The role of glucose 6-phosphate in mediating the effects
of glucokinase overexpression on hepatic glucose
metabolism
Linda Ha
¨
rndahl
1
, Dieter Schmoll
2
, Andreas W. Herling
2
and Loranne Agius
1
1 School of Clinical Medical Sciences-Diabetes, The University of Newcastle upon Tyne, Medical School, Newcastle upon Tyne, UK
2 Aventis Pharma Deutschland GmbH, TD Metabolism, Frankfurt, Germany
Glucose 6-phosphate (Glc6P) is the first intermediate
in glucose metabolism and is generated by the hexo-
kinase-catalysed reaction. Hepatocytes express all four
hexokinase isoenzymes (EC 2.7.1.1), but predominantly
hexokinase IV, commonly known as glucokinase [1].
Glucokinase differs from the other isoenzymes by its
lack of inhibition by physiological concentrations of
Glc6P [2] and by its low affinity for glucose and sig-
moidal kinetics [1]. Accordingly, in the hepatocyte, the
cellular content of Glc6P responds to changes in both
glucose concentration and glucokinase activity [3]. The
latter is regulated by insulin and glucagon at the tran-
scriptional level [4], and by glucose, precursors of fruc-
tose 1-phosphate and hormones at post-transcriptional
Keywords
glucokinase activators; glucokinase; glucose

Glc6P (2.4–1.0) and glucokinase (3.7–1.8) on glycogen synthesis decreased
with glucose concentration. The high sensitivity of glycogenic flux to Glc6P
at low glucose concentration is consistent with covalent modification by
Glc6P of both phosphorylase and glycogen synthase. The high control
strength of glucokinase on glycogenic flux is explained by its concentration
control coefficient on Glc6P and the high control strength of Glc6P on gly-
cogen synthesis. It is suggested that the regulatory strength of pharmacolo-
gical glucokinase activators on glycogen metabolism can be predicted from
their effect on the Glc6P content.
Abbreviations
Fru2,6P
2
, fructose 2,6-bisphosphate; Glc6P, glucose 6-phosphate; GKRP, glucokinase regulatory protein; MEM, minimal essential medium;
PP-1, protein phosphatase-1; S4048, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-
acryloyloxy)-cyclohexanecarboxylic acid.
336 FEBS Journal 273 (2006) 336–346 ª 2005 The Authors Journal compilation ª 2005 FEBS
levels through the interaction of glucokinase with bind-
ing proteins [5,6]. The glucokinase regulatory protein
(GKRP) is a specific inhibitor of glucokinase [5] and
also a nuclear receptor that sequesters glucokinase in
the nucleus at low concentrations of glucose [7]. Its
affinity for glucokinase is decreased by fructose
1-phosphate, which causes translocation of glucokinase
from the nucleus to the cytoplasm [5,7]. Conditions
that cause dissociation of glucokinase from GKRP are
associated with a parallel increase in the cell content
of Glc6P, confirming the regulatory role of GKRP on
glucokinase activity [8]. Recent studies have identified
small molecule activators of glucokinase that bind to
an allosteric site and markedly increase the affinity

[17]. Glc6P, like glucose, favours the T conformation
[18] and regulates phosphorylase-a by allosteric inhibi-
tion and promoting its dephosphorylation [14]. The
actions of glucose and Glc6P on dephosphorylation
are synergistic [19], indicating an enhancing role for
Glc6P. Phosphorylase-a is a potent allosteric inhibitor
of glycogen synthase phosphatase activity by binding
to the glycogen targeting protein of PP-1, designated
GL [17]. Thus, Glc6P-mediated depletion of phos-
phorylase-a by dephosphorylation impacts glycogen
synthase by a cascade mechanism (Fig. 1).
Adenoviral vectors for the overexpression of gluco-
kinase and hexokinase I in hepatocytes have been very
useful tools for demonstrating the impact of over-
expression of these isoenzymes on Glc6P and glycogen
synthesis [3,20,21] and for applying metabolic control
analysis to titrated enzyme overexpression to determine
the control exerted by glucokinase on hepatic glucose
metabolism [22,23]. Although both glucokinase and
hexokinase I cause an increase in the cell content of
Glc6P, the former, but not the latter, causes activation
of glycogen synthase [3]. This anomaly can be
explained by sequestration of Glc6P derived from glu-
cokinase and hexokinase I in distinct pools [3,24], or
by the involvement of mechanisms, additional to
Glc6P, in mediating the effects of glucokinase over-
expression. As glucokinase binds to a dual-specificity
phosphatase [25], and also associates with PP1 in a
multiprotein complex [26], it might promote dephos-
phorylation of glycogen synthase through scaffolding

and phosphorylated, respectively. Phos-a is a potent inhibitor of
activation of glycogen synthase (GS) by binding to the glycogen tar-
geting protein, G
L
, in association with protein phosphatase-1 (PP-1),
which converts less active GSB to more active GSA by dephospho-
rylation. Glc6P (G6P) activates glycogen synthase by a cascade
mechanism, whereby (a) it favours the conversion of phos-a to
phos-b by a substrate-directed mechanism that is synergistic with
glucose, (b) depletion of phos-a relieves the inhibition of G
L
, and (c)
Glc6P stimulates the conversion of GSB to GSA by a substrate-
directed mechanism.
L. Ha
¨
rndahl et al. Control strength of Glc6P on glycogenesis
FEBS Journal 273 (2006) 336–346 ª 2005 The Authors Journal compilation ª 2005 FEBS 337
metabolic effects of small-molecule glucokinase activa-
tors, which may be a future treatment for type-2 diabe-
tes [9–11]. A wide range of these molecules have been
reported in patents [27,28]. However, there have been
few published studies of their metabolic effects in
hepatocytes [10], leading to extrapolation of their
expected effects from studies of glucokinase overex-
pression [29,30].
The aim of the present study was to provide a better
understanding of the contribution of Glc6P to the
metabolic effects of glucokinase overexpression in
hepatocytes and to test for evidence for additional

concentration, which was significant at 10–35 mm glu-
cose after 1 h (Fig. 3A) and at 15–35 mm glucose after
3 h (results not shown). S4048 increased the fructose
2,6-bisphosphate (Fru2,6P
2
) content by twofold at
5–15 mm glucose and by 50–70% at higher glucose
concentrations (Fig. 3B). Glycogen synthesis was
increased by S4048 by between two- and threefold at
glucose concentrations of > 10 mm (Fig. 3C), whereas
glycolysis was modestly increased (24–48%) at
10–20 mm glucose, but not at higher glucose concen-
trations (Fig. 3D). S4048 had no effect on either
glucokinase binding or on the metabolism of
[2-
3
H]glucose, which approximates the rate of glucose
phosphorylation (Fig. 3E,F). The distribution of glu-
cokinase between the nucleus and the cytoplasm,
expressed as the nuclear ⁄ cytoplasmic ratio, was also
not affected by S4048 (2 lm) at either 5 mm glucose
(2.0 ± 0.1 vs. 2.0 ± 0.1, mean ± SE; n ¼ 18) or
20 mm glucose (1.5 ± 0.1 vs. 1.5 ± 0.1). These results
suggest that the increase of Glc6P in the presence of
S4048 was caused by the inhibition of Glc6P hydro-
lysis in the absence of detectable changes in glucose
phosphorylation.
S4048 causes inactivation of phosphorylase
As the elevation of Glc6P causes inactivation of phos-
phorylase in hepatocytes [14], the increase in Glc6P

(control, 0.4 ± 0.1; 2 lm S4048, 2.2 ± 0.6 nmolÆmg
)1
of protein), but not in its absence (from 0.3 ±
0.1 nmolÆmg
)1
of protein to 0.2 ± 0.1 nmolÆmg
)1
of
protein) and, likewise, S4048 (0.05–2 lm) caused inac-
tivation of phosphorylase in the presence, but not in
the absence, of dihydroxyacetone (Fig. 5A), confirming
that the inactivation by S4048 is caused by the elevated
Glc6P. A plot of phosphorylase-a activity against
the respective Glc6P content in the incubations with
dihydroxyacetone and varying concentrations of S4048
showed saturation of phosphorylase inactivation at
 1.4 nmolÆmg
)1
of Glc6P and a half-maximal effect at
0.48 ± 0.15 nmol
)1
Æmg
)1
(Fig. 5B). The saturation of
phosphorylase inactivation by Glc6P could explain the
lack of further inactivation by S4048 at 35 mm glucose
(Fig. 4).
Comparison of the metabolic effects of
glucokinase overexpression and S4048
As the effects of S4048 on glucose metabolism are not

010203040
0
100
200
300
400
*
***
*
***
***
***
***
**
**
**
***
**
0
2
4
6
8
10
Control
S4048
***
**
**
**

).
(D) Glycolysis (nmolÆ3h
)1
Æmg
)1
). (E) Free
glucokinase activity (% total), 3 h. (F)
Glucose phosphorylation (nmolÆ3h
)1
Æmg
)1
).
Data represent the mean ± SE from four to
six experiments. *P < 0.05; **P < 0.01;
***P < 0.001, effect of S4048.
Fig. 4. 1-[2-(4-Chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-
5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarb-
oxylic acid (S4048) potentiates the inactivation of phosphorylase-a
by glucose. Hepatocytes were incubated for 3 h with the concen-
trations of glucose shown, in the absence or presence of 2 l
M
S4048. Phosphorylase-a is expressed as munitsÆmg
)1
of protein.
Data are the mean ± SE from five experiments. *P < 0.05; **P <
0.01, effect of S4048.
L. Ha
¨
rndahl et al. Control strength of Glc6P on glycogenesis
FEBS Journal 273 (2006) 336–346 ª 2005 The Authors Journal compilation ª 2005 FEBS 339

J
G6P
), and (c) the sensi-
tivity of glycogen synthesis to glucokinase activity,
expressed as the flux control coefficient of glucokinase
(C
J
GK
) (Table 1).
The concentration control coefficient of glucokinase
on Glc6P, determined from the slope of double log
plots of Glc6P against the respective glucokinase activ-
ity, was relatively independent of glucose concentration
(1.4–1.7). However, the flux control coefficient of
Glc6P on glycogen synthesis was more than twofold
higher at 5 mm compared with 20 mm glucose, as was
the flux control coefficient of glucokinase on glycogen
synthesis (Table 1).
Dual control of glycogen synthase by Glc6P and
phosphorylase
The higher flux control coefficient of Glc6P on glyco-
gen synthesis at 5 mm compared with 20 mm glucose
(Table 1) may be in part caused by a dual effect of
Glc6P on phosphorylase and glycogen synthase at low
Glc6P and by an effect on glycogen synthase at eleva-
ted Glc6P. To test the latter possibility, we determined
the separate and combined effects of S4048 and gluco-
kinase overexpression on phosphorylase inactivation and
glycogen synthase activation and compared this with
the effects of a phosphorylase inhibitor (CP-91149),

tion of phosphorylase-a (0.5 nmolÆmg
)1
). It is notewor-
thy that activation of glycogen synthase by CP-91149
was independent of changes in Glc6P, consistent with
the role of phosphorylase-a as a negative modulator of
synthase phosphatase [17].
Discussion
Glc6P is an allosteric regulator of glycogen synthase
and phosphorylase and it also promotes the de-
phosphorylation of both enzymes, causing activation
of synthase and inactivation of phosphorylase (con-
version of phosphorylase-a to phosphorylase-b,
Fig. 1). As phosphorylase-a is a potent allosteric
inhibitor of glycogen synthase phosphatase, depletion
of phosphorylase-a by Glc6P leads to the further
activation of glycogen synthase through a cascade
Fig. 6. Glycogen synthesis correlates with
glucose 6-phosphate (Glc6P), and glycolysis
correlates with glucose phosphorylation.
Hepatocytes were either untreated (control,
S0.02, S0.5) or treated with varying titres of
adenoviral vector encoding rat liver glucokin-
ase (Ad-LGK) for overexpression of glucokin-
ase by 1.7-fold (GK1.7), 2.5-fold (GK2.5) and
4.2-fold (GK4.2). They were incubated with
5, 10, 15 or 20 m
M glucose, in the absence
(Con) or presence of 1-[2-(4-chloro-phenyl)-
cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-

correlation studies [13]. However, evidence that the
overexpression of hexokinase I causes a smaller acti-
vation of glycogen synthase than expected from the
increment in Glc6P [3] has raised questions on the
extent by which the steady-state cell content of
Glc6P, as distinct from either glucose phosphoryla-
tion or glucokinase protein through macromolecular
interactions, accounts for the metabolic effects of glu-
cokinase overexpresssion.
In this study we established the validity of S4048, a
potent inhibitor of Glc6P transport into the endoplas-
mic reticulum and thereby of Glc6P hydrolysis [31], as
an experimental tool to modulate the Glc6P content in
the absence of changes in rates of glucose phosphory-
lation. We show, using S4048 to modulate Glc6P, that
during glucokinase overexpression, glycogenic flux cor-
relates closely with Glc6P content rather than glucose
phosphorylation, whereas the converse is true for gly-
colysis. We used metabolic control analysis to deter-
mine the quantitative relationship between glucokinase
and the cell content of Glc6P, and also between Glc6P
and metabolic flux.
S4048 causes large perturbations in Glc6P concen-
tration in liver cells [31,32,38], as well as secondary
changes in gene expression, including up-regulation of
lipogenic enzymes and down-regulation of glucokinase
mRNA levels [38]. In the experimental conditions used
in this study, involving incubation for up to 3 h, S4048
Table 1. Control coefficients of glucokinase and glucose 6-phos-
phate (Glc6P ) on glycogen synthesis. Coefficients were determined

G6P
C
J
GK
5 0.2–1.1 1.4 1.7 (2.4) 2.6 (3.7)
10 0.4–2.2 1.4 1.3 (1.9) 2.0 (2.8)
15 0.5–3.7 1.7 0.8 (1.1) 1.7 (2.4)
20 0.6–4.3 1.7 0.7 (1.0) 1.1 (1.8)
Fig. 7. Combined effects of glucokinase overexpression and 1-[2-(4-
chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-
b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid
(S4048) on phosphorylase and glycogen synthase. Hepatocytes
were either untreated (Control, S, CP) or treated with adenoviral
vector encoding rat liver glucokinase (Ad-LGK) (glucokinase or
GKS), and incubated for 1 h without or with 2 l
M S4048 (S)
or 10 l
M CP-91149 (CP) at either 10 mM (open bars ⁄ symbols) or
25 m
M glucose (solid bars ⁄ symbols), as indicated. Phosphorylase-a
(A), glycogen synthase and Glc6P were determined as described in
the Experimental procedures. (B) Glycogen synthase (activity ratio)
relative to the corresponding Glc6P. Data represent the mean ± SE
of three experiments. *P < 0.05, **P < 0.01, relative to the
control.
Control strength of Glc6P on glycogenesis L. Ha
¨
rndahl et al.
342 FEBS Journal 273 (2006) 336–346 ª 2005 The Authors Journal compilation ª 2005 FEBS
did not affect the total glucokinase activity, suggesting

accessible to the regulatory mechanisms involved in sti-
mulation of glycogen synthesis [13]. Another possibility
is that hexokinase I may have an inhibitory effect on
glycogen synthesis.
Glycolysis, unlike glycogen synthesis, correlated
more strongly with glucose phosphorylation than with
Glc6P concentration. Glc6P has a dual role in the con-
trol of glycolysis through changes in the cell content of
Fru2,6P
2
, an activator of phosphofructokinase-1 [37],
and up-regulation of pyruvate kinase gene expression
[38]. Although S4048 increased the Fru2,6P
2
content,
as expected [37], the stimulation of glycolysis was small
and only observed at low glucose concentrations. This
is consistent with recent findings that the overexpres-
sion of phosphofructokinase-2 ⁄ fructose bisphospha-
tase-2 does not increase glycolytic flux, despite the
increase in Fru2,6P
2
concentration, indicating that the
endogenous Fru2,6P
2
concentration is saturating [6].
Changes in protein expression are probably minimal
under the short-term incubation conditions of the
present study (see above). It can be inferred therefore
that under short-term conditions, the rate of glucose

sigmoidal function of Glc6P concentration when deter-
mined over a range of glucose concentrations and
activities of glucokinase; and, third, the flux control
coefficient of Glc6P on glycogen synthesis shows very
similar trends to the flux control coefficient of gluco-
kinase on glycogen synthesis and is more than twofold
higher at 5 mm glucose than at 20 mm glucose.
The large increase in Glc6P content (5.5–7-fold) dur-
ing glucokinase overexpression (fourfold) is consistent
with the prediction that perturbations in enzyme activ-
ity cause large changes in metabolite concentrations
[40] and occurs despite the presence of multiple mecha-
nisms that buffer the Glc6P concentration, including
glucose 6-phosphate hydrolysis.
The sigmoidal relationship between the rate of gly-
cogen synthesis and Glc6P indicates a high sensitivity
at low Glc6P concentration. This can be explained by
the combined effects of Glc6P on phosphorylase and
glycogen synthase (Fig. 1). Glc6P causes allosteric
activation and enhances dephosphorylation of glyco-
gen synthase through a substrate-directed mechanism.
The affinity of glycogen synthase for Glc6P as an
allosteric activator is a sigmoidal function of the
degree of dephosphorylation [16]. However, the sensi-
tivity of glycogen synthase to Glc6P as an allosteric
L. Ha
¨
rndahl et al. Control strength of Glc6P on glycogenesis
FEBS Journal 273 (2006) 336–346 ª 2005 The Authors Journal compilation ª 2005 FEBS 343
activator is an inverse function of the affinity for

hepatocyte for glucose and a similar stimulation of
glycogenesis as that induced by physiological stimuli
(precursors of fructose 1-phosphate) but a larger sti-
mulation of glucose phosphorylation and glycolysis
[10]. In contrast, glucokinase overexpression causes a
larger increase in both glycogen synthesis and Glc6P
content than either physiological or pharmacological
activation of endogenous glucokinase [3]. This can be
explained by the progressive increase in Glc6P
caused by increasing glucokinase expression and by
the high flux control coefficient of Glc6P on glyco-
gen synthesis. Based on the results of the present
study, it is suggested that the regulatory strength of
glucokinase activators on glycogen metabolism can
be predicted from the magnitude of their effect on
the cell content of Glc6P. If dysregulation of lipid
metabolism by glucokinase overexpression in vivo
[30,43] is caused by Glc6P, as suggested by the lipid
accumulation caused by S4048 [38], then it could be
argued that the impact of glucokinase activators on
lipid metabolism can also be predicted from the
Glc6P content.
Experimental procedures
Materials
S4048 [31] was synthesized at Aventis (Pharma GmbH,
Frankfurt, Germany), and CP-91149 [35] was a kind gift
from Pfizer Global Research and Development (Pfizer, New
London Laboratories, Groton, CT, USA). Sources of other
reagents were as described previously [6].
Hepatocyte isolation and monolayer culture

glycolysis or glucose phosphorylation, respectively. Glyco-
gen synthesis was determined from the incorporation of
14
C
label into glycogen [22], and glycolysis and glucose phos-
phorylation from the formation of
3
H
2
O [8]. Parallel incuba-
tions were performed to determine enzyme activities and
metabolites. Rates of metabolic flux were similar in 1 h and
3 h incubations, and the cell content of Glc6P and the activ-
ity of phosphorylase were also similar after 1 h and 3 h.
Metabolite and enzyme assays
Glc6P and Fru2,6P
2
were determined as described previ-
ously [8]. Glucokinase (free and bound activity) was deter-
mined by the digitonin permeabilization assay and free
activity is expressed as the percentage total [22]. Phosphory-
lase-a, assayed spectrometrically [42], is expressed as munitsÆ
mg
)1
protein, and glycogen synthase, assayed in the
absence or presence of Glc6P [42], is expressed as the activ-
ity ratio.
Control strength of Glc6P on glycogenesis L. Ha
¨
rndahl et al.

UK and the Medical Research Council (Joint Research
Equipment Initiative) for equipment grants to LA; Dr
Chris Newgard for the adenoviral vector and Dr
Judith Treadway for helpful advice.
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