A Karyopherin a2 Nuclear Transport Pathway isRegulated by Glucose in Hepatic and Pancreatic Cells

Aurelia Cassany1, Ghislaine Guillemain1,Christophe Klein2, Veronique Dalet1, EdithBrot-Laroche1 and Armelle Leturque1,

*

1 Inserm U505, UPMC and2 Service commun d’imagerie cellulaire IFR58,15 rue del’Ecole de Medecine, F-75006 Paris, France* Corresponding author: Armelle Leturque,leturque@infobiogen.fr

We studied the role of the karyopherin a2 nuclear importcarrier (also known as importin a2) in glucose signaling.In mhAT3F hepatoma cells, GFP-karyopherin a2 accumu-lated massively in the cytoplasm within minutes of glu-cose extracellular addition and returned to the nucleusafter glucose removal. In contrast, GFP-karyopherina1 distribution was unaffected regardless of glucoseconcentration. Glucose increased GFP-karyopherin a2nuclear efflux by a factor 80 and its shuttling by a factor4. These glucose-induced movements were not due toglycolytic ATP production. The mechanism involved wasleptomycin B-insensitive, but phosphatase- and energy-dependent. HepG2 and COS-7 cells displayed no glucose-induced GFP-karyopherin a2 movements. In pancreaticMIN-6 cells, the glucose-induced movements of karyo-pherin a2 and the stimulation of glucose-induced genetranscription were simultaneously lost between pas-sages 28 and 33. Thus, extracellular glucose regulates anuclear transport pathway by increasing the nuclearefflux and shuttling of karyopherin a2 in cells in whichglucose can stimulate the transcription of sugar-responsivegenes.

Key words: glucose signal transduction, importin a2,nucleocytoplasmic transports, nutrient regulation ofgene expression, protein nuclear efflux, protein shuttling

Received 6 August 2003, revised and accepted for pub-lication 23 September 2003

Gene expression in cells can be modulated by extracellular

signals. This requires the transfer of information from the

plasma membrane to the nucleus by the transport of

molecules. Small molecules can diffuse freely through

the nuclear pore, but larger proteins (>40–50 kDa) require

karyopherin receptors, localization signals and a RanGTP

gradient (1,2). There are at least 20 different karyopherin

receptors in humans (3), resulting in considerable diversity

in nucleocytoplasmic transport pathways. The import

machinery therefore constitutes a potential target for the

regulation of gene transcription (4).

Glucose repression of gene transcription is present in all

cells (5). In contrast, the stimulation of gene transcription

by glucose is restricted to cells that can detect high extra-

cellular glucose concentration. The mechanism by which

sugars stimulate gene transcription is less well understood

in mammals (6) than in yeast (7). GLUT2, a member of

the mammalian facilitative glucose transporters family

(8) may be involved in glucose detection in hepatic cells.

Indeed, we have shown that the GLUT2 protein, via an

intracytoplasmic domain, transduces the activation signal

for glucose-sensitive gene transcription (9). Moreover,

karyopherin a2 interacts transiently with this specific

intracytoplasmic domain of GLUT2 and a truncated isoform

of this import receptor, that cannot migrate into the

nucleus, blocks the stimulation of glucose-sensitive gene

transcription (10).

Nuclear import receptors are thought to be involved in

diabetic nephropathy because hyperglycemic rats have

abnormally large amounts of several isoforms of karyo-

pherin alpha proteins in the kidney (11). This feature may

be involved in the changes in gene expression associated

with diabetes in tissues that express GLUT2, such as the

liver, pancreas, kidney and intestine.

We investigated the role of karyopherin a2 in a glucose

signaling pathway, by studying the location of karyopherin

a2 in mhAT3F, a hepatoma cell line that expressed GLUT2

(12). We followed the intracellular movements of fluores-

cent-karyopherin a2 protein, in living cells in response to

glucose. To elucidate the mechanism involved in this pro-

cess, we analyzed some of the molecular and biochemical

prerequisites necessary for the response to sugars of

karyopherin a2, in several recipient cells.

Results

Changes in GFP-karyopherin a2 location in mhAT3F

cells, in response to glucose

No clear distribution of GFP-karyopherin a2 was identified

in mhAT3F hepatoma cells cultured for 24 h in medium

with or without glucose. Indeed, depending on the cell,

fluorescence was observed either mainly in the nucleus, or

mainly in the cytoplasm. We therefore incubated mhAT3F

cells for short periods of time (15min) in PBS with various

supplements and recorded GFP- karyopherin a2 movements

in real time by confocal microscopy. The fluorescence

intensity in the nucleus and the cytoplasm was quantified

(Figure 1).

Traffic 2004; 5: 10–19Copyright # Blackwell Munksgaard 2004

Blackwell Munksgaard doi: 10.1046/j.1600-0854.2003.00143.x

10

After addition of 17mM glucose, most of the GFP-

karyopherin a2 accumulated in the cytoplasm, and a con-

comitant decrease in fluorescence was observed in the

nucleus. Once the glucose was removed, overexpressed

GFP-karyopherin a2 left the cytoplasm and accumulated

massively in the nucleus (Figure 1A). Net nuclear export

Figure 1: Changes in the distribution of GFP-karyopherin a2 in response to glucose addition in mhAT3F cells. Confocal

microscopy images were captured every 20 s in living mhAT3F cells transfected with a GFP-karyopherin a2. (A) The images were acquired

at the indicated time, incubation in glucose-free medium at times started 0, 14, and 48min and in 17mM glucose at times 4 and 30min.

The white bar represents 10mm. (B) Quantification of GFP-karyopherin a2 intracellular movements in a single cell. Fluorescence intensity,

expressed as mean gray-scale level, was measured in the region of the nucleus (dark blue) and a region of the cytoplasm (dotted pink).

Blue areas indicate the periods of time for which the cell was in the presence of 17mM glucose (þ), whereas the white area indicates the

periods of time without glucose (–). These results are representative of more than 10 experiments. (C) GFP-karyopherin a2 fluxes in

response to glucose in single cell. The images were analyzed using the model described in the Materials and Methods section. Nuclear

efflux from the nucleus (Jex red line) and influx (Jin yellow dotted line) were calculated. Near equilibrium, efflux was very close to influx,

and corresponded to the shuttling of karyopherin a2. (D) Quantification of the nuclear efflux and influx rates in several cells (n¼7). The

results were expressed as mean AU�SEM of GFP-karyopherin a2 arbitrary unit molecules/s. The shuttling rates at equilibrium were

increased 4–5-fold by glucose (p< 0.0001). The initial nuclear efflux rates, immediately after glucose removal (white bars), were almost

abolished, whereas they were maximal immediately after glucose addition (blue bars) (p< 3E-10). Meanwhile, the initial nuclear influx

rates remained unaffected whatever the glucose concentration, NS (not statistically significant). Statistical analysis was performed by the

analysis of variance.

Glucose Induces Karyopherin a2 Nuclear Export

Traffic 2004; 5: 10–19 11

was faster (5min) than net nuclear import (10min). We

were able to induce several successive changes in GFP-

karyopherin a2 location of similar magnitude, and following

a similar time-course, in the same cell (Figure 1B). In all

cases, an increase in fluorescence intensity in one com-

partment was accompanied by a reciprocal decrease in the

other (Figure 1B). The movement of GFP-karyopherin a2was not detected at room temperature (not shown). This

process required a minimal temperature of 28 �C, suggest-ing the involvement of an active mechanism. Transient

labeling of the nuclear membrane was observed during

both the influx and the efflux of GFP-karyopherin a2,presumably due to the physiological bottleneck of the

pore (Figure 1A) (see the movie in the video gallery at

www.traffic.dk). Thus, extracellular addition of glucose

seems to regulate a nuclear transport pathway mediated

by the net nuclear entry and exit of karyopherin a2.

Computational analysis of glucose-induced

karyopherin a2 movements in cells

We investigated whether the glucose-induced accumula-

tion of GFP-karyopherin a2 in the cytoplasm was due to an

increase in nuclear efflux, a decrease in nuclear influx or

some other process. Nuclear and cytoplasmic fluores-

cence intensities were recorded over time. Considering

that the fluorescence levels are directly related to GFP

concentrations, we fitted the experimental curves of fluor-

escence intensity to the equations of a simple two-

compartment model. From this fit, we estimated the

kinetic constants of nuclear efflux kex or influx kin in the

presence or absence of glucose. We then calculated, from

these kinetic constants and the GFP-karyopherin a2 con-

centrations, the nuclear influx and efflux rates as the num-

ber of GFP-karyopherin a2 molecules that enter or exit the

nucleus per second.

A steady-state was approached after more than 10min in

the presence or absence of glucose, at which time the net

flux of GFP-karyopherin a2 (Jnet¼ Jin� Jex) was almost

abolished. At that time, nuclear efflux (Jex) was therefore

very close to nuclear influx (Jin). These values corres-

ponded to the shuttling of GFP-karyopherin a2, in and out

of the nucleus when no net flux can be observed (Figure

1C).

In the absence of glucose, when fluorescence levels

in the nucleus remained stable, Jex and Jin remained low

(Figure 1C). In the presence of glucose, when fluores-

cence levels remained stable in the cytoplasm, Jex and

Jin increased the shuttling of karyopherin a2 by a factor

of 3–4.

Immediately after glucose addition, initial efflux (Jex) was

increased by a factor of 7, whereas influx was only slightly

affected (Jin). Immediately after glucose removal, efflux

was blocked whereas influx increased slightly. Glucose

caused Jex to vary by a factor of almost 80.

Similar analysis was conducted on 7 cells in which the high-

est flux values were taken to be 100. The shuttling rates

were increased 4–5-fold by glucose. Nuclear influx [26� 4

arbitrary units (AU) of GFP-karyopherin a2/s] remained

unaffected whatever the glucose concentration, whereas

nuclear efflux rates were increased dramatically by glucose

(87� 8 AU/s) and almost abolished in the absence of glu-

cose (4� 2 AU/s) (Figure 1D).

Thus, glucose increases the rate of karyopherin a2 shut-

tling at equilibrium. This is achieved by means of a major

effect of glucose on efflux that differed considerably

between the two experimental conditions.

Specificity of glucose-induced karyopherin a2movement

We investigated the movement of a GFP-karyopherin a2deleted of its IBB (importin beta binding) domain. This

truncated protein was present in the cytoplasm and was

not translocated into the nucleus, regardless of glucose

concentration (Figure 2A). This suggests that the binding

Figure 2: Location of various karyopherin alpha molecules in

response to glucose. A: No change in distribution of GFP-

karyopherin a2 deleted of its IBB: importin (karyopherin) b binding

domain in transfected mhAT3F cells. B: No change in GFP-

karyopherin a1 distribution in response to glucose in transfected

mhAT3F cells. The confocal microscopy analysis was conducted

as in Figure 1, except that images were captured every 30 s.

Fluorescence intensity, expressed as mean gray- scale level, was

measured in a region of the nucleus (solid line) and a region of the

cytoplasm (dotted line). The images on the right were acquired at

5 and 12min, respectively. Results are representative of 3

independent experiments. Bar equals 10 mm.

Cassany et al.

12 Traffic 2004; 5: 10–19

to karyopherin b (importin b) is required during transloca-

tion, as in a classical import pathway.

We investigated the distribution of GFP-karyopherin a1following the addition of glucose, in mhAT3F (Figure 2B),

COS-7 and HepG2 cells (not shown). The a1 isoform

had a similar cellular distribution to the a2 isoform after

24 h in medium with or without glucose. No significant

change in the cellular distribution of GFP-karyopherin a1was observed in any of these cells at any glucose concen-

tration (Figure 2B). The glucose effect therefore targets a

transport pathway that uses karyopherin a2 as an adaptor

and karyopherin b as a second partner.

Substrates triggering changes in the distribution

of GFP-karyopherin a2We analyzed some of the biochemical requirements for

changes of karyopherin a2 distribution. A concentration

of 5mM glucose, corresponding to physiological blood

glucose levels, was able to produce an accumulation in

the cytoplasm of GFP-karyopherin a2 (Table 1). Fructose,

another specific substrate of GLUT2, induced the accumu-

lation of fluorescence in the cytoplasm, whereas xylose, a

pentose not transported by GLUT2, induced no net nuclear

export of GFP-karyopherin a2 (Table 1). Furthermore, GFP-

karyopherin a2 did not accumulate in the cytoplasm after

the addition of a glucose analog, 3-O-methylglucose

(3-OMG) (Table 1). As this glucose analog does not provide

energy to the cells, we investigated the role of energy

provision in glucose-induced protein movement. We sup-

plied the cells with lactate/pyruvate, substrates that feed

into the final steps of glycolysis. Lactate/pyruvate did not

promote cytoplasmic accumulation of GFP-karyopherin a2,suggesting that energy provision through glycolysis did not

drive glucose-induced karyopherin a2 movement (Table 1).

The GFP-karyopherin a2 movements, induced by glucose

were not affected by an inhibitor of glucose metabolism

(N-acetylglucosamine, a hexokinase inhibitor) (Table 2).

Thus, the glucose-induced movements of karyopherin a2seem to involve neither a metabolite nor energy provided

by glucose metabolism.

It is known that energy from GTP hydrolysis by Ran is

required for the recycling in the cytoplasm of most import

carriers. Profound ATP depletion has been shown to

induce RanGTP depletion. The addition of sodium azide

(10mM) was used to deprive the cells of ATP by inhibiting

oxidative phosphorylation. This inhibitor of mitochondrial

phosphorylation induced the sequestration of GFP-

karyopherin a2 in the nucleus, despite the presence of

glucose (Table 2). This suggests that glucose-induced

movements of karyopherin a2 involve an energy-dependent

step.

Mechanisms of changes in the distribution

of GFP-karyopherin a2We investigated the effects of an inhibitor of GLUT activity

(phloretin), together with glucose. Glucose transport inhib-

ition blocked the net efflux of GFP-karyopherin a2 from the

Table 1: Effect of various substrates on GFP-karyopherin a2redistribution in mhAT3F cells. After 15min of incubation, GFP-

karyopherin a2 accumulated in the cell compartment. The results

are expressed as the ratio of fluorescence intensities in nucleus vs.

cytoplasm, which is arbitrarily taken as 1 in the absence of glucose,

before the addition of the effector. A ratio close to 1 indicates that

the effector is inefficient in altering karyopherin a2 location,

whereas a ratio lower than 1 indicates cytoplasmic accumulation

of GFP-karyopherin a2. Several effectors were tested: no glucose

corresponds to 17mM mannitol to keep osmolarity constant,

3-OMG stands for 3-O-methylglucose. Each experimentwas carried

out at least four times. Results are expressed as mean�SEM

Effectors GFP-karyopherin a2nucleus/cytoplasm ratio

No glucose 1.08� 0.07 (n¼ 49)

Glucose (17mM) 0.43� 0.03 (n¼ 21) ***

Glucose (5mM) 0.44� 0.04 (n¼ 9) ***

Fructose (17mM) 0.53� 0.08 (n¼ 5) *

Xylose (17mM) 1.09� 0.20 (n¼ 5) NS

3-OMG (17mM) 1.10� 0.20 (n¼ 4) NS

Lactate/pyruvate (10/1mM) 1.05� 0.08 (n¼ 4) NS

Statistical significance was tested using Student’s t-test: NS not

statistically different; * p<0.05; *** p<0.001 when compared to

the ratios obtained in the absence of glucose.

Table 2: Effect of various inhibitors on glucose-induced GFP-

karyopherin a2 redistribution in mhAT3F cells. Inhibitors were

tested in the presence or absence of glucose. Fluorescence inten-

sities nucleocytoplasmic ratio N/C was arbitrarily taken as 1

(1.08�0.07, n¼49) in the absence of glucose. A ratio close to

1=indicates that the effector is altering karyopherin a2 movement

in response to glucose, whereas a ratio lower than 1 indicates that

the effector is inefficient, since GFP-karyopherin a2 cytoplasmic

accumulation is observed like in the presence of glucose alone.

We used N-acetylglucosamine as a hexokinase inhibitor, sodium

azide as an oxidative phosphorylation inhibitor, phloretin as a

glucose transport inhibitor, leptomycin B as an inhibitor of Crm1-

mediated export, wortmannin as a PI3kinase inhibitor, staurospor-

ine as a protein kinase A, G, and C inhibitor. Each experiment was

carried out independently at least 3 times. The results are

expressed as mean�SEM

Inhibitors�glucose Pre-incubation GFP-karyopherin a2nucleus/cytoplasm ratio

N-acetylglucosamine

(50mM)

30min 0.50�0.05 (n¼11) **

Sodium azide (10mM) 20min 0.97�0.12 (n¼5) NS

Phloretin (50mM) no 1.80�0.10 (n¼3) *

Leptomycin B (100nM) 2 h 0.50�0.06 (n¼4) *

Wortmannin (100nM) 20min 0.33�0.02 (n¼4) **

Staurosporine (20 nM) 30min 0.48�0.15 (n¼3) *

The statistical significance was tested using Student’s t-test: NS

not statistically different; * p<0.05; ** p<0.01; *** p< 0.001

when compared to the ratios obtained in the absence of glucose.

Glucose Induces Karyopherin a2 Nuclear Export

Traffic 2004; 5: 10–19 13

nucleus (Table 2). Glucose transport through the plasma

membrane is therefore necessary.

The net nuclear efflux of GFP-karyopherin a2, observed in

response to glucose, was not blocked by an inhibitor of

protein export, leptomycin B (Table 2). Thus, the glucose-

regulated export does not involve the exportin CRM1.

Kinases and phosphatases are involved in numerous

cellular signaling pathways, including protein trafficking.

Neither the addition of wortmannin (a PI3 kinase inhibitor),

nor that of staurosporine (a Protein Kinase A and C inhibi-

tor), modified the glucose-induced movements of GFP-

karyopherin a2 (Table 2). Finally, we showed that okadaic

acid (a phosphatase inhibitor) prevented nuclear import of

GFP-karyopherin a2 in the absence of glucose, suggesting

a phosphatase-dependent mechanism (Figure 3).

Glucose-induced movements of GFP-karyopherin a2in several cell lines

The GLUT2 hexose transporter was shown to be required.

The location of GFP-karyopherin a2 was not affected by

glucose in COS-7 cells (Figure 4) or in hepatoma HepG2

cells (Figure 4); both cell lines are devoid of GLUT2, and

display no stimulation of glucose-sensitive gene expres-

sion (not shown). A GLUT2 expressing cell line, MIN-6

pancreatic cells, at passage 28, displayed glucose-induced

GFP-karyopherin a2 movements similar to mhAT3F cells

(Figure 5A). This suggests that GLUT2 is required to medi-

ate karyopherin a2 movement, in response to glucose in

MIN-6 pancreatic cells. However, when we used MIN-6

pancreatic cells at a later passage (33 vs. 28), GLUT2

mRNA (measured to assess the presence of the GLUT2)

was constitutively and abundantly expressed (Figure 5B).

Nevertheless, the cells were no longer sensitive to the

effect of glucose on karyopherin a2 movement (Figure

5A) or on the stimulation of gene transcription (Figure

5C). Thus, the presence of GLUT2 is necessary, but not

sufficient for glucose-induced protein translocation. Intra-

cellular proteins from the host cells may therefore be

crucial.

Discussion

Karyopherins a have been described as adaptor-receptors

of the machinery that imports cytoplasmic proteins bearing

an NLS (nuclear localization signal) into the nucleus. We

showed that GFP-karyopherin a2 and a1 were located in

the nucleus, but also to some extent in the cytoplasm in

mhAT3F, MIN-6, HepG2 and COS-7 cells. These receptors

are known to shuttle in and out of the nucleus, but the

regulation of this translocation has not been described.

Most of the overexpressed GFP-karyopherin a2 seemed

to be subject to such regulation. We found that extracel-

lular addition of nutrient sugars regulated the efflux of GFP-

karyopherin a2 within seconds and increase the shuttling

of the import carrier within minutes. This suggests that

glucose regulates a karyopherin a2-specific pathway of the

nucleocytoplasmic transport machinery, together with

translocation of karyopherin a2 protein cargoes.

This process displayed several characteristics of a classical

nuclear import pathway. Since the process was not

observed with a karyopherin a2 deleted of its IBB, the

binding to karyopherin b was required as in most import

pathways (13). The glucose-regulated export of karyo-

pherin a2 was not inhibited by leptomycin B, suggesting

that this processwasCRM1-independent (14). Ran-dependent

Figure 3: Effect of okadaic acid as a PP1, PP2A phosphatase inhibitor on GFP-karyopherin a2 import in mhAT3F. The first image

shows GFP-karyopherin a2 nuclear accumulation in mhATF cells incubated for 20min in the presence of okadaic acid but without glucose.

In the second image, acquired 3min after glucose addition, the presence of okadaic acid did not prevent GFP-karyopherin a2 cytoplasmic

accumulation, suggesting that the inhibitor was inefficient in controlling glucose-induced nuclear efflux of GFP-karyopherin a2. The cells

were then placed in a medium containing okadaic acid but without any glucose, and in the third image acquired after 15min of incubation,

the GFP-karyopherin a2 remained in the cytoplasm suggesting that the inhibitor was blocking GFP-karyopherin a2 nuclear import. The last

image, acquired 15min after inhibitor removal, is presented to show that these cells were still able to accumulate GFP-karyopherin a2 in

the nucleus. The inhibitory effect of okadaic acid was thus reversible.

Cassany et al.

14 Traffic 2004; 5: 10–19

nuclear transport requires energy provided by the

hydrolysis of GTP, for the recycling of import carriers

(15–17). Profound ATP depletion has been reported to

cause the depletion of free GTP, and thus of RanGTP,

and can lead to the inhibition in vivo of Ran-dependent

nuclear transport (18). The use of sodium azide inhibits

ATP production and blocks export of several carriers

(18,19). Here, the sequestration of the carrier into the

nucleus following treatment with sodium azide shows

that glucose-induced export of karyopherin a2 depends

on energy, probably from free GTP, and might be

mediated by a Ran-dependent step.

Okadaic acid blocked the nuclear accumulation of karyo-

pherin a2 induced by glucose removal. Consistent with

this, okadaic acid has been reported to inhibit nucleocyto-

plasmic import in vitro, suggesting that a phosphatase-

dependent step is involved in nuclear import (20). Recently,

a serine/threonine phosphatase PP2A, inhibited by okadaic

acid, was implicated in nucleocytoplasmic transport on the

basis of its binding to karyopherin b (21). Interestingly,

okadaic acid efficiently controlled the nuclear efflux of

karyopherin a2 in response to the glucose addition. Glucose,

via one of its metabolite, xylulose 5- phosphate, has been

shown to activate a protein phosphatase of the PP2A family

(22). This xylulose-activated phosphatase was recovered

from both the cytoplasm and nucleus of liver cells (22). It

is currently unclear whether this xylulose-activated phos-

phatase is the target of okadaic acid involved in the glu-

cose-sensitive redistribution of karyopherin a2.

Glucose specifically regulates not only the efflux of karyo-

pherin a2, but also its nucleocytoplasmic shuttling. Karyo-

pherin a2 fluxes in nucleocytoplasmic transport are currently

thought to be conditioned by numerous factors. Those

include the number of protein complexes that can pass

through nuclear pores and the permeability of the nuclear

envelope to these complexes (pore property and number)

(23). The differences in kinetic constants obtained in this

study probably reflect changes in the formation of these

import and export complexes. The nuclear availability of

Ran GTP has also been reported to limit the formation of

complexes that can cross the nuclear envelope (24). As the

composition of import and export complexes is not entirely

clear (25), we did not introduce these factors into our model.

A simple two-compartment model was used to calculate

GFP protein fluxes. The increased nuclear efflux in response

to glucose leading to the massive cytoplasmic accumulation

of karyopherin a2 is a specific feature of this process.

The existence of numerous isoforms of import receptors

suggests that highly selective nucleocytoplasmic transport

process may occur (26). Indeed, we report here that glu-

cose induced redistribution of the karyopherin a2 isoform

but not of another isoform, karyopherin a1. We have

shown that the GLUT2 glucose transporter binds specific-

ally and transiently to karyopherin a2, but not to karyo-

pherin a1 (10). Thus, the GLUT2 binding capacity appears

to be related to the induction of karyopherin a2 move-

ments in response to glucose.

The metabolism of glucose generates energy, but the glu-

cose-induced movement of karyopherin a2 seemed to be

independent of glycolysis. Indeed, the redistribution of pro-

tein was induced by glucose or fructose addition, but was

not blocked by an inhibitor of hexose metabolism or stimu-

lated by insulin, a hormone that accelerates metabolism

(not shown). The protein movement seems also to be inde-

pendent of the ATP produced during glucose metabolism,

since lactate/pyruvate that provides ATP in the Krebs cycle,

did not induce the translocation. Hepatoma cells possess

intracellular stores of energy providing substrates that are

mobilized in the absence of glucose, which could fulfill the

energy requirement of the import machinery. We therefore

suggest that glucose or fructose triggers a non-metabolic

signal leading to the nuclear efflux of karyopherin a2. Pro-tein signals generated by glucose in yeast are initiated by

plasma membrane sensors (5,7,27).

GLUT2 is required to promote the process of GFP-

karyopherin a2 accumulation in the cytoplasm after glucose

addition. This conclusion is supported by arguments based

on the substrate specificity of this isoform of glucose

Figure 4: Location of GFP-karyopherin a2 in response to

glucose in transfected COS-7 cells (A) and HepG2 cells (B).

The confocal microscopy analysis was conducted as in Figure 2.

Fluorescence intensity, expressed as mean gray-scale level,

was measured in a region of the nucleus (blue solid line) and a

region of the cytoplasm (pink dotted line). The images on the right

were acquired at 10min. These results are representative of 4

experiments. Bar equals 10mm.

Glucose Induces Karyopherin a2 Nuclear Export

Traffic 2004; 5: 10–19 15

transporters (28), and on the use of cell lines that do or do

not express endogenous GLUT2 (8,12). GLUT2 is also

required for the stimulation of glucose-sensitive gene

expression in hepatoma cell lines (12). This highlights the

possibility of a functional relationship between glucose-

induced karyopherin a2 movements and GLUT2 expression

in cells.

However, although GLUT2 expression is required, it is

not sufficient for karyopherin a2 movements in response

to extracellular glucose. Indeed, a glucose analog trans-

ported by GLUT2 did not induce karyopherin a2 redistribu-

tion, suggesting that stereospecificity may be involved

in this process. Moreover, a late passage of MIN-6 cells,

that constitutively express GLUT2 mRNA (29) but dis-

played no glucose-sensitive gene transcription, showed

no glucose-induced karyopherin a2 redistribution. Two

sublines of insulin-secreting MIN-6 cells from two pas-

sages have been studied using high-density microarrays;

one that has retained glucose-induced secretion, and

one that has not. A long list of genes involved in metabo-

lism, intracellular signaling, and cell architecture were sig-

nificantly affected by cell passage, but none of the

candidate genes involved in glucose sensing in the pan-

creas was modified (30). Therefore, intracellular effectors,

present in mhAT3F and MIN-6 passage 28, and absent in

HepG2, in COS-7 cells and MIN-6 passage 33, may be

required to mediate glucose-induced cytoplasmic accumu-

lation of karyopherin a2.

The cargoes that bind specifically to karyopherin a2, may

constitute a set of factors involved in a glucose signaling

pathway downstream from GLUT2. Glucose has been

reported to induce nuclear accumulation of several

gene-regulating factors. Factors stimulating the tran-

scription of glucose-sensitive genes, such as an AMP-

kinase subunit in yeast (31), and ChREBP in mammalian

liver cells (32), and a factor inhibiting the transcription

of genes repressed by glucose, Mig1, a yeast repressor

(33), have been recovered in the nucleus after glucose

addition. As glucose increases the rate of karyopherin

a2 shuttling, we suggest that karyopherin a2 increases

the import of glucose- sensitive transcription activators.

However, if the main action of glucose is to stimulate the

massive translocation of karyopherin a2 into the

cytoplasm, then karyopherin a2 may mediate an inhibitory

process that is relieved by glucose. To address this

point, we are now investigating the partners of karyo-

pherin a2.

In conclusion, extracellular addition of glucose increases

the nuclear efflux and the shuttling rate of GFP-karyopherin

a2 in cells displaying stimulation of glucose-sensitive gene

transcription. Glucose may induce the transcription of

sugar-responsive genes, via karyopherin a2 nuclear efflux

or shuttling.

Materials and Methods

Cell culture

The mhAT3F hepatoma cell line was derived from trans-

genic mice expressing the SV40 large T and small t

Figure 5: Redistribution of GFP-

karyopherin a2 in transfected

MIN-6 pancreatic cells. (A) The

experiment was conducted as in

Figure 2. In MIN-6 cells at passage

28 (early) or 33 (late), fluorescence

intensity, expressed as mean gray-

scale level, was measured in a

region of the nucleus (blue solid

line) and a region of the cytoplasm

(pink dotted line). (B) The ratio

GLUT2/L19 mRNA abundance was

estimated by RT-PCR in MIN-6 at

passage 28 (early) and 33 (late) and

compared to that in mhAT3F. (C)

Human GLUT2 promoter activity

was assessed by measuring lucif-

erase activity, as a reporter gene, in

cells cultured in media with high

and low glucose concentrations.

We determined the statistical sig-

nificance (p<0.05) of differences

from values obtained at high and

low glucose concentrations. These

data are representative of 3–4 inde-

pendent experiments.

Cassany et al.

16 Traffic 2004; 5: 10–19

antigens under the control of the antithrombin III promoter.

The mhAT3F cells (34) were generously provided by B.

Antoine (INSERM U129, Paris, France). Cells were cul-

tured in Dulbecco’s modified Eagle’s medium/Ham-F12,

Glutamax I, with 17mM glucose (Life Technologies,

InVitrogen, Cergy Pontoise, France), supplemented with

penicillin, streptomycin, 0.1mM insulin, 1mM dexamethasone,

1mM triiodothyronine, 0.03 mM selenium and 5% fetal

calf serum. The COS-7 and HepG2 cell lines were

cultured in Dulbecco’s modified Eagle’s medium/-

Ham-F12, Glutamax I (Life Technologies) supplemented

with penicillin, streptomycin and 10% fetal calf serum.

It should be noted that fetal calf serum adds about

1mM glucose to the medium. MIN-6 cells were

generously provided by Philippe Ravassard (CNRS

UMR 9923, Paris, France). MIN-6 at passages 28–33

were cultured in Dulbecco’s modified Eagle’s medium,

with 25mM glucose, Glutamax I (Life Technologies) sup-

plemented with penicillin, streptomycin, 0.25 ml/100ml b-mercaptoethanol and 15% decomplemented fetal calf

serum.

Transfection

We plated 2� 105 cells in single-chambered Labtek

coverslips (Nunc, Merck Strasbourg, France). The follow-

ing day, cells were transiently transfected with 7 mg of

the rat full-length karyopherin a2 cDNA in frame in

the pEGFPn1 and pEGFPc3 vectors (Clontech, Ozyme,

Saint-Quentin en Yvelines, France) (10). The position

of the tag, at the N- or C-terminus of karyopherin a2 had

no effect on protein redistribution in response to

glucose. We therefore chose arbitrarily to use the GFP-N-

karyopherin a2 in this study. The pEGFPc2-karyopherin

a1 was constructed from pGEX3X-karyopherin a1vector which was supplied by M. Prieve (35). The

mhAT3F, HepG2 and COS-7 cells were transfected

with 5 mg of pEGFPc3-karyopherin a1. We also deleted

the importin b binding domain of GFP-karyopherin a2 (10)

to prevent nuclear translocation of the truncated protein

(36). Transfections were performed with Lipofectin (Life

Technologies).

Confocal microscopy on living cells

One day after transfection, cells were transferred in glu-

cose-containing or glucose-free medium for 24 h and then

placed at 28 �C in circulating PBS (137mM NaCl, 2.7mM

KCl, 4.3mM Na2HPO4 7H2O, 1.4mM KH2PO4). The PBS

was continually renewed by gravitational flow, ensuring

the total replacement of the medium in the culture cham-

ber within 1min. Mannitol was added to the PBS, to a

concentration of up to 17mM to keep osmolarity constant.

Substrates were added at the indicated concentration

in PBS. We used a Zeiss LSM510 confocal microscope

equipped with a C-Apochromat� 63 objective lens. Green

cells were selected for their unsaturated fluorescence

intensities. Images were acquired every 20 or 30 s. For

each image in the time series, the green fluorescence

intensity was measured in two areas of identical size,

one within the nucleus and the other within the cytoplasm.

Fluorescence intensities are expressed as mean gray-

scale level/pixel.

Computational analysis of the images

At steady-state, the GFP-karyopherin a2 influx into the

nucleus (Jin, influx, molecules/s) and its efflux out of the

nucleus (Jex, efflux) are equal, and correspond to the shut-

tling rate of karyopherin a2. The resulting net flux is equal

to zero (Jnet¼ Jin� Jex¼ 0).

The following system of kinetic equations was used to

model nuclear influx and efflux:

Jin ¼ kin � GFP Ka2CJex ¼ kex � GFP Ka2NJnet ¼ Jin � Jex ¼ kin � GFP Ka2� kex � GFP Ka2

When the glucose concentration of the medium is chan-

ged, influx, efflux or both may be modified and the net flux

will vary (J net 6¼ 0) until a new-steady state (Jnet¼ 0) is

reached. A change in fluorescence is caused by changes

in the amounts of GFP-karyopherin a2 in the nucleus and

cytoplasm over time. This can be described by the follow-

ing differential equations:

J ¼ � dGFP Ka2C ðtÞdt

¼ kin � GFP Ka2C ðtÞ

� kex � GFP Ka2NðtÞ

�J ¼ � dGFP Ka2NðtÞdt

¼ kex � GFP K�2NðtÞ

� kin � GFP Ka2C ðtÞ

Finally, the total amount of GFP-karyopherin a2 is con-

served throughout the experiment:

GFP Ka2Nðt0Þ þ GFP Ka2C ðt0Þ ¼ GFP Ka2NðtÞþ GFP Ka2CðtÞ

We measured fluorescence intensities (mean gray-

scale level/pixel), which are related to concentration,

whereas fluxes are expressed in molecules/s. We mod-

eled variations in concentration to obtain kinetic cons-

tants (kin and kex) and to calculate fluxes (Jin and Jex).

The previous equations were rewritten in term of concen-

trations, with VN the nuclear and VC the cytoplasmic

volumes:

Glucose Induces Karyopherin a2 Nuclear Export

Traffic 2004; 5: 10–19 17

� d ½GFP Ka2C �ðtÞdt

¼ kin � ½GFP Ka2C �ðtÞ � kex �VN

VC

½GFP Ka2N �ðtÞ

� d ½GFP Ka2N �ðtÞdt

¼ kex � ½GFP Ka2N �ðtÞ � kin �VC

VN

½GFP Ka2C �ðtÞ1

VC

½GFP Ka2N �ðt0Þ þ1

VN

½GFP Ka2C �ðt0Þ ¼1

VC

½GFP Ka2N �ðtÞ þ1

VN

½GFP Ka2C �ðtÞ

From this set of equations, analytical solutions can be

found for [GFP Ka2C](t) and [GFP Ka2N](t)

½GFP K�2C �ðtÞ ¼ ½GFP Ka2C �ðt0Þexp�ðkinþkex Þt

þ kex

kin þ kex

½GFP Ka2N �ðt0ÞR

þ�

½GFP Ka2C �ðt0Þ�

1� exp�ðkinþkex Þt� �

½GFP Ka2N �ðtÞ ¼ ½GFP Ka2N �ðt0Þexp�ðkinþkex Þt

þ kin

kin þ kex

�½GFP Ka2N �ðt0Þ

þR � ½GFP Ka2C �ðt0Þ�

1� exp�ðkinþkex Þt� �

with:

R ¼ VC

VN

The experimental plots of fluorescence levels were used

to fit simultaneously these two functions to give us kin, kexand R. Data were fitted with the solver function of Excel

software (Microsoft) by minimizing the sum of squared

differences between experimental and calculated points,

with kin, kex, [GFP Ka2N](t0), [GFP Ka2C](t0), and R as

variables. If we arbitrarily set nuclear volume to 1, fluxes

can be calculated as:

JinðtÞ ¼ kin � R � ½GFP Ka2C �ðtÞJexðtÞ ¼ kex � ½GFP Ka2N �ðtÞ

Moreover, the extrapolation of the fits allowed us to

estimate Jin and Jex at equilibrium and to provide a shut-

tling value. Assuming that cytoplasmic and nuclear volumes

(rather than just their ratio) are constant throughout the

experiment, we can compare fluxes for different conditions

in one cell. However, to compare the variation in fluxes from

cell to cell, the highest value of fluxes was taken to be 100

for each cell. In fact, the absolute values of fluxes varied

from 0.667 to 2.524 (n¼ 7) due to the transient transfection

protocol used to express the GFP-protein.

Quantitative RT-PCR

We cultured mhAT3F andMIN-6 cells in a medium contain-

ing a high glucose concentration. Levels of mRNA for

GLUT2 and L19 were determined by real-time PCR (Light

Cycler system, Roche, Meylan, France). The ribosomal

L19 mRNA was used as a control for RNA extraction.

Thus, the results are expressed in an arbitrary unit as the

ratio GLUT2/L19 mRNA abundance (10).

Promoter activity

The �1100/þ300 region of the human GLUT2 promoter

was inserted into the episomal replicative p205-luc plas-

mid driving the luciferase reporter gene. Luciferase activ-

ities were measured in homogenates of cells cultured in

the presence or absence of glucose, using the Luciferase

Assay System (Promega, Madison, USA) in a Lumat

LB9501 luminometer (Berthold). The human GLUT2 gene

was kindly provided by G.I. Bell (Howard Hughes Institute,

Chicago, USA) (37). The results, in an arbitrary unit of

luciferase activity/mg protein of cells cultured at high glu-

cose concentration, are expressed as a fraction of those

cultured at low glucose concentration, which was taken as

1. Protein content was determined by a BCA assay

(Ultima, Interchim, Montlucon, France).

Statistical analysis

Student’s t-test and two-way analysis of variance were

performed using Prism Software.

Acknowledgments

We thank Jean Chambaz, Alain Ktorza and members of U505 for helpful

discussions and Adrien Klapisz for the film.

A.C. received a grant from La Ligue Contre Le Cancer (France); G.G.

received a grant from the Ministere de la Recherche et de la Technologie

(France); A.L. is supported by grants n�9303 and n�5531 from ARC (Asso-

ciation pour la Recherche contre le Cancer, France).

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