Cell biology assessment of glucokinase mutations V62M and G72R in pancreatic
beta-cells: evidence for cellular instability of catalytic activity.
Received for publication 16 August 2006 and accepted in revised form 21 March
Catherine Arden1, Alison Trainer1, Nuria de la Iglesia1, Kathleen T.Scougall1, Anna
L. Gloyn2, Alex J. Lange3, James AM Shaw1, Franz M. Matschinsky4 and Loranne
Short title: Catalytic instability of glucokinase V62M and G72R
1Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2
2 Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology &
Metabolism, University of Oxford, Oxford OX3 7LJ
3Department of Biochemistry, Molecular Biology and Biophysics, University of
Minnesota, Minneapolis, MN55455
4 Department of Biochemistry & Biophysics and Diabetes Research Center,
University of Pennsylvania School of Medicine, Philadelphia 19104
(#) Correspondence address:
School of Clinical Medical Sciences,
The Medical School,
Newcastle upon Tyne, NE2 4HH, UK
Abbreviations: Ad-LGK, adenoviral vector expressing liver GK; Ad-GKRP,
adenoviral vector expressing GKRP; GFP, green fluorescent protein; GK,
glucokinase; GKA, glucokinase activator; GKRP, liver glucokinase regulatory
protein, 68kDa; GST, glutathione S-transferase; MG132, carbobenzoxy-L-leucyl-
Leucyl-L-leucinal; MODY, Maturity Onset Diabetes of the Young; PFK2/FDP2, 6-
phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PNDM, Permanent Neonatal
Diabetes Mellitus; WT, wild-type.
Diabetes In Press, published online March 27, 2007
Copyright American Diabetes Association, Inc., 2007
Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis.
In some cases (V62M and G72R) the phenotype cannot be explained by altered
enzyme kinetics or protein instability. We used transient and stable expression of
GFP-GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and
G72R. GK activity in lysates of MIN6 cell lines stably expressing GFP-GK wild-type
or mutants showed the expected affinity for glucose and response to pharmacological
activators indicating expression of catalytically active enzyme. MIN6 cells stably
expressing GFP-V62M or GFP-G72R had a lower GK-activity / GK-
immunoreactivity ratio and GK activity / GK-mRNA ratio but not GK-
immunoreactivity / GK-mRNA ratio than GFP-GK-wildtype. Heterologous
expression of liver PFK2-FDP2 in the cell lines increased GK activity for GK-wild-
type and V62M but not for G72R whereas expression of GKRP (liver GK regulatory
protein) increased GK activity for wild-type but not V62M or G72R. Lack of
interaction of these mutants with GKRP was also evident in hepatocyte transfections
from the lack of nuclear accumulation. These results suggest that cellular loss of GK
catalytic activity rather than impaired translation or enhanced protein degradation may
account for the hyperglycaemia in subjects with V62M and G72R mutations.
Glucokinase (hexokinase IV,
GK) is the predominant hexokinase
expressed in hepatocytes
pancreatic beta-cells where it functions
as the glucose sensor for insulin
secretion (1,2). It is also expressed in
cells of the gut, hypothalamus and
anterior pituitary (3-5). The role of GK
in the control of blood glucose
homeostasis is supported by the impact
of naturally occurring mutations, which
cause either diabetes (GK-MODY and
hyperinsulinaemic hypoglycaemia of
infancy (PHHI) (1, 6).
GK is regulated
transcriptional and post-transcriptional
mechanisms (7). Post-transcriptional
regulation differs in hepatocytes and
pancreatic β-cells as demonstrated by
the different sub-cellular location of GK.
In hepatocytes, regulation involves an
inhibitory regulatory protein (GKRP)
that functions as a nuclear receptor,
sequestering GK in an inactive state in
the nucleus at low glucose (8-10). An
increase in extracellular
concentration causes translocation of
GK to the cytoplasm, enabling rapid
stimulation of glucose phosphorylation.
Translocation of GK between the
nucleus and the cytoplasm is also
dependent on other proteins that
function as cytoplasmic receptors or
bifunctional protein PFK2/FDP2, which
catalyses the formation and degradation
of fructose 2,6-bisphosphate (11,12).
Islet β-cells do not express
hepatic GKRP and accordingly GK does
not accumulate in the nucleus (5,10,13).
Whether they express a truncated form
of GKRP lacking the nuclear localising
signal (14) is not known. PFK2/FDP2
serves as a GK binding protein in β-cells
and has a role in the regulation of GK
activity (11, 15, 16). Additional binding
proteins of GK have also been identified
such as the
in β-cells, including neuronal nitric
oxide and others (17-19).
To date approximately 200
naturally occurring GK mutations have
been identified that cause either diabetes
or hypoglycaemia (1,20). Detailed
kinetic analysis is available for about
one fifth of these (20,21) and whilst in
most cases the defective glycaemia can
be explained by the enzyme kinetics,
some mutations which co-segregate with
hyperglycaemia in GK-MODY have
near normal enzyme kinetics or even a
mild increase in affinity for glucose.
Some mutants (H137R,
C213R) are enzymatically unstable in
heat-stability assays (21,22), whilst the
mutant E300K also showed protein
instability when expressed in pancreatic
β-HC9 cells using an adenoviral
expression system (23),
particular interest are V62M and G72R
(24, 25). These mutants segregate with
hyperglycaemia in three families but are
moderately activating and show only
mild thermolability. Both mutants show
lack of inhibition by liver GKRP and
lack of activation by pharmacological
GK activators that bind to the novel
allosteric site (26). Because liver GKRP
is not expressed in pancreatic β-cells
and no physiological ligands have been
identified that mimic the action of
explanation is available as yet for the
mechanism by which V62M and G72R
The aim of this study was to
develop a cell-based approach to
investigate the activity and expression of
GK-MODY mutants, such as V62M and
G72R, where the clinical phenotype
cannot be explained by the kinetic
properties of the enzyme.
that are of
Generation of constructs: Constructs for
human wild-type pancreatic β-cell GK
were generated with the GFP-tag at
either the N- or C- terminus. For
generation of N-terminal GFP-tagged
GK, GK was excised from the
pT73.36His vector (kindly provided by
K. Brocklehurst, AstraZeneca, Cheshire,
UK) using the BamHI/XhoI restriction
sites for ligation into BglII/SalI of the
pEGFP-C1 vector (Gibco Invitrogen Co,
Paisley, UK). For generation of C-
terminal tagged GK, GK was excised
from the pT73.36His vector using the
BamHI/NotI restriction sites for ligation
into the PCR3 vector (Promega,
Southampton, UK) with addition of a C-
terminal GFP-tag generated by PCR.
The mutant constructs GST-V62M and
GST-G72R were excised from the
pGEX-3X plasmid and inserted into the
pEGFP-C3 vector for addition of the N-
terminal GFP tag as above. Correct
insertion into the
confirmed by DNA sequencing.
Cell culture and
adenoviral vectors. MIN6 cells (p20–
27) were cultured in Dulbecco’s
modified Eagle’s medium containing
15% (vol/vol) fetal bovine serum.
Adenoviral vectors for expression of
untagged GK (Ad-LGK), GKRP (Ad-
GKRP) and PFK2/FDP2
PFK2/FDP2) were described previously
(27,28, 29). MIN6 monolayers were
incubated with the adenoviral vectors
for 4-6 h and then cultured for 24-48 h
to allow protein expression.
Transfection and generation of stable
cell lines. For transient transfection,
MIN6 were transfected with 1-3µg
plasmid DNA and 1-3µl Lipofectamine
2000 for 8h and then cultured for 24-
48h. Stable cell lines were generated
after transient transfection by selection
with 300µg/ml G418 for 3-4 weeks. For
non-clonal cell lines (GFP-GK and GK-
GFP), the cells were cultured in G418-
containing medium. For clonal cell
lines (GFP-GK, GFP-V62M and GFP-
G72R), clones were isolated and
expanded in the presence of 300µg/ml
expression of GFP-GK were selected for
further study based on GK activity,
mRNA expression. The clones used in
this study were WT3B and WT10B for
GFP-GK, V62M 3C for GFP-V62M and
G72R 25A for GFP-G72R. For transient
transfection of hepatocytes, freshly
isolated rat hepatocytes (30) were
transfected 3h after attachment with 4µg
plasmid DNA and 12µl jet-PEI-Gal
(Qbiogene) and cultured for 24h.
GK activity, GK immunoreactivity and
GK kinetics. GK activity of MIN6 and
determined on 13,000g supernatants as
described in (31).
phosphorylating activity was determined
at 0.5mmol/l and 50mmol/l glucose,
hexokinase activity, respectively and
GK activity determined from the
difference between total hexokinases
and low Km
immunoreactivity was determined by
SDS-PAGE and immunoblotting with a
GK antibody (H-88 Santa Cruz Biotech,
Santa Cruz, CA) as described previously
(31). For kinetic analysis, GK activity
concentrations of glucose and a GKA
(GKA1, ref (32), a gift from M.
Coghlan, AstraZeneca, Cheshire, UK).
The S0.5 for glucose was determined
from Hill plots.
Real time RT-PCR. Cellular RNA was
extracted using TRIzol (Invitrogen) and
treated with DNaseI (Roche, East
Sussex, UK). Single strand cDNA was
synthesized from 1µg of total RNA with
and human GK
cell lines was
at the indicated
random hexamers and Superscript II
(Invitrogen). Real-time RT-PCR was
performed in a total volume of 10µl
containing 20ng of reverse transcribed
total RNA for determination of murine
GK, human GK and cyclophilin,
5µmol/l primers (Murine GK: forward
5’- GCA GAA GGG AAC AAC ATC
GT, reverse 5’- CAC ATT CTG CAT
CTC CTC CA; Human GK: forward 5’-
ATC TCC GAC TTC CTG GAC AA,
reverse 5’- CAC TCG GTA TTG ACG
CAC AT; Cyclophilin: forward 5’-
ATG GCA CTG GTG GCA AGT CC -
3’, reverse 5’- TTG CCA TTC CTG
GAC CCA AA -3’ (33)), 2mmol/l
MgCl2 and 1µl Sybr Green (Roche).
The reactions were carried out in
capillaries in a Light Cycler (Roche)
with initial denaturation at 95°C for
10min followed by 40 cycles consisting
of 95°C for 15sec, 58°C for 7sec and
72°C for 15sec. Murine and human GK
mRNA were expressed relative to
cyclophilin mRNA and as percentage of
Isolation of mouse pancreatic islets.
Pancreatic islets were isolated from
male C57/BL6 mice. The pancreas was
shaken with 1.5mg/ml liberase (Roche)
for 6 min, with further digestion with
washing in Krebs-Ringer Phosphate
Buffer (10mmol/l Hepes (pH 7.4),
90mmol/l NaCl, 5mmol/l NaHCO3,
4.8mmol/l KCl, 0.7mmol/l KH2PO4,
CaCl2, 0.1% BSA, 5.5mmol/l glucose).
Islets were picked
overnight in RPMI.
Insulin secretion. MIN6 monolayers
were cultured in 24-well plates and
washed with Krebs-Ringer buffer (34).
They were preincubated for 30 min in
followed by 1 h incubation at the
indicated concentrations of glucose and
a GKA (35), a gift from J. McCormack,
OSI Prosidion, Oxford, UK). After
overnight culture, islets were pre-
incubated with Krebs Ringer Phosphate
buffer (3 islets/200 µl) at 3 mmol/l
glucose for 30 min, followed by 2h at
the indicated concentrations of glucose
and GKA. Medium insulin was
determined using a rat insulin assay kit
(Mercodia, Uppsala, Sweden)
coverslips were washed with PBS and
fixed using 4% paraformaldehyde in
PBS. Immunostaining for endogenous
GK and insulin was as described
previously (31). Cells were visualised
using a Leica TCS-SP2-UV microscope
with an x63, NA 1.3 oil immersion
objective or a LSM Zeiss microscope
with an x63 1.4 oil immersion objective.
Statistical Analysis. This was by
ANOVA followed by the Bonferroni
test using the Prism analysis program.
GFP-tagged GK is catalytically active
when expressed in MIN6. Transient
transfection of MIN6 with GK tagged
with GFP at either the N-terminus or C-
immunoreactivity to GK at 62-64 kDa as
expected, and with a 7-10 fold increase
in GK activity, confirming that GFP-
tagged GK is catalytically active (Fig.
1A). Because, fluorescence microscopy
showed large intercellular heterogeneity
in GFP expression after transient
transfection we generated stable non-
clonal cell lines by G418 selection. GK
activity conferred by GFP-tagged GK
was stable during early passages (< p9)
but declined in later passages (Fig. 1B).
Likewise human GK-mRNA decreased
with progressive passage (5-8 fold
between p9 and p19). Expression of
endogenous (mouse) GK mRNA was
similar in the GK-GFP and GFP-GK cell
lines (125% and 93%) as in non-
transfected MIN6. The affinity of GK
for glucose (mmol/l) was similar in
extracts of GK-GFP and GFP-GK cells
(8.9 ± 0.3 and 8.9 ± 0.8) as in MIN6
overexpressing untagged GK with an
adenoviral vector (8.5 ± 0.6) and was
similarly increased by a GKA (32) in the
cell lines (2.3 ± 0.3 and 2.1 ± 0.1) as in
the untransfected MIN6 (2.8 ± 0.3)
indicating that the GFP tag does not
compromise affinity for glucose or
activation by a GKA.
Effects of GK
overexpression on insulin secretion.
Glucose-induced insulin secretion was
similar in GK-GFP and GFP-GK cell
lines as in untransfected early passage
MIN6 (Fig. 2A). Insulin secretion in β-
cells is dependent on GK activity (36)
and on the differentiated state, which in
MIN6 declines with passage (37).
Clonal cell lines transfected with GFP
alone were considered an inappropriate
control because they showed a greater
increase in low-Km hexokinase with
increasing passage than the GFP-GK
cell lines indicating
dedifferentiation (results not shown).
Conversely, untransfected early passage
MIN6 may represent
differentiated state than the GFP-GK
cell lines, which could explain the
secretion (Fig. 2A). To investigate the
effect of GK activity and concentration
on insulin secretion independently of
passage number or the differentiated
state of MIN6, we compared the effect
of titrated adenoviral overexpression of
untagged GK with pharmacological GK
activation. Whereas GK overexpression
had little effect on insulin secretion
irrespective of the glucose concentration
(Fig. 2A), the GKA caused a large
stimulation at 3
(Fig.2B). Similar results were obtained
when insulin secretion was determined
in mouse islets. The GKA caused a 2.6
fold stimulation (P < 0.01) whereas GK
immunoblotting) had no significant
effect (Fig. 2C).
Subcellular location of GFP-tagged GK
chimeras in MIN6 and hepatocytes.
Co-localisation of GK with insulin
granules in pancreatic β-cells has been
shown by immunofluorescence
microscopy ((38-41), Fig 3A), electron
microscopy (38, 41) and subcellular
fractionation (31). To determine
whether GFP-tagged GK colocalises
with insulin granules, GFP-GK cell lines
and MIN6 cells transiently transfected
with GFP-GK were stained for insulin
(red) and visualised by confocal
microscopy. Endogenous GK showed
clear co-localisation with endogenous
insulin (Fig. 3A) whereas GFP-GK
expressed transiently showed negligible
co-localisation (Fig. 3B) and stably
expressed GFP-GK showed partial co-
localisation with insulin (Fig. 3C).
When the GK-GFP and GFP-GK
constructs were transiently transfected
accumulated in the nucleus during
incubation with 5 mmol/l glucose
(results not shown) indicating that the
GFP tag does not interfere with binding
to nuclear GKRP, in agreement with
previous findings with an N-terminal
Effect of MG132 on expression of GFP-
tagged GK chimeras. We tested
whether the decrease in transgene
expression in late passages could be
prevented by inhibition of proteosomal
degradation using MG132. MG132 had
no effect in either untransfected MIN6
or MIN6 overexpressing untagged GK
but it increased GK activity and
immunoreactivity (64kDa) in the GK-
GFP cell lines (Fig. 4A). To test
whether this increase in GK activity and
immunoreactivity was due to inhibition
of protein degradation, we determined
the effect of MG132 in the presence of
synthesis. Although GK activity
decreased in the
cycloheximide, this decrease was not
prevented by MG132, excluding an
effect on protein degradation (Fig. 4B).
MG132 caused a large increase in
human GK-mRNA in the GFP-GK cell
lines (100-200 fold, Fig 6A below).
This was abolished
transcriptional inhibitor, actinomycin D
(Fig. 4C), indicating that MG132
increased transgene expression. MG132
was used in the rest of the study to
increase transgene expression.
Pharmacological activation of GK.
Previous enzyme kinetic studies on
showed that V62M and G72R mutants
have a lower S0.5 for glucose than wild-
type GK and were not activated by a
GKA (24,25). We determined the
affinity for glucose of GFP-V62M and
GFP-G72R in cell extracts of the clonal
cell lines pre-cultured with MG132 (see
above). Both mutants had a lower S0.5
for glucose than wild-type (wild-type,
9.0 ± 1.0; V62M 5.2 ± 0.4; G72R 4.6 ±
1.0 mmol/l) and
significantly activated by the GKA (32)
(Fig. 5A-C). There was no effect of
MG132 on the affinity for glucose (Fig.
GK activity and immunoreactivity of
GFP-V62M and GFP-G72R in clonal
cell lines. The catalytic activity and GK
immunoreactivity of the mutants were
determined in the clonal cell lines GFP-
V62M and GFP-G72R and compared
with two clonal cell lines expressing
wildtype GFP-GK (WT3B, WT10B).
GK activity, immunoreactivity and
mRNA were determined after pre-
culture without or with MG132, which
caused a large increase in transgene
mRNA, GK protein and activity (Fig.
6A-C). GFP-V62M and GFP-G72R had
a lower GK activity than the wild-type
clones when expressed relative to either
immunoreactivity or mRNA (Fig. 6D-
E). There was no significant difference
in GK immunoreactivity relative to
mRNA levels for
activity but not immunoreactive protein.
Transient transfection studies with GFP-
V62M and GFP-G72R.
activity/immunoreactivity ratio was also
determined after transient transfection of
MIN6 with varying plasmid amounts of
GFP-GK (wild-type), GFP-V62M and
GFP-G72R (Fig. 7A-B). Plots of GK
showed a lower
immunoreactivity ratio for both GFP-
V62M and GFP-G72R compared with
wild-type GFP-GK (Fig. 7C) consistent
with the findings from the clonal cell
The effect of the GKA (35) on insulin
secretion was determined in MIN6 that
were either untransfected or transiently
transfected with the GFP-constructs.
The GKA increased (P< 0.05) insulin
secretion at 5mmol/l glucose in all four
conditions tested (untransfected: 1.7 ±
0.2 to 2.7 ± 0.1; GFP-GK, 1.8 to ± 0.1 to
3.6 ± 0.1; GFP-V62M, 1.7 to ± 0.1 to
3.0 ± 0.1; GFP-G72R, 1.5 to ± 0.1 to
3.7 ± 0.2 µg/h per mg protein).
Stabilisation of GK by liver PFK2-FDP2
and GKRP. The GK-binding proteins
PFK2/FDP2 and GKRP modulate GK
activity in pancreatic β-cells and liver,
respectively. We therefore tested
whether the mutants show altered
activation or stabilisation when these
proteins are heterologously expressed in
MIN6 cells. Expression of PFK2/FDP2
using an adenoviral vector in the clonal
cell lines increased GK activity in the
wild-type clones and in GFP-V62M but
not in GFP-G72R or in the non-
transfected cells (Fig. 8A). The lack of
effect in the non-transfected cells may
be due to a saturating effect of the
endogenous PFK2/FDP2 on endogenous
Overexpression of the liver GKRP
protein resulted in a small but significant
increase in GK activity in the wild-type
GFP-GK but not in the mutant cell lines
or in untransfected MIN6 (Fig. 8B).
Interaction of the mutants with GKRP
was also tested by transient transfection
of the GFP constructs in hepatocytes.
Unlike GFP-GK which accumulated in
the nucleus at 5 mmol/l glucose and
translocated to the cytoplasm at 25
mmol/l glucose, neither GFP-V62M nor
GFP-G72R accumulated in the nucleus
at 5 mmol/l glucose (Fig. 8C).
Mutations in the GK gene
identified in MODY-2 subjects are
associated with a similar phenotype of
elevated fasting blood glucose (> 5.5
mmol/l) and a 2-hour increment in blood
glucose after an oral glucose tolerance
test of ~ 2 mmol/l, irrespective of
whether the mutation
compromises GK kinetics or has
negligible effect or is mildly activating
(21,24,25). The apparent paradox of
elevated blood glucose in MODY2
subjects with mildly activating GK
mutations is as yet unexplained. In this
study we used MIN6 cells either
expressing GFP-GK chimaeras to study
the expression of the MODY mutants
V62M and G72R, which are mildly
activating. The limitations of transient
heterogeneity of expression, which
necessitates high levels of transgene
transfection and also the variability in
mRNA and protein expression with
time. Conversely, the limitation of cell
lines is the progressive loss in
differentiated state with increasing
passage, which may differ between
wild-type and mutant clones. A
transient and stable transfection methods
identifies possible artefacts that may be
inherent in either technique.
We show in this study that GFP-
tagged GK expressed either transiently
in MIN6 cells or after clonal selection
with G418 is catalytically active and
shows similar kinetic properties as the
untagged enzyme or recombinant GST-
GK fusion enzymes (43). Accordingly,
GFP-tagged GK is predicted to be
functional when expressed in MIN6.
However, although clonal cell lines
increased GK activity, they showed
secretion as untransfected MIN6. This
cannot be attributed to mistargeting of
the GFP-tagged GK, because similar
results were obtained when untagged
GK was overexpressed
adenoviral vector in MIN6. The latter
finding is not a unique property of the
MIN6 β-cell model because lack of
stimulation of insulin secretion after GK
overexpression was also reported in rat
islets (27). In this study we show that in
both mouse islets and MIN6 cells there
is lack of stimulation of insulin secretion
by titrated GK overexpression, despite
stimulation by a pharmacological GKA.
A tentative explanation is that coupling
between GK activity and stimulation of
insulin secretion is dependent on
subcellular location or targeting through
Saturation of these
endogenous GK may explain the lack of
stimulation of insulin secretion by GK
overexpression despite stimulation by
the GKA, which mimics the effect of
We could not detect a difference in sub-
cellular localisation between mutants
and wild-type. It is noteworthy,
however, that colocalisation between
GK and insulin in MIN6 cells is most
clearly observed at endogenous levels of
GK (31,38,39). Expression of GFP-GK
wild-type resulted in the accumulation
of GK in the cytoplasm with partial co-
localisation with the insulin granules.
This may be explained by saturation of
GK binding proteins
saturation of GKRP in hepatocytes when
GK is overxpressed by 2-fold, which
results in a marked decrease in nuclear /
cytoplasmic distribution (44). Although,
we could not detect a difference in co-
localisation between mutants and wild-
type we also cannot unequivocally rule
out a difference in targeting.
We show in this study using
clonal cell lines expressing mutant
(V62M and G72R) or wild-type GFP-
GK that the
immunoreactivity ratio is lower for the
mutants than for wild-type GK, despite a
similar protein expression based on
immunoreactivity / mRNA ratio. The
transient transfection experiments in
MIN6 also confirmed a lower activity /
immunoreactivity ratio for V62M and
G72R (60% and 85% decrease in the
slope of the linear plots). Two possible
mechanisms can be considered for the
lower “specific activity” of the mutants.
First that GK binding proteins have a
positive effect on the wild-type enzyme
by either an “activation” mechanism as
proposed for PFK/FDP2
protecting GK from inactivation by
oxidation of thiol groups (45,46).
Second, that the mutants are more
unstable than the wild-type in the
cellular environment because their
susceptible to inactivation by oxidation
of thiol groups or other mechanisms.
The lack of catalytic instability of the
mutants when tested in thermal stability
assays using the purified recombinant
enzyme (24,25, Arden, C and Agius, L,
GK activity /
unpublished) argues against the latter
A role for binding proteins in
stabilising GK activity has been clearly
demonstrated in liver. Although GKRP
was initially identified as an inhibitor
and nuclear receptor for GK (8-10)
studies on GKRP-/- mice established a
major role for this protein in stabilising
hepatic GK at the post-transcritpional
level (47,48). However, less is known
about stabilising proteins in pancreatic
β-cells. Furthermore, whether MIN6
cells express the full complement of GK
binding proteins as pancreatic islets is
unknown. The present finding that
heterologous expression of the liver
protein GKRP in β-cells increased GK
activity for GFP-GK wild-type but not
V62M or G72R is consistent with the
lack of inhibition of catalytic activity of
the recombinant mutants by GKRP
(24,25), but is also indicative of a high
catalytic or protein instability of wild-
type GK in β-cells. The lack of effect
on GK activity of GKRP overexpression
in untransfected MIN6 indicates a role
for endogenous proteins in β-cells that
may have a homologous or analogous
function to GKRP. A role for
PFK2/FDP2 in activating GK in β-cells
has been demonstrated (11,15,16). In
this study expression of PFK2/FDP2
increased GK activity in cells expressing
GFP-GK wild-type and GFP-V62M but
not in non-transfected cells. This
supports a role for the endogenous
PFK2/FDP2 in MIN6, which was also
confirmed by down-regulation
PFK2/FDP2 by siRNA in MIN6 (C.
Arden & L. Agius, unpublished). The
lack of effect of PFK2/FDP2 on GK
activity in cells expressing GFP-G72R
suggests that this mutation affects the
indicates that the decrease in catalytic
activity / immunoreactivity for G72R is
at least in part explained by lack of
activation by PFK2/FDP2.
Although the V62M and G72R
mutants are relatively unresponsive to
activation by pharmacological GKAs
compared with wild-type-GK (24,25),
overexpressing GFP-V62M or GFP-
G72R was stimulated by the GKA to a
similar extent as in untransfected MIN6
or in cells expressing GFP-GK wild-
type. This shows that the mutants do
not have a dominant negative effect or
Accordingly, the explanation for the
phenotype of these mutations is the low
In summary, we show in this
study using a cell-based approach that
in MIN6 cells
cellular instability of GK catalytic
activity in pancreatic β-cells rather than
increased mRNA degradation, impaired
translation or enhanced
degradation may be the key factor that
accounts for the
associated with the V62M and G72R
ACKNOWLEDGEMENTS We thank
Drs Jun-ichi Miyazaki for the MIN6
cells. This work was funded by project
and equipment grant support from
Diabetes UK (BDA RD01/0002364;
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LEGENDS Download full-text
FIG. 1. Transient and stable expression of GFP-GK and GK-GFP in MIN6.
A. MIN6 were transiently transfected with GK-GFP and GFP-GK. After 48 h GK
activity (expressed as % of non-transfected cells, NT) and immunoreactivity (50 and
62-64 kDa) were determined. Results are representative of 2 experiments. B-D.
Non-clonal GK-GFP or GFP-GK cell lines. B GK activity at increasing passage
number. C,D. GK activity at varying glucose without (C dashed lines, D open bars)
or with (C solid lines, D solid bars) 5µmol/l GKA (32) in GK-GFP and GFP-GK cell
lines and MIN6 cells that were either untreated or pre-treated (18h) with Ad-LGK for
expression untagged liver GK (+GK). C. GK activity as % 30 mmol/l glucose, effects
of GKA were significant for all 4 cell types: * P < 0.05; ** P < 0.01. D. GK activity
at 30 mmol/l glucose * P < 0.05, ** P < 0.01 relative to untreated MIN6, n=6.