of glucose metabolism than in hepatocytes
coefficients for glycogen degradation were similar. For the PPP flux the (Fig. 6).
control was exerted by GPx and NAPDH consumption in AS-30D cells
and by GPx in rat fed hepatocytes (Fig. 5 and Table S5). The H2O2 3.8. Flux control distribution of glucose central metabolism in human
concentration (GPx substrate) was fixed at 55 nM (hepatocyte model) cancer cells
or 65 nM (AS-30D model) under non-oxidative stress conditions.
These results indicated that HPI and PGM exert high control on To extend our knowledge on how an essential metabolic pathway
glycolysis and glycogen synthesis, respectively, in AS-30D cells (Fig. 5). can be modified in cancer, kinetic parameters of GP, PGM and UGP
Afterwards, the relationship between pathway fluxes and HPI and PGM were also determined in cytosolic fractions of human cervix HeLa
activities was analyzed using the kinetic models. Significant decreases cancer cells, one of the most commonly studied cancer cell lines. In fact,
in the fluxes of glycolysis and glycogen synthesis were observed in AS- AS-30D and HeLa cells show similar glycolytic control patterns
30D when the HPI and PGM activities were simultaneously decreased; [13,14,27]. The values of the kinetic parameters in HeLa cells were
whereas, the pathway fluxes in rat fed hepatocytes were not affected similar to those obtained for the enzymes from AS-30D cells and fasted
(Fig. 6). In contrast, when GLUT and HK were simultaneously de- hepatocytes, with AMP behaving as a non-essential mixed type acti-
creased, both pathway fluxes were drastically inhibited in both types of vator of HeLa GP (Table 1). In contrast, the level of serine phosphor-
cells (Fig. 6). ylation of HeLa GP was lower than that of the AS-30D and fasted

10
Á. Marín-Hernández, et al. BBA - General Subjects 1864 (2020) 129687

Fig. 6. Modeled dependence of glycogen synthesis

A) Hepatocytes C) (A,C) and glycolytic (B,D) fluxes on HPI-PGM or on
GLUT-HK activities in rat fed hepatocytes and AS-
JGycogen synthesis %

100 100
AS-30D cells
30D cells. The reference 100% enzyme activity va-
80 80 lues were those corresponding to the respective
Vmax values (Table S2) whereas 100% fluxes were
60 those predicted by each model. To determine the
60 AS-30D cells
dependence of glycogen synthesis and glycolytic
40 Hepatocytes fluxes on HPI-PGM or on GLUT-HK, the HPI-PGM or
40
GLUT-HK activities were simultaneously decreased
20 by the same percentage. For HPI and PGM, a de-
20 crease of the Vmaxf value was accompanied by a
0 proportional decrease in the Vmaxr value.
0 20 40 60 80 100 0 20 40 60 80 100

B) Hepatocytes
D)
100 100
AS-30D cells
80
JGlycolysis %

80
60
60 AS-30D cells
40 Hepatocytes
40
20

20 0
0 20 40 60 80 100 0 20 40 60 80 100
% of simultaneous decrease of % of simultaneous decrease of
HPI and PGM GLUT and HK

Table 4
Effect of oxamate on glycolytic and glycogen synthesis fluxes in AS-30D cancer cells and rat fed hepatocytes.
AS-30D Rat fed hepatocytes

Control + Oxamate Control + Oxamate

% glycolytic flux 100 (5) 48 ± 9 (5)** 100 (4) 97 ± 21 (4)

% glycogen synthesis flux 100 (4) 52 ± 8 (4)** 100 (3) 107 ± 29 (3)
% ATP 100 (4) 45 ± 8 (4)** 100 (5) 94 ± 17 (5)
Fru1,6BP (mM) 4 ± 2 (3) 11 ± 3 (3)* 1 ± 0.2 (4) 0.3 ± 0.1** (4)

The oxamate and glucose concentrations were 10 and 5 mM, respectively, during cell incubations. The glycolysis/glycogen fluxes and ATP con-
centration in the absence of oxamate were 9.2 ± 3 (n = 5)/5.6 ± 1.8 (n = 4) nmol/min*mg of cellular protein and 6.1 ± 2.6 (n = 4) mM in AS-
30D cells; and 0.3 ± 0.03 (n = 4)/ 0.04 ± 0.02 (n = 3) nmol/min*mg of cellular protein and 2.4 ± 1 (n = 5) mM in rat fed hepatocytes, re-
spectively. Values are the mean ± SD; the number of preparations assayed is shown in parentheses. *P < .05, **P ≤ .001 vs. control cells.

hepatocytes enzymes, but similar to that of rat fed hepatocytes GP
(Fig. 1B).
HeLa cells grown under hypoglycemia (2.5 mM glucose) and AS-
30D growing in the rat intra-peritoneal cavity under low glucose con-
centration (0.026 mM) show similar higher expression of GLUT3 and
HKI isoforms as well as glycolytic flux control distribution [13,14,27].
Therefore, the previously published HeLa kinetic models of glycolysis
were updated to establish the main control steps in the glycogen me-
tabolism and PPP pathway. Thus, for modeling HeLa glucose central
metabolism, PGM, UGP, GS, GP, Glc6PDH, 6PGDH, GR, GPx, NADPH
consumption and NDK reactions were incorporated to the previous ki-
netic model of glycolysis [14] using the kinetic parameters experi-
mentally determined here (Tables 1, S2, S4 and S6) or previously re-
ported by our group [14,24]. In HeLa models under non-oxidative stress
conditions, the H2O2 concentration was fixed between 60 and 153 nM,
which was higher than that fixed in hepatocytes (Table S3). This feature
correlated with the generalized observation of greater ROS levels in
human cancer cells as compared to normal cells [56]. The models were
able to closely simulate the experimentally determined metabolite
concentrations and fluxes attained at 5 mM external glucose in short-
term incubations in hyperglycemic- and hypoglycemic-grown HeLa
cells, except for Fru6P, Fru1,6BP, G3P and PEP (Table S6).
The main controlling steps of glycolysis were GP > HPI
> ATPases > HK in hyperglycemic HeLa cells and GLUT > HK > HPI
in hypoglycemic HeLa cells (Table S7). For glycogen synthesis flux, the
main controlling steps were GS = GP and GS > > GLUT, in hy-
perglycemic and hypoglycemic cells, respectively (Table S6). In both
types of HeLa cells, GP and ATPases were the main controlling steps for
the glycogen degradation, whereas GPx and NADPH consumption were
the main controlling steps in the PPP. The distribution of control of
glycolysis of hypoglycemic cells was similar to that in AS-30D cells,
which grown in the rat intra-peritoneal cavity, under hypoglycemic
conditions, but not for glycogen synthesis. It remains to be established
whether a similar flux control distribution is observed in normal human
cells.
4. Discussion
The study of central glucose metabolism in AS-30D cells showed
that AMP is an essential activator of GP and Glc6P is a non-essential

11
Á. Marín-Hernández, et al.
activator of GS, which contrast with their lack of effect on rat fed he-
patocytes. It also became evident that PGM modulation by Glc1,6BP
(activator) and Fru1,6BP (inhibitor) as well as alanine, phenylalanine
and lactate inhibition of PYK are important regulatory mechanisms that
impact pathway fluxes and control. Inclusion of these kinetic para-
meters in a kinetic model of AS-30D enabled to predict that HPI and
PGM were the main controlling steps of glycolysis and glycogen
synthesis in cancer cells but not in fed hepatocytes. This prediction was
experimentally validated by using oxamate that inhibited the glycolytic
and glycogen synthesis fluxes in AS-30D through accumulation of
Fru16BP, a physiological inhibitor of HPI and PGM.
4.1. Content of protein and activities of glycogen metabolism enzymes
There was a lack of correlation between protein levels and enzyme
activities, which now seems a common feature for enzymes of the
cancer glucose metabolism [14] mainly as a consequence of wide-
spread covalent regulation. In particular, GP, GS and UGP activities
may be regulated by phosphorylation or acetylation [16,57]. The ac-
tivities of other glycolytic proteins may also be regulated by phos-
phorylation (PGM, HPI, GLUT1, HKI, HPI, PFK1, ENO1), or in the case
of HKII, phosphorylation may regulate its binding to the external mi-
tochondrial membrane [58–62].
The Km values determined for PGM and UGP from hepatocytes and
cancer cells were similar to values reported for enzymes from rat he-
patomas, calf, rabbit and human liver tissues [33,34,39]. Moreover, the
Km Glc1P value suggested the expression of the PGM2 isoform in hepa-
tocytes, AS-30D and HeLa cells [33].
The effect of insulin and glucagon was not evaluated in this study.
However, it has been reported that insulin does not modify the protein
levels of GLUT 4 (insulin sensitive) neither the rate of glucose transport
in AS-30D cells [63], suggesting that insulin might not affect the gly-
cogen metabolism in AS-30D cells. In contrast, insulin stimulates gly-
cogen synthesis in HeLa cells [64]. The effects of glucagon on AS-30D
and HeLa cells have not yet been systematically examined.
GP of hepatocytes, AS-30D and HeLa cells showed similar affinity
(1/Km) values for glycogen and Pi. The Km values were similar to those
previously reported for human liver recombinant enzyme and rat liver
and kidney native enzymes [35,36]. For the rabbit GP isoforms Km
values for Pi of 1.5 mM (brain), 3 mM (liver) and 4.2 mM (muscle) have
been reported [37]; then, the values found in AS-30D and HeLa cells for
the GP Km Pi suggested that the muscle isoform was expressed. GP
isoforms can also be distinguished by their regulatory properties. Liver
GP is mainly regulated by phosphorylation, triggering glycogen de-
gradation in response to hormones such as glucagon. In contrast,
muscle and brain GP isoforms are regulated by non-covalent mod-
ulators such as AMP. Furthermore, muscle GP is cooperatively regu-
lated by AMP, whereas brain GP is non-cooperatively (hyperbolic) ac-
tivated by AMP and is poorly activated by phosphorylation [16]. In the
present study it was observed that AMP behaved as an essential hy-
perbolic activator of AS-30D GP (data not shown), although the enzyme
was phosphorylated, whereas AMP was a non-essential activator of
HeLa GP. These last data suggested expression of muscle or brain iso-
form in both cancer cells. The brain GP isoform is also mainly expressed
in several human cancer cells lines [6] and its expression has been
proposed as an early biomarker in human colorectal cancer [5].
It has been previously suggested that GP is a controlling step of
glycogen degradation flux [65], although no experimental analyses in
that regard was performed. Indeed, the kinetic modeling carried out in
the present study in three different cells types indicated that GP was the
main control step of the glycogen degradation flux. GP exerts positive
control on glycogen synthesis fluxes in AS-30D cells because its activity
generates Glc1P, which may re-feed glycogen synthesis (glycogen re-
cycling). In turn, in hyperglycemic HeLa cells, GP exerts control on
glycolytic flux because these cells express low-affinity GLUT isoforms
and their glycolytic flux becomes partially dependent on glycogen
12
BBA - General Subjects 1864 (2020) 129687
degradation, when they are exposed to low glucose concentrations
[14].
4.2. Control of glucose central metabolism in normal cells
Identification of the differences at the genetic and biochemical
(functional) levels between normal and cancer cells is essential in the
search for new therapeutic targets, which should be specific for pa-
thological cells and non-harmful for normal cells. Kinetic modeling of a
metabolic pathway allows the identification of the steps with the
highest control in cancer cells, and low control in normal cells, thus
becoming the best therapeutic targets. Furthermore, kinetic modeling
provides mechanistic understanding of why a given enzyme or trans-
porter exerts significant control on a pathway [13,14].
In general, the kinetic model predictions were robust to changes in
the kinetic parameters of most of the pathway enzymes, which gives
certainty about the validity of the results obtained. The majority of the
metabolite concentrations and fluxes predicted by the model correlated
well with the in vivo values, with the exception of Fru6P in AS30D cells.
This indicated that additional kinetic experimentation-based refine-
ment is necessary at the level of PFK-1, because low levels of Fru6P are
attributed to the strong activation by F2,6BP that overcomes the in-
hibitory effect of citrate and ATP [27]. In contrast, in the rat fed he-
patocyte model, the Fru6P levels are not low because PFK-1 activity is
very low.
Similarly to other normal cells (human erythrocytes, baby mouse
kidney cells, mouse fibroblasts) and organs/tissues (rat liver, rat ske-
letal muscle, mouse heart) [20,52–54,66], it was here found that GLUT,
HK, PFK1, GP, GS and NADPH demand (as GPx and other pathways that
consume NADPH) were the main controlling steps of the glucose central
metabolism in hepatocytes. Furthermore, the flux-control coefficients
predicted by the present model were similar to values reported in
human skeletal muscle and rat heart for GLUT/HK (0.5–1) and GS
(0.01–0.4), when control of glycogen synthesis was determined by
elasticity analysis [54]; and GLUT (0.25–0.5), HK (0.69–0.77) and PFK
(0.24–0.65), when glycolysis was analyzed in human erythrocytes and
mouse heart (by kinetic modeling), in rat liver (by titration with pur-
ified enzyme in homogenates) and baby mouse kidney cells and mouse
fibroblasts (by varying the expression level by genetic means)
[20,52,53,66].
However, in some of these previous analyses of glycolysis flux
control distribution in normal cells, the GLUT reaction was not included
and hence the control of flux estimated for HK most likely also involved
the contribution of GLUT. Now, with the construction, and experi-
mental validation, of the present kinetic model of glucose central me-
tabolism of hepatocytes, the relevant GLUT step was explicitly included
as well as the glycogen metabolism and PPP branches. The high control
exerted by GLUT and HK on the fluxes of glycolysis and glycogen
synthesis correlated with increased levels of glycogen and/or lactate
production when GLUT and/or HK activities were increased in mice
skeletal muscle, baby mouse kidney cells and mouse fibroblasts
[66,67]. These previous observations in normal cells supported the
predictions of the present model of hepatocytes.
Through kinetic modeling of PPP in hepatocytes it was previously
reported that Glc6PDH and HK were controlling steps; however, such
model did not include the reactions of NADPH consumption [19]. In
contrast, in in vivo studies in Xenopus laevis oocytes where reactions of
NADPH demand are present, Glc6PDH and HK exhibited null control on
the PPP flux [68]. The latter authors argued that the NADPH demand
might exert control on PPP. Here, it was determined that NADPH de-
mand (as GPx and other NADPH consuming pathways such as, amino
acids, nucleotides, cholesterol and fatty acid syntheses) exerted control
on the PPP flux. As in the present models the peroxide management
system was included (as another NADPH demand), the levels of H2O2
did significantly affect the PPP flux.
Á. Marín-Hernández, et al.
4.3. MCA-based selection of therapeutic targets
The novel insight provided by the present kinetic modeling of
central glucose metabolism is that HPI exerted significant control on
glycolytic flux of AS-30D cells [13,14] but interestingly it showed
negligible control on glycolysis of normal cells [52–54,66]. Moreover, it
was also established that PGM exerted significant control on glycogen
synthesis flux in AS-30D cells but not in rat fed hepatocytes. These
observations indicated that both enzymes are suitable therapeutic tar-
gets in tumor cells. In contrast, other controlling steps such as GLUT,
HK and GP are unspecific targets because glycolysis and glycogen
synthesis/degradation would be inhibited in both normal and AS-30D
cells. Such an effect was certainly observed in lung cancer cells (A549)
and normal fibroblasts (HSF55) after treatment with CP-91,149, a GP
inhibitor that causes accumulation of glycogen in normal and cancer
cells in comparison with non-treated cells [6]. In addition, 2-deoxy-
glucose (2-DG), an inhibitor of GLUT, HK and HPI [69], decreases
glycolysis in normal and cancer cells and tissues [70]. This is one of the
possible causes of adverse events reported in 2-DG treated cancer pa-
tients [69].
HPI exerted control on the glycolytic flux and PGM on glycogen
synthesis flux in cancer cells because their activities were strongly
regulated by several metabolites. In the case of HPI, erythrose-4P, 6-
phosphogluconate, Fru1,6BP and DHAP are potent competitive in-
hibitors at physiological concentrations [13,23,55]. In turn, Fru1,6BP is
also a potent competitive inhibitor of PGM activity at physiological
concentrations. On the other hand, the physiological levels of these
modulators in rat fed hepatocytes were not sufficient to modulate the
activity of both HPI and PGM.
The model simulations suggested that simultaneous inhibition of
HPI and PGM (Fig. 6) may be a novel strategy to decrease specifically
the glucose metabolism in cancer cells. As there are not specific in-
hibitors of HPI and PGM, the increase in the levels of the HPI and PGM
physiological inhibitors such as Fru1,6BP could be an alternative me-
chanism to decrease their activities in some cancer cells specifically.
Oxamate is an inhibitor of the glycolytic downstream steps LDH, PYK
and ENO [55]. Direct inhibition of these enzymes does not decrease
glycolysis, because the steps of the last segment of glycolysis in cancer
cells exert low control on glycolytic flux, as determined by elasticity
analysis and kinetic modeling studies [13,14,27]. Consequently, their
activities have to be inhibited by more than 90% to decrease the
pathway flux by 1–5% [13,14,27,55]. Kinetic modeling of cancer gly-
colysis revealed that oxamate inhibition of LDH, PYK and ENO was
insufficient to achieve glycolysis flux inhibition [23,55]. However, their
inhibition by oxamate induced an increase in the levels of the upstream
metabolites Fru1,6BP and DHAP, because PYK and ENO exert the
highest control on the concentrations of these metabolites [23].
Therefore, the high levels of these glycolytic metabolites induce a de-
crease in the glycolytic flux in cancer cells because HK and HPI (the
main controlling steps of cancer glycolysis) become inhibited
[13,14,23,55]. Oxamate does not affect glycolysis of non-cancer cells
because the Fru1,6BP and DHAP levels reached are low and insufficient
to inhibit HK and HPI. Likewise, kinetic modeling also indicated that
only when the Fru1,6BP and DHAP inhibitory effects on HPI and HK
activities were included in their respective rate equations, the glycolytic
flux became affected by oxamate [23,55].
The oxamate effects on glycogen metabolism have not been so far
characterized. Here, it was observed that oxamate inhibited glycogen
synthesis and glycolysis in AS-30D cells, but not in rat fed hepatocytes.
In other models of non-cancer cells (immortalized human B lympho-
cytes, normal human skin fibroblasts and mouse podocytes), it has also
been reported that oxamate (10–15 mM) induces slight decreases
(8–10%) in lactate production and ATP levels [71–73]. Moreover, ox-
amate attenuates growth of several cancer cell lines (human breast
cancer, MDA-MB-231and BT474; human chondrosarcoma, Hs819T and
SW1353; human nasopharyngeal carcinoma, CNE1 and CNE2; murine
13
BBA - General Subjects 1864 (2020) 129687
melanoma, B16F10) in nude mice [74–78] and shows low toxicity in
human and murine normal cells [74,77–79]. Then, according to the
results of the present study, it is possible that this apparent specificity of
oxamate on human and murine cancer cells derives from the non-con-
trolling role of HPI and PGM in normal cells. Further, in lung adeno-
carcinoma patients, the expression of HPI correlates with poor prog-
nosis and decreased overall survival [80].
Lithium, used to treat bipolar disorder, has been shown to inhibit
PGM activity and decrease the levels of glycogen in astrocytes [81].
Lithium has also shown anticancer activity in combination with cis-
platin and paclitaxel in ovarian cancer cells [82]. Therefore, based on
their effects on HPI and PGM, the combination of oxamate plus lithium
might be a strategy to decrease the glycogen synthesis and glycolytic
fluxes in cancer cells. The effects of this drug combination should be in
parallel determined, particularly on other pathways such as the Krebs
cycle, β-oxidation and OxPhos to search for collateral effects. Thus, to
solidly propose a mechanism-based multisite therapy, the present study
showed that it was necessary to determine the main controlling steps of
the targeted metabolic pathways in cancer and normal cells.
Author contributions
AM-H, JCG-P, MAR-G and MS-G performed the experiments. AM-H,
RM-S and ES conceived the study, designed the experiments, refined the
kinetic model, analyzed data and wrote the paper. AM-H, ES, MM-S, SR-
E, RM-S: revision and approval of the submitted manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The present work was partially supported by grants from CONACyT-
México to AMH (A1-S-40481), SRE (283144), RMS (239930 and
281428) and ES (282663).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bbagen.2020.129687.
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