Download GLUT2 intracytoplasmic loop transmits glucose signaling

841
Journal of Cell Science 113, 841-847 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS0902
The large intracytoplasmic loop of the glucose transporter GLUT2 is involved
in glucose signaling in hepatic cells
Ghislaine Guillemain1,2, Martine Loizeau1, Martine Pinçon-Raymond2, Jean Girard1 and Armelle Leturque1,2,*
1Endocrinologie Métabolisme et Développement, CNRS – UPR1524,
2Institut Biomédical des Cordeliers, INSERM-U505, 15 rue de l’Ecole
9 rue Jules Hetzel, 92190 Meudon, France
de Médecine, 75006 Paris, France
*Author for correspondence at address 2 (e-mail: [email protected])
Accepted 14 December 1999; published on WWW 14 February 2000
SUMMARY
The hypothesis that the glucose transporter GLUT2 can
function as a protein mediating transcriptional glucose
signaling was addressed. To divert the putative interacting
proteins from a glucose signaling pathway, two
intracytoplasmic domains of GLUT2, the C terminus and
the large loop located between transmembrane domains 6
and 7, were transfected into mhAT3F hepatoma cells.
Glucose-induced accumulation of two hepatic gene mRNAs
(GLUT2 and L-pyruvate kinase) was specifically inhibited
in cells transfected with the GLUT2 loop and not with the
GLUT2 C terminus. The dual effects of glucose were
dissociated in cells expressing the GLUT2 loop; in fact a
normal glucose metabolism into glycogen occurred
concomitantly with the inhibition of the glucose-induced
transcription. This inhibition by the GLUT2 loop could be
due to competitive binding of a protein that normally
interacts with endogenous GLUT2. In addition, the GLUT2
loop, tagged with green fluorescent protein (GFP), was
located within the nucleus, whereas the GFP and GFPGLUT2 C-terminal proteins remained in the cytoplasm. In
living cells, a fraction (50%) of the expressed GFP-GLUT2
loop translocated rapidly from the cytoplasm to the nucleus
in response to high glucose concentration and conversely in
the absence of glucose. We conclude that, via protein
interactions with its large loop, GLUT2 may transduce a
glucose signal from the plasma membrane to the nucleus.
INTRODUCTION
interactions (Johnston, 1999). Extracellular glucose is detected
by two nutrient sensors of low and high concentrations, Snf3p
and Rgt2p, cloned in Saccharomyces cerevisiae (Özcan et al.,
1996). These proteins are structurally similar to mammalian
and yeast glucose transporters, except for a long cytoplasmic
C terminus (Celenza et al., 1988; Özcan et al., 1996) that may
serve as a signaling domain (Ko et al., 1993; Schmidt et al.,
1999; Vagnoli et al., 1998). The nature of the glucose induction
signal is unknown, but it is not generated by glucose
metabolism, contrary to the glucose repression signal
(Johnston, 1999).
The aim of this study was to test the hypothesis that, in
addition to its transport activity, GLUT2 can function as a
glucose sensor, triggering glucose-regulated gene expression.
GLUT2 is a member of a family of proteins (GLUTs) whose
predicted structure consists of 12 transmembrane domains, and
N- and C-terminal cytoplasmic domains. A large loop between
transmembrane domains 6 and 7 is predicted to reside in the
cytoplasm. The specificity of GLUT isoforms is determined by
the intracytoplasmic domains, which are not conserved in
terms of their amino acid sequences (Bell et al., 1990). Signal
transduction from a transmembrane receptor protein requires
the activation of its intracytoplasmic domain. If GLUT2
transmits a glucose signal, one of its intracytoplasmic domains
must be part of the signaling pathway. In order to identify the
Glucose signaling in eukaryotic cells can be regarded as a
process involving the detection of extracellular glucose levels,
followed by the transduction to the nucleus of a glucose
repression or induction signal. Hepatocytes and pancreatic β
cells both stimulate gene transcription in response to high
glucose concentrations (Chen et al., 1990; Girard et al., 1997;
Towle and Kaytor, 1997; Vaulont and Kahn, 1994). In these
cells, glucose transport is mediated by the GLUT2 glucose
transporter (Thorens et al., 1988). A close correlation between
GLUT2 levels and glucose-regulated gene expression is
observed in hepatoma cells (Antoine et al., 1997), engineered
β cells (Newgard et al., 1997) and GLUT2-null mice (Guillam
et al., 1997), suggesting that in liver and pancreatic β cells, the
presence of GLUT2 is required for the transcriptional effect of
glucose.
The primary role of GLUT2 is to transport glucose inside
the cell, where it is metabolized. The prevailing idea is that the
glucose signal in eukaryotic cells originates from its own
metabolism (Girard et al., 1997; Towle and Kaytor, 1997;
Vaulont and Kahn, 1994). A recent report, however, provides
evidence that the glucose-signaling pathway can be triggered
by other means. Originating from a plasma membrane sensor,
this pathway, described in yeast, is mediated by protein-protein
Key words: GLUT2, Glucose signaling, Living cell, Liver, Glucose
metabolism, Glucose-regulated transcription
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G. Guillemain and others
GLUT2 domain that affects glucose sensing in the cell, the C
terminus and large intracellular loop of GLUT2 were expressed
in a hepatoma cell line. In large amounts, the intracytoplasmic
domains would appear to compete with endogenous GLUT2
for binding to proteins involved in the glucose signaling
pathway and thereby modify glucose-regulated gene
expression. We assessed the impact of expression of the two
domains on both a metabolic and a transcriptional effect of
glucose.
MATERIALS AND METHODS
Cell culture
The mhAT3F hepatoma cell line was derived from transgenic mice
synthesizing the SV40 large T and small t antigens under the control
of the antithrombin III promoter (Antoine et al., 1992). mhAT3F cells
were grown in Dulbecco’s modified Eagle’s medium/Ham-F12,
Glutamax (Life Technologies) supplemented with 100 ui/ml
penicillin, streptomycin, 0.1 µM insulin, 1 µM dexamethasone, 1 µM
triiodothyronine and 5% fetal calf serum. The mhAT3F cell line was
chosen because GLUT2 is abundantly expressed and the stimulation
of gene transcription by glucose is preserved (Antoine et al., 1997).
The cells were cultured in 17 mM glucose unless otherwise stated.
Cells were also cultured in glucose-free medium for 24 hours (it
should be noted that some glucose was present in the fetal calf serum).
When mhAT3F cells were cultured in the absence of glucose, the
residual glucose concentration in the medium after 24 hours was
0.03±0.01 mM. When mhAT3F cells were cultured with an initial
glucose concentration of 17 mM, the residual glucose concentration
after 24 hours was 8.65±0.10 mM.
Plasmid constructs
The coding regions of the intracellular loop between transmembrane
domains 6 and 7 of the rat GLUT2 glucose transporter (amino acids
237-301) and the C terminus GLUT2 domain (amino acids 481-521)
were amplified by means of PCR using a plasmid containing the fulllength GLUT2 cDNA (Thorens et al., 1988) as template. The
fragments were then inserted, in-frame, into the pEGFPc vector
(Clontech), in fusion with green fluorescent protein (GFP).
Transfection experiments
mhAT3F cells were plated and transfected by using the calcium
phosphate-DNA precipitation method (Chen and Okayama, 1988).
The GLUT2 loop and GLUT2 C terminus in pEGFP vectors, and the
vector alone, were transfected and harvested after 2 days for transient
expression measurements. Transfected cells were selected with
geneticin sulphate G-418 (0.4 mg/ml) (Gibco-BRL) for 3 weeks.
Pooled colonies of stably transfected cell lines were established, and
individual clones were then obtained by clonal dilution.
Western blot analysis
Proteins were separated by SDS-PAGE (10% acrylamide) and
electrotransferred to a nitrocellulose membrane. After overnight
incubation in 5% (w/v) non-fat milk in Tris-buffered saline (TBS) (20
mM Tris-HCl, pH 7.5, 150 mM NaCl), the blots were washed in TBS
containing 0.1% (v/v) Triton X-100 and incubated with a monoclonal
anti-GFP (Clontech) or anti-GLUT2 C terminus antibody (EastAcres). Immune complexes were detected by ECL (Amersham).
Northern blot analysis
Total RNA was purified from confluent mhAT3F cells after 24 hours
of culture in the various experimental conditions, and northern
analysis was performed as previously described (Postic et al., 1993).
The northern blots were hybridized with radiolabeled liver-pyruvate
Fig. 1. (A) Schematic representation of the secondary structure of
GLUT2 in the plasma membrane. (B) Transfection of mhAT3F with
intracytoplasmic domains of GLUT2 tagged at the N terminus with
GFP. (C) Western blot analysis of the fusion proteins GFP-GLUT2
loop and GFP-GLUT2 C terminus domain, revealed by an anti-GFP
antibody. Lanes 1,2: 10 µg of homogenate of mhAT3F cells
constitutively expressing GFP; lanes 3,4: 10 µg of homogenate of
mhAT3F cells constitutively expressing GFP-GLUT2 C terminus;
lanes 5,6: 10 µg of homogenate of mhAT3F cells constitutively
expressing GFP-GLUT2 loop. In lanes 1, 3 and 5 the cells were
cultured in the absence of glucose and in lanes 2, 4 and 6 with 17
mM glucose. The positions of molecular mass marker proteins are
shown.
kinase and GLUT2 cDNAs and mRNAs were quantified by means of
computer-based scan analysis. An 18S rRNA probe was used to
visualize RNA loading (Postic et al., 1993).
Determination of radiolabeled glucose incorporation into
glycogen
[14C]glucose (2 µCi, Amersham Pharmacia) incorporation into
glycogen was measured in confluent cells cultured in 30 mm dishes,
after 24 hours in the different experimental conditions, as previously
described (Kasus-Jacobi et al., 1997). Cells were washed twice with
ice-cold 0.9% NaCl and scraped free in 30% KOH lysis buffer.
Glycogen was precipitated with ethanol and radioactivity was
counted. Results are expressed as nmoles glucose incorporated into
glycogen/106 cells.
Fluorescence analysis
Stable clones of mhAT3F cells were grown on Permanox fourchamber slides (Lab-Tek, Nunc) with 17 mM glucose. Cells were
washed three times with PBS (137 mM NaCl, 1.3 mM KCl, 16.1 mM
Na2HPO4, 1.5 mM KH2PO4), fixed for 30 minutes in 4%
paraformaldehyde in PBS, and quenched for 10 minutes in 50 mM
NH4Cl. The nuclei were stained bright blue using Hoechst 33258 (0.5
µg/ml) for 5 minutes. After extensive washes in PBS the coverslips
were mounted in Mowiol (Hoechst). Fluorescence microscopy was
performed using an Olympus IMT2 inverted microscope, a
GLUT2 intracytoplasmic loop transmits glucose signaling
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Fig. 2. Effect of glucose on GLUT2
and liver-pyruvate kinase RNA
accumulation in mhAT3F cells
stably transfected with GFP, GFPGLUT2 C terminus and GFPGLUT2 loop. (A) This
representative northern blot was
hybridized with GLUT2 cDNA.
After hybridization with L-PK
cDNA, two bands were detected
corresponding to liver-pyruvate
kinase (L-PK) and muscle-pyruvate
kinase (m-PK).The blots were also
hybridized with an 18S rRNA probe
as control. In mhAT3F cells, m-PK
mRNA was constitutively expressed
and served as a control.
(B) Quantification was performed
by scanning densitometry.
*Significant increase in mRNA
(P<0.05) in cells cultured with
glucose (+) compared to cells (−)
cultured in the absence of glucose;
NS, no significant difference
between the two groups (n=4).
fluorescence immersion lens (40×) and an FITC filter. Photographs
(Kodak Panther P1600 film) were taken with an Olympus MO-4Ti
camera adapted to the microscope. The location of the fusion proteins
was quantified by using the Imagetool Software.
Fluorescence imaging of GFP-GLUT2 loop
A stable clone of GFP-GLUT2 loop was grown on glass coverslips
Labtek (Nunc), with or without glucose added in the culture medium
for 24 hours. The experiments were performed at 25°C in circulating
PBS with or without glucose (17 mM). The microscopy was
conducted using a confocal Zeiss LSM 510 microscope and a 63×
objective lens. One 1 µm slice located in the nucleus of the cell was
observed, and images were captured every 30 seconds during the
periods of the experiments. The PBS was circulating constantly; the
total replacement of PBS in the culture chamber took 1 minute.
Captured images were then analysed using the provider software.
Quantification of green fluorescence was established on a scale of grey
intensity (256 grey levels). The mean grey intensities were measured
in a circle (diameter of the region of interest: 5 µm) arbitrarily
positioned inside the nucleus and in the cytoplasm of the cell. The
results, expressed as mean grey intensities, were recorded in each
captured image during the experimental period. We verified that the
replacement of the circulating medium by a medium of the same
composition did not affect fluorescence location (results not shown).
Statistical analysis
Results are expressed as means ± s.e.m. Statistical analysis was
performed by using Student’s t-test for unpaired data.
RESULTS
Expression of GLUT2 intracytoplasmic domains in
mhAT3F cells
The production of fusion proteins in clonal stable mhAT3F
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G. Guillemain and others
GLUT2 mRNA in mhAT3F
Arbitrary Units
160
* wild type
*
120
GFP-GLUT2 loop
80
40
no
glucose
fructose
xylose
no
glucose
fructose
xylose
0
Fig. 3. Effect of glucose, fructose and xylose on GLUT2 mRNA
accumulation in wild-type mhAT3F cells and mhAT3F cells stably
transfected with the GFP-GLUT2 loop. Northern blots were
hybridized with GLUT2 cDNA and quantified by scanning
densitometry. *Significant increase (P<0.05) compared cells cultured
for 24 hours in the absence (no) or presence of 17 mM sugar (n=4).
GLUT2 mRNA amounts in mhAT3F cells expressing the GFPGLUT2 loop were always statistically lower than GLUT2 mRNA
amounts in wild-type cells (P<0.01; n=4).
GLYCOGEN
14
6
(nmol [U- C ]glucose/10 cell / 24h)
GFP
GFP- loop
GFP- C term
*
60
Fig. 5. Location of fluorescent proteins in cells stably transfected
with GFP (A,B), GFP-GLUT2 C terminus (C,D) and GFP-GLUT2
loop (E,F). (B,D,F) The nuclei were stained with Hoechst dye.
(A,C,E) The green fluorescent fusion proteins are visualized by
epifluorescence with a light microscope.
40
*
20
0
0
5
17
0
5
17
0
5 17 mM glucose
Fig. 4. Effect of the glucose concentration on glycogen synthesis in
mhAT3F cells stably transfected with GFP or GFP fusions of
intracytoplasmic domains of GLUT2. Results are expressed in
nmoles U14C-glucose incorporated into glycogen by 106 cells over
24 hours. *Significant increase in cells transfected with GFP-GLUT2
C terminus (P<0.05) compared to cells transfected with GFP-GLUT2
loop (n=5-6 determinations).
transfectants was analyzed by using an antibody directed
against green fluorescent protein. The fusion proteins
recognized by the anti-GFP antibody were of the expected
sizes, i.e. 29 kDa for GFP alone, 32.5 kDa for GFP-GLUT2 C
terminus and 34.8 kDa for GFP-GLUT2 loop (Fig. 1). The
expression levels of the three fusion proteins were similar. As
necessary for this study, the expression of the fusion proteins
was not affected by the presence of glucose in the culture
medium.
Expression of the GFP-GLUT2 C terminus and loop was 3-
4 times higher than that of endogenous GLUT2 in
untransfected cells, as estimated by northern blotting (not
shown). All mRNA signals were detected using the
radiolabeled full-length GLUT2 cDNA as probe. The probe
hybridized with about 10% of the mRNAs for GFP-GLUT2 C
terminus and loop compared with 100% of GLUT2 mRNA,
leading to a probable underestimation of the transgene
amounts.
Accumulation of GLUT2 mRNA in mhAT3F cells
The transcriptional effect of glucose on two glucose-dependent
liver genes, encoding GLUT2 and L-pyruvate kinase, was
assayed in clonal stable mhAT3F transfectants (Fig. 2). The
cells were cultured in the absence or presence of 17 mM
glucose for 24 hours. In mhAT3F cells expressing GFP alone,
the level of GLUT2 and L-pyruvate kinase mRNAs increased
three- to fourfold in response to glucose, a level similar to that
observed in wild-type cells. Expression of the GFP-GLUT2 C
terminus did not affect the accumulation of GLUT2 and Lpyruvate kinase mRNA in response to glucose. In contrast,
expression of the GFP-GLUT2 loop specifically abolished both
GLUT2 and L-pyruvate kinase mRNA accumulation in
response to glucose. After hybridization with L-PK cDNA, two
bands were detected: a 2.8-kb signal corresponding to liver
pyruvate kinase (L-PK) and a 2.4-kb signal corresponding to
GLUT2 intracytoplasmic loop transmits glucose signaling
845
Fig. 6. Intracellular movements of
GFP-GLUT2 loop in one living
mhAT3F cell. These results were
obtained by confocal microscopy.
Bar, 10 µm. In the same cell,
images of the GFP-GLUT2 loop
were recorded in the absence (A)
and presence (B) of 17 mM
glucose. The fluorescence was
quantified (see Materials and
Methods), in the nucleus (black
squares) and the cytoplasm (open
squares) as mean grey intensity in
an area of 5 µm diameter (right
part). (A,B) Two captured images
from the same cell at the times
indicated on the right. This
experiment was representative of
three different experiments.
muscle pyruvate kinase (M-PK). m-PK is constitutively
expressed in mhAT3F cells and served as a control (Fig. 2).
Inhibition of the glucose effect was also observed in a pool
of stable mhAT3F cells constitutively expressing GFP-GLUT2
loop (not shown), indicating that the inhibition was due solely
to the fusion protein, and not to a peculiar insertion of the GFPGLUT2 loop construct in the mhAT3F cell genome.
The intracellular loop of GLUT2 was thus identified as the
protein domain that interferes with the transcriptional signaling
triggered by glucose.
Effect of glucose, fructose and xylose on GLUT2
mRNA accumulation
To determine the nature of the triggers that stimulate the
glucose signaling pathway, several sugars were tested. In wildtype mhAT3F cells, 17 mM fructose, a hexose transported by
GLUT2 (although less efficiently than glucose; Gould et al.,
1991), mimicked the effect of glucose on GLUT2 mRNA
accumulation. With 17 mM xylose, a pentose poorly
transported by GLUT2 (Gould et al., 1991), no significant
stimulation of GLUT2 mRNA accumulation was detected in
wild-type mhAT3F cells (Fig. 3). A correlation between the
amount of transported sugar and GLUT2 mRNA accumulation
was thus observed. No stimulation in GLUT2 mRNA
accumulation was observed in mhAT3F cells expressing GPFGLUT2 loop with any type of sugar (Fig. 3).
Incorporation of radioactive glucose into glycogen
in mhAT3F cells
To assess the impact of GLUT2 domains upon a metabolic
pathway, glycogen synthesis was studied using glucose as a
substrate. When mhAT3F cells, transfected with GFP, GFP-
MEAN GREY INTENSITY IN 6 NUCLEI
with
250
without
glucose
200
150
100
50
0
0
2
4
6
8 10 12 14 min
Fig. 7. Intracellular movements of GFP-GLUT2 loop in six nuclei
from living transfected mhAT3F cells. These results were obtained
by confocal microscopy. Images were captured in the GFP-GLUT2
loop cells incubated for 7 minutes in the presence of 17 mM glucose
and then for 7 minutes in the absence of glucose. GFP-GLUT2 loop
fluorescence in the nuclei was quantified as mean grey intensity (see
Materials and Methods), the dark line represents the mean values of
six different cells (pale lines). This experiment was reproduced three
times.
GLUT2 C terminus or GFP-GLUT2 loop, were cultured for 24
hours with increasing glucose concentrations (0, 5 and 17
mM), incorporation of radioactive glucose into glycogen was
dependent upon the concentration of glucose (Fig. 4). When
compared to GFP-transfected cells, the incorporation of
radioactive glucose into glycogen did not vary in the presence
846
G. Guillemain and others
of GFP-GLUT2 loop or GFP-GLUT2 C terminus in transfected
cells, thus demonstrating that the metabolic pathway was not
altered (Fig. 4). In contrast, a slight increase in glucose
incorporation into glycogen was observed in cells transfected
with GFP-GLUT2 C terminus when compared to GFP-GLUT2
loop (Fig. 4). The reason for this difference remained
unexplained but could be due to the intrinsic property of
GLUT2 C terminus, which is responsible for the high Km for
glucose of GLUT2 protein (Katagiri et al., 1992).
Location of the overexpressed proteins in mhAT3F
cells
The subcellular location of the fusion proteins was studied
using the green fluorescence of GFP (Fig. 5). GFP and GFPGLUT2 C terminus were distributed uniformly in mhAT3F
cells. In contrast, the GFP-GLUT2 loop was concentrated in
the nuclei, as visualized by superimposition of the green (GFP)
and bright blue (nuclear) fluorescence. This specific location
was observed regardeless of the position of the tag, i.e. GFPGLUT2 loop (Fig. 5) or GLUT2 loop -GFP (result not shown).
The fluorescence of the fusion proteins was carefully
quantified. The ratio of mean grey intensity of identical areas in
the nucleus and cytoplasm showed that the GFP alone and GFPC terminus proteins had the same cellular location. Their mean
grey ratios were, respectively, 1.51±0.05, n=10 and 1.51±0.10,
n=10. In contrast, the mean grey ratio of GFP-GLUT2 loop in
AT3F cell was 2.15±0.11, n=12, showing a nuclear accumulation
of the fusion protein. This specific location was observed with
the light microscope (Fig. 5) and further confirmed by confocal
(Fig. 6) microscopic observations.
Intracellular movements of GFP- GLUT2 loop in
living cells in response to glucose
Using confocal microscopy in living cells, intracellular
movements of the GFP-GLUT2 loop were detected despite the
high level of transgene expression. The translocations of GFPGLUT2 loop in mhAT3F were followed in response to
switches of glucose concentrations in the culture medium. In
cells equilibrated at 25°C in PBS, the GFP-GLUT2 loop was
located in the nucleus and to a lesser extent in the cytoplasm
(Fig. 6). Upon a switch from PBS to PBS containing 17 mM
glucose, the fluorescence from GFP-GLUT2 loop decreased in
the cytoplasm and increased in the nucleus during the 14
minutes experimental period (Fig. 6). When the cells
equilibrated in PBS containing 17 mM glucose were switched
to PBS, the fluorescence from GFP-GLUT2 loop decreased by
twofold in the nuclei of six recorded cells, during the
experimental period (Fig. 7). A fraction (50%) of the GFPGLUT2 loop translocated within a few minutes from the
cytoplasm to the nucleus in response to high glucose, and
conversely during glucose deprivation.
DISCUSSION
The presence of the glucose transporter isoform GLUT2 is
required for appropriate responses to glucose in both pancreatic
β cells and hepatocytes. We studied the role of two GLUT2
protein domains in the transduction of the glucose signal in
hepatic cells. Specific functions have been attributed to
domains of glucose transporters. For example, subcellular
targeting of GLUTs has been attributed to the N- and Cterminal domains, whereas the substrate binding site and its
specificity are mainly due to transmembrane domains
(Saravolac and Holman, 1997). Expression of intracellular
domains of GLUT4, the insulin-sensitive glucose transporter,
demonstrated that the C terminus of GLUT4 transfected into
adipocytes could cause insulin-like stimulation of glucose
transport (Lee and Jung, 1997).
The potential signaling functions of intracytoplasmic
domains of GLUT2 were studied in an mhAT3F cell line. The
effect of glucose on the accumulation of mRNA encoding two
glucose-sensitive hepatic genes, GLUT2 and L-pyruvate
kinase, was abolished in cells expressing the large loop but not
the C terminus of GLUT2. The GLUT2 loop was thus
identified as a GLUT2 domain mediating the transcriptional
glucose signal. Overexpression of the GLUT2 loop relative to
endogenous GLUT2 was sufficient to divert the glucose signal
from its usual pathway, suggesting that the GFP-GLUT2 loop
competitively binds a protein that normally binds to
endogenous GLUT2. We therefore created a dominantnegative mutant mammalian cell, allowing the investigation of
the effects of glucose on gene transcription.
The effect of glucose on GLUT2 mRNA accumulation was
mimicked by sugars that are efficiently transported by GLUT2
in wild-type cells. Therefore, sugar transport by GLUT2
appears to be necessary to initiate the pathway and control its
amplitude. In contrast, in cells expressing the GFP-GLUT2
loop, the entry of glucose and fructose mediated by
endogenous GLUT2 was not sufficient to trigger the
transcription signal.
In bacteria, yeast and plants, complex signal transduction
networks are activated in response to glucose, and the
transcriptional effects of glucose appear to be dissociated from
the metabolic effects. In mammalian cells, the transcriptional
and metabolic processes have always been closely related in
glucose signaling. By expressing the intracytoplasmic domain
of GLUT2, we discriminated between the dual effects of
glucose.
Indeed,
increasing
extracellular
glucose
concentrations modulated glucose incorporation into glycogen
in both wild-type and transfected cells. This showed that
glucose metabolism via glucose-6-phosphate was preserved
when the loop domain was transfected. Taken together, these
results show that the transfected intracytoplasmic loop of
GLUT2 inhibits the transcriptional effect of glucose on
glucose-sensitive genes independently of glucose storage into
glycogen and suggests the existence of a parallel pathway in
mammalian cells, as in other cells.
The intracellular location of the fusion protein might help to
clarify its inhibitory role on glucose-induced transcription.
Indeed, we showed that the GFP-GLUT2 loop specifically
accumulated in the nucleus of mhAT3F cells, whereas the C
terminus remained in the cytoplasm. It is known that a globular
protein under 50 kDa (about 9 nm) can freely cross the nuclear
pore. The nuclear location of the GFP-GLUT2 loop was
probably not size-dependent, as the GFP and GFP-GLUT2 C
terminus are smaller than the GFP-GLUT2 loop. Using the
PSORT II program, the GLUT2 loop (not the C terminus) was
shown to contain 20% basic amino acids, which are a target
for nuclear import machinery. The location of the fusion
protein might be affected by extracellular glucose
concentrations. In response to a high glucose concentration and
GLUT2 intracytoplasmic loop transmits glucose signaling
within a few minutes, a fraction of the expressed GFP-GLUT2
loop translocated from the cytoplasm to the nucleus in living
mhAT3F cells. These regulated movements favor the
hypothesis that GFP-GLUT2 loop translocates via a complex
mechanism involving other proteins. In these cells, the ratio of
GFP-GLUT2 loop versus endogenous GLUT2 favored the
former in the competition for protein binding. The proteins that
would normally interact with the wild-type GLUT2 protein in
the plasma membrane would now bind cytoplasmic GFPGLUT2 loop. The higher nuclear location of the GFP-GLUT2
loop in response to glucose accompanied inactivation of
transcription of glucose-sensitive genes in transfected cells.
This suggests that, in wild-type cells, the protein that binds to
endogenous GLUT2 has to be released from its anchoring site
on the plasma membrane to activate the transcription of
glucose-sensitive genes inside the nucleus.
There are several mechanisms by which the large intracellular
loop of GLUT2 may transduce a signal from the plasma
membrane to the nucleus. A recent discovery in the Notch-1
pathway shows that growth factors stimulate intracytoplasmic
cleavage of a transmembrane receptor, and the fragment peptide
released mediates the Notch-1 signaling pathway (Schroeter et
al., 1998). Cleavage of the intracellular loop of the endogenous
glucose transporter by the action of an external signal is unlikely,
since the glucose transporter GLUT2 is an integral plasma
membrane protein with 12 transmembrane domains, and has
never been found in the cytoplasm or nucleus of hepatocytes or
pancreatic β cells. Alternatively, endogenous GLUT2 could
sequester a cytoplasmic protein, on receipt of a sugar signal; the
protein would then be released from the plasma membrane,
unmasking a site for mediation of the transcriptional effect of
glucose. The external signal that provokes glucose signaling from
glucose transporter GLUT2 might be the sugar itself. Indeed,
glucose crosses the cell membrane by means of structural
changes (inward- or outward-facing substrate binding sites) of its
transporters (Gould and Seatter, 1997). In this study, however, we
cannot rule out a role for one of the early glucose metabolites.
In conclusion, we provide evidence that GLUT2, through its
large intracytoplasmic loop, mediates a glucose signaling
pathway from the plasma membrane to the nucleus of
hepatoma cells.
We thank Edith Brot-Laroche and Anne-Françoise Burnol for
helpful discussions. We also thank Dominique Perdereau for DNA
sequencing, and Françoise Vialat (CNRS UPR 9068, Nogent) and
Christophe Klein (IFR 58, Paris) for the illustrations. GLUT2 cDNA
was a kind gift from B. Thorens (Institute of Pharmacology and
Toxicology, University of Lausanne, Switzerland) and the liver
pyruvate kinase cDNA was from A. Kahn (INSERM U129, Paris,
France). mhAT3F cells were established and generously provided by
B. Antoine (INSERM 129, Paris, France). G.G. is the recipient of a
grant from Ministère de la Recherche et de la Technologie (France);
J.G. is supported by Fondation pour la Recherche Médicale; A.L. is
supported by Association pour la Recherche sur le Cancer, Grant no.
9303.
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