1 School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2
9JT, UK
2 Centro de Estudios Científicos CECS, Casilla 1469, Valdivia,
Chile
3 Biochemistry Department, Dundee University, Dundee DD1 5EH, Scotland, UK
4 Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College School
of Medicine, Hammersmith Hospital, London W12 0NN, UK
5 U465 INSERM, Centre de Recherches Biomédical des Cordeliers, 75270
Paris Cedex 06, France
* Author for correspondence (e-mail: K.Barnes{at}leeds.ac.uk )
Accepted 18 March 2002
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Summary |
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Key words: Glucose, Transport, GLUT1, Stress, AMP-activated protein kinase
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Introduction |
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The signal transduction pathways linking metabolic and osmotic stress to
glucose transport are also unclear. However, a key feature of metabolic
stresses such as hypoxia and exposure to inhibitors of oxidative
phosphorylation is ATP depletion, which leads to the elevation of cellular
AMP:ATP ratios and thus to stimulation of AMP-activated protein kinase (AMPK)
activity (Hardie et al.,
1998). A major function of AMPK appears to be as a protective
device, switching off ATP-utilising biosynthetic pathways and switching on
ATP-generating metabolic pathways so as to preserve ATP levels
(Corton et al., 1994
). Its
activation not only by stress but also by physiological processes that are
associated with stimulation of glucose uptake, such as muscle contraction
(Winder and Hardie, 1996
;
Hutber et al., 1997
),
suggested that AMPK might also be involved in the regulation of transport
(Baldwin et al., 1997
).
Evidence supporting such an involvement has come from the use of the adenosine
analogue 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which,
following uptake into cells and conversion to the monophosphorylated
derivative 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP), mimics AMP in
activating AMPK (Corton et al.,
1995
). Numerous studies have recently reported that AMPK
activation by AICAR leads to translocation of the glucose transporter isoform
GLUT4 to the plasma membrane and consequently to stimulation of glucose
transport in skeletal muscle and cardiomyocytes
(Merrill et al., 1997
;
Hayashi et al., 1998
;
Kurth-Kraczek et al., 1999
;
Russell et al., 1999
;
Hayashi et al., 2000
).
Similarly, we have shown that AICAR treatment of BHK cells leads to
translocation of GLUT1 and concomitant stimulation of transport
(Baldwin et al., 1997
). In
addition, Abbud and co-workers have more recently reported that AICAR
stimulates glucose uptake in the GLUT1-containing cell line Clone 9, where
transport stimulation does not appear to involve translocation of transporters
(Abbud et al., 2000
).
Although the studies described above suggest a role for AMPK in the signal
transduction pathways involved in regulation of glucose transport by stress,
AICAR has many cellular effects in addition to activation of this kinase
(Kemp et al., 1999). A key
objective of the present study was therefore to obtain additional evidence for
the role of AMPK in the response of Clone 9 cells to stress. A second
objective was to discover the identity of other components of the signalling
pathways involved. These have so far been little investigated, although we
have previously shown that stimulation of transport by metabolic and osmotic
stresses involves neither the p38 MAP kinase pathway nor the enzyme
phosphatidylinositol 3-kinase (PI 3-kinase), which plays an essential role in
the stimulation of transport by insulin
(Barros et al., 1995
;
Barros et al., 1997
). The
similar sensitivity of both insulin- and stress-induced transport stimulation
to inhibition by ML-9, an inhibitor of several kinases, including myosin light
chain kinase and protein kinase B (Smith
et al., 2000
), suggests that the pathways might share some
elements (Barros et al., 1995
).
Recent studies have implicated not only protein kinase B but also atypical
diacylglycerol-insensitive protein kinase C (PKC) isoforms
and
as components downstream of PI 3-kinase in the insulin signalling pathway
(Bandyopadhyay et al., 1997a
;
Standaert et al., 1997
;
Kotani et al., 1998
;
Hill et al., 1999
;
Bandyopadhyay et al., 2000
).
Similarly, conventional PKC isoforms have been implicated in the stimulation
of glucose uptake by metabolic stress in L6 myotubes
(Khayat et al., 1998
), whereas
the results of studies on H-2Kb myotubes suggest that stimulation
of glucose uptake in skeletal muscle by exercise involves activation of nitric
oxide synthase (NOS) by AMPK (Fryer et
al., 2000
). Although stimulation of transport by exposure of Clone
9 cells to alkaline pH has been reported not to involve phorbol-sensitive PKC
isoforms (Hakimian and Ismail-Beigi,
1991
), to date a possible role for PKC isoforms in the response to
metabolic and osmotic stresses has not been investigated in these cells.
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Materials and Methods |
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Adenoviral infection of Clone 9 cells
Clone 9 cells were grown to confluence in 10 cm dishes. The medium was then
aspirated and the cells washed once in phosphate-buffered saline (PBS). In
order to express a constitutively active form of AMPK, cells were infected
with a recombinant adenovirus harbouring cDNA encoding residues 1-312 of
AMPK1, containing a mutation of threonine 172 to aspartic acid and
bearing a myc-tag (Stein et al.,
2000
; Woods et al.,
2000
). An adenovirus in which the expression cassette contains the
major late promoter but no exogenous gene was used to infect cells as a
control. Adenovirus stocks were diluted in serum-free medium to give a
multiplicity of infection of 10, and they were incubated with the cells for a
period of 2.5 hours at 37°C. An equal volume of 2% fetal calf serum in
medium was then added to the cells, which were incubated for a further period
of 48 hours at 37°C. The cells were washed twice with PBS prior to
exposure to stress in preparation for sugar transport assays or
immunoblotting.
Cell culture and sugar transport assays
Clone 9 cells were cultured in 6-well plates or in 57 cm2 dishes
as described previously (Barros et al.,
1995). In some instances cells were infected with recombinant
adenovirus constructs as described above. Initial rates of radiolabelled sugar
uptake were estimated in confluent cell cultures using a modification of a
previously described protocol (Barros et
al., 1995
). For 3-0-methyl-D-glucose transport
measurements, uptake was initiated by addition to each well of 0.5 ml 0.1 mM
3-O-[methyl-3H]-D-glucose in Krebs Ringer Hepes buffer
(KRH; 136 mM NaCl, 20 mM Hepes, 4.7 mM KCl, 1.25 mM MgSO4, 1.25 mM
CaCl2, pH 7.4) at 22°C, and the uptake was routinely measured
for a period of 5 minutes. Preliminary experiments showed that uptake was
linear for this period and did not exceed 20% of the equilibrium uptake value
(data not shown). However, for determination of the kinetic parameters of
transport, uptake was measured over a period of 30 seconds at 6°C to be
certain that initial rates of uptake were being measured at all
concentrations. Carrier-mediated uptake rates were calculated by subtracting
non-specific uptake measured in the presence of 20 µM cytochalasin B from
the initial rates. After stopping transport using three washes in 50 µM
phloretin in ice-cold PBS, cells were lysed with 1% Triton X-100, and the
radioactivity was measured by liquid scintillation counting. 2-Deoxy-D-glucose
uptake measurements were performed in a similar fashion, except that 0.2 mM
[3H]2-deoxy-D-glucose was used and uptake was measured for 5
minutes at 37°C. Pre-incubations of cells with appropriate concentrations
of stress-inducing agents (e.g. sodium azide) or inhibitors in KRHG (KRH plus
25 mM glucose) were carried out for 30-60 minutes at 37°C as indicated in
the figure legends. Data are presented as the means±s.e.m. (n) (n is
the number of experiments carried out). Significance was assessed using a
Student's t test for two groups and was taken at
P<0.05.
The AMPK assay
AMPK activity was measured as described previously
(Hardie et al., 2000).
Briefly, cell lysates were prepared from confluent uninfected or
adenovirus-infected Clone 9 cells, grown in 10 cm culture dishes by treatment
with 0.5 ml lysis buffer (1% Triton X-100 in 50 mM Tris/HCl, pH 7.4 at
4°C, containing 0.25 M mannitol, 1 mM EGTA, 1 mM DL-dithiothreitol (DTT),
0.1 mM 4-(2-aminoethyl)benzenesulphonylfluoride (AEBSF), 1 mM benzamidine, 5
µg/ml soya bean trypsin inhibitor, 5 mM Na pyrophosphate and 50 mM NaF).
Cells were scraped and transferred to a microfuge tube, and after
centrifugation (13000 g, 4°C for 3 minutes) they were
immunoprecipitated using protein G-sepharose coupled to affinitypurified sheep
antibodies against the
1 or
2 isoform catalytic subunits of AMPK
or against a mixture of these antibodies
(Woods et al., 1996
). The
immunocomplexes were centrifuged (18000 g, 1 minute for 4°C), and
the pellets washed with 5x1 ml ice-cold immunoprecipitation buffer (50
mM Tris/HCl, pH 7.4 at 4°C, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.1 mM
AEBSF, 1 mM benzamidine, 5 µg/ml soya bean trypsin inhibitor, 5 mM Na
pyrophosphate and 50 mM NaF) containing 1 M NaCl. After further washing with
lysate buffer, the pellets were resuspended in 30 µl HEPES-Brij buffer (50
mM Na Hepes, pH 7.4, 1 mM DDT, 0.02% Brij-35) prior to the assay in reaction
mixture (5 µl 1 mM [
-32P]ATP, 25 mM MgCl2
[specific activity 250 to 500 cpm.pmol-1], 5 µl 1 mM AMP in
HEPES-Brij buffer, 5 µl 1 mM AMARA peptide [AMARAASAAASARRR] in HEPES-Brij
buffer, 5 µl HEPES-Brij buffer; total volume 25 µl) at 30°C for 10
minutes. A 15 µl aliquot was then spotted onto P81 paper (Whatman). After
stopping the reaction with 1% (v/v) phosphoric acid, radioactivity was
measured by liquid scintillation counting. One unit of AMPK is the amount that
incorporates 1 nmol phosphate into substrate peptide per minute.
The protein assay
The amount of protein present was determined by the bicinchoninic acid
method with bovine serum albumin as the standard
(Smith et al., 1985).
Photolabelling
Dishes (4x10 cm diameter) of confluent Clone 9 cells were treated
with or without (control) 5 mM sodium azide in KRHG for 30 minutes. The cells
were then washed twice in KRH to remove the glucose before photolabelling with
500 µM Bio-LC-ATB-BMPA (Koumanov et
al., 1998) by irradiation in open dishes twice for 1 minute
periods using a Rayonet RPR 100 photochemical reactor (RPR-3000 lamps) at
22°C. After labelling, cells were washed three times in ice-cold KRH and
then lysed in 200 µl 1% Triton X-100 containing 2 µM pepstatin A, 10
µM leupeptin and 100 µM AEBSF for 20 minutes on ice. Debris was removed
from the lysates by centrifugation (100,000 g for 30 minutes), and
biotinylated proteins from 1 ml (
1 mg protein) of the pooled lysate from
each test condition were then precipitated by continuous mixing with 75 µl
of a 50% (w/v) streptavidin-agarose bead slurry (Pierce and Warriner)
overnight at 4°C. The precipitates were washed three times in 0.1% (w/v)
Triton X-100 in PBS, twice in PBS and then eluted by heating at 95°C in 30
µl of sample buffer containing 2% (w/v) SDS and 10 mM DTT. Samples of the
eluted protein (20 µl) and of the cell lysates (20 µg) were then
subjected to western blotting to quantify GLUT1.
Western blotting and immunocytochemistry
For western blotting, proteins were electrophoresed on 10% (w/v)
polyacrylamide/SDS gels and then transferred to nitrocellulose membranes.
Blots were probed with affinity-purified rabbit antibodies against residues
477-492 of rat GLUT1 (0.5 µg/ml;
[Davies et al., 1990]) or with
mouse monoclonal antibodies to c-myc (0.25 µg/ml; Clone 9E10, Oncogene
Research Products) overnight at 4°C, followed by goat anti-rabbit or goat
anti-mouse IgG horseradish peroxidase conjugate (1/40,000; Jackson
ImmunoResearch Laboratories, Inc.), as appropriate, for 1 hour. In the case of
AMPK, blots of Clone 9 cell lysates were probed with sheep antibodies specific
for the phosphorylated form of the AMPK
subunit or with the antibodies
specific for the
1 or
2 isoforms
(Hardie et al., 2000
),
followed by an anti-sheep IgG horseradish peroxidase conjugate. The antigen
was then visualised by addition of enhanced chemiluminescence detection
reagent (Supersignal® Chemiluminescent substrate, Pierce Chemical
Company). Staining intensity was quantified using a Bio-Rad Fluor-S gel
documentation system and Multi-analyst software. Plasma membrane lawns were
prepared and immunostained for GLUT1 as previously described
(Barros et al., 2001
). Images
were obtained and quantified using a Zeiss LSM Pascal 5 Confocal
microscope.
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Results |
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Fig. 1 shows that although
the total cellular content of GLUT1 was unaffected, the level of photolabelled
cell-surface GLUT1 was increased 4.1±1.0 (4)fold following exposure of
the cells to azide. However, as previously reported by ourselves and others
(Shetty et al., 1993;
Barros et al., 1995
),
quantification of GLUT1 by immunocytochemistry of plasma membrane lawns
revealed no significant change in the total cell-surface concentration of this
transporter (data not shown). In parallel experiments, exposure to azide
increased the initial rate of 0.2 mM 2-deoxy-D-glucose uptake by
4.2±0.6 (4)fold. These results suggest that a substantial part of the
effect of azide on glucose transport in Clone 9 cells stems from the unmasking
of previously inactive GLUT1 molecules that were already present at the cell
surface.
A possible role for AMPK in the activation of glucose transport in
Clone 9 cells by inhibition of oxidative phosphorylation and by osmotic
stress
In other cell types, exposure to osmotic stress or elevation of cellular
AMP:ATP ratios as a result of inhibition of oxidative phosphorylation has been
reported to lead to the stimulation of AMPK activity
(Hardie et al., 1998). A
possible role for AMPK in the signal transduction pathways leading to
stimulation of glucose uptake in Clone 9 cells was suggested by our finding
that treatment of these cells with 500 µM AICAR for 60 minutes increased
the initial rate of uptake of 0.1 mM 3-O-methly-D-glucose at 6°C
by 5.0±0.8 (3) fold. Similar results have recently been reported by
another laboratory (Abbud et al.,
2000
). A detailed investigation of the kinetics of this phenomenon
showed that, like inhibition of oxidative phosphorylation
(Shetty et al., 1993
;
Barros et al., 1995
) and
exposure to hypertonic conditions (Barros
et al., 2001
), AICAR treatment increased the Vmax for
transport with little effect on Km. In four paired experiments, one
of which is illustrated in Fig.
2, exposure to 500 µM AICAR for 60 minutes increased the
Vmax for 3-O-methyl-D-glucose uptake from 114±9 (4)
to 427±61 (4) pmol/minute/106 cells, whereas the
Km values of 2.6±0.8 (4) and 2.1±0.7 (4) mM measured
in the control and AICAR-stimulated cells, respectively, were not
significantly different. AICAR stimulation of transport also resembled that
resulting from inhibition of oxidative phosphorylation and/or exposure to
hypertonic conditions in that immunocytochemistry of plasma membrane lawns
revealed no significant change in the total cell-surface concentration of
GLUT1 (Fig. 3a,b). These
findings confirmed, by an independent method, those recently reported by Abbud
et al. (Abbud et al., 2000
).
The capacity of the plasma membrane lawn method to detect increases in cell
surface GLUT1 was confirmed in our experiments by exposing the cells to
CoCl2 (Fig. 3c), a
treatment known to increase both the expression of GLUT1 and the uptake of
hexoses in Clone 9 cells (Behrooz and
Ismailbeigi, 1997
).
|
|
Given the similarity of the responses to AICAR, osmotic and metabolic
stress, we next sought to assess the possibility that AMPK is involved in the
signal transduction pathways leading to stimulation of glucose uptake in Clone
9 cells by these stress stimuli. To this end we examined the effects of
metabolic and osmotic stress on the activity of this enzyme. The effects on
hexose uptake rates and on AMPK activity produced by exposure to 5 mM azide,
500 µM AICAR or hypertonic (0.4 M) sorbitol for 60 minutes were
investigated in parallel using the same batch of cells. Following such
treatments the initial rate of uptake of 0.1 mM 3-O-methyl-D-glucose
increased 7.9±1.8-, 5.0±0.4- and 2.6±0.1-fold,
respectively. Total AMPK activities in immunoprecipitates captured from
lysates of the treated cells using a mixture of antibodies against both the
1 and
2 subunit isoforms of AMPK were 8.6±0.9-,
4.9±1.1- and 2.5±0.2-fold greater, respectively, than basal
values (Table 1). Western blots
of the lysates probed with antibodies specific for the phosphorylated form of
the AMPK
-subunit revealed that changes in the phosphorylation state of
the kinase paralleled the changes in enzymatic activity in each experimental
condition, that is, phosphorylation of the
-subunit was lowest in
untreated cells and highest in azide-treated cells
(Fig. 4c). The slightly lower
electrophoretic mobility of the bands stained in western blots by antibodies
specific for the
1 and
2 subunits in samples from azide- and
AICAR-treated cells compared with untreated cells, likewise reflects their
phosphorylation (Fig. 4a,b).
Measurement of AMPK activities in immunoprecipitates prepared using individual
isoform-specific antibodies revealed that the
1 isoform accounted for
most of the activity, both in the basal and stimulated states
(Table 1). Western blotting
confirmed that levels of the
1 subunit were greater than those of the
2 subunit in Clone 9 cells (Fig.
4a,b). These results differ from those of Abbud and colleagues,
who reported that the predominant
-subunit in Clone 9 cells is
2
and suggested that changes in the activity of this subunit might be primarily
responsible for the stimulation of glucose transport by AICAR
(Abbud et al., 2000
). We are
uncertain of the reason for this discrepancy, but it does not reflect an
inability of the antibodies used in the present study to precipitate the
2 subunits: in previous studies these were used to show that the
1 and
2 isoforms contribute equally to total AMPK activity in
rat liver (Woods et al.,
1996
).
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|
To investigate further the possible involvement of AMPK in the stimulation of glucose uptake by metabolic stress, Clone 9 cells were infected with recombinant adenovirus encoding a constitutively active (ca), myc-tagged mutant of AMPK (ca-AMPK). The initial rate of uptake of 0.1 mM 3-O-methyl-D-glucose uptake by unstressed cells infected with an adenovirus encoding ca-AMPK was 5.8±0.4-fold greater than that seen in uninfected cells (Fig. 5a). By contrast, infection of cells with an adenovirus lacking AMPK increased transport only 1.9±0.3-fold (Fig. 5a). Successful expression of the kinase was confirmed by staining western blots of cell lysates with antibodies to c-myc: a band with the expected size for ca-AMPK (35 kDa) was evident in cells infected with an adenovirus encoding myc-tagged ca-AMPK but not in uninfected cells or those infected with an adenovirus lacking AMPK (Fig. 5b). No changes in the total levels of GLUT1 expressed in the cells were detectable by western blotting following infection with either type of adenovirus (data not shown). Exposure to metabolic stress had little additional effect on transport in cells expressing ca-AMPK: the fold increase in the initial rate of 0.1 mM 3-O-methyl-D-glucose uptake following exposure to 5 mM azide was 5.4±0.3, 1.1±0.1 and 3.1±0.1 for uninfected cells, cells infected with adenovirus encoding ca-AMPK and cells infected with adenovirus lacking AMPK, respectively (Fig. 5a).
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Effect of NOS and PKC inhibitors on stimulation of glucose uptake by
metabolic stress or by AICAR
The effects of ca-AMPK expression and of AICAR on hexose uptake by Clone 9
cells, together with the observed stimulation of AMPK activity in these cells
by both AICAR and stress stimuli, strongly suggested that this enzyme lies on
the signal transduction pathway linking stress stimuli to GLUT1 activation.
The targets of AMPK in Clone 9 cells have not been described, but the results
of experiments performed on the muscle cell line H-2Kb and on rat
muscle (Fryer et al., 2000)
identified NOS as a candidate. To probe the involvement of nitric oxide in the
stress response, we therefore examined the effects of the NOS inhibitors
L-NMMA and L-NAME. Stimulation of glucose uptake by AICAR was not
significantly inhibited by either compound
(Table 2) when they were used
at concentrations (400 µM and 2 mM, respectively) that prevented transport
stimulation by AICAR in H-2Kb myotubes and in rat skeletal muscle
strips (Fryer et al., 2000
).
Similarly, when used at these concentrations, L-NMMA and L-NAME decreased
transport stimulation by azide by only approximately 30%
(Table 2). These findings
indicate that, in contrast to the situation in skeletal muscle, NOS probably
does not lie on the pathway linking metabolic stress to transport stimulation
in Clone 9 cells.
|
Stimulation of glucose uptake in L6 myotubes stressed by acute exposure to
mitochondrial uncouplers has been associated with translocation to the plasma
membrane and activation of conventional protein kinase C isoforms
(Khayat et al., 1998). In
addition, atypical protein kinase C isoforms have been implicated in the
stimulation of glucose transport in adipocytes by insulin
(Bandyopadhyay et al., 1997a
;
Standaert et al., 1997
;
Kotani et al., 1998
;
Bandyopadhyay et al., 2000
).
Although stimulation of transport by exposure of Clone 9 cells to alkaline pH
has been reported not to involve phorbol-sensitive PKC isoforms, phorbol
esters do stimulate transport (Hakimian
and Ismail-Beigi, 1991
). Protein kinase C isoforms therefore
represent another set of signalling components potentially involved in the
regulation of glucose uptake in Clone 9 cells in response to stress. To
examine their involvement in the increase of glucose uptake provoked by
metabolic stress or by AICAR in Clone 9 cells, the effects of a series of PKC
inhibitors were therefore investigated.
The indolocarbozole compound Gö 6976, with IC50 values in
the low nanomolar range (Martiny-Baron et
al., 1993), selectively inhibits conventional protein kinase C
isoforms but had no significant effect on the stimulation of transport
resulting either from azide-induced metabolic stress
(Fig. 6a) or from exposure of
cells to AICAR (data not shown), even at inhibitor concentrations as high as
20 µM. Similarly, the bisindolymaleimide compound Gö 6850 (GF 109203X;
Bisindolymaleimide I), which inhibits both conventional and novel PKC
isoforms, the latter with IC50 values
250 nM
(Martiny-Baron et al., 1993
;
Wilkinson et al., 1993
), was
without effect at concentrations up to 2 µM and inhibited azide-stimulated
hexose transport by only
30% even at 20 µM
(Fig. 6b). Neither Gö 6976
nor Gö 6850 had any effect on the basal rate of hexose uptake into
unstressed cells at concentrations up to and including 20 µM (data not
shown). These findings indicate that, in contrast to the reported situation in
L6 myoblasts (Khayat et al.,
1998
), neither conventional nor novel protein kinase C isoforms
are likely to be involved in metabolic stress-regulated signal transduction
pathways in Clone 9 cells.
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![]() |
Discussion |
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The molecular mechanism responsible for the inability of most of the GLUT1
molecules present at the Clone 9 cell surface to be labelled by
Bio-LC-ATB-BMPA or to transport glucose in the basal state
(Hamrahian et al., 1999)
remains unclear. However, catalytically inactive GLUT1 molecules, which are
similarly refractory to labelling by exofacial affinity reagents, have also
been detected in adipocytes, where they appear to be `unmasked' in response to
anisomycin (Harrison et al.,
1992
). Interestingly, measurements of the binding of the transport
inhibitor cytochalasin B to Clone 9 cell plasma membranes have revealed the
presence of fewer binding sites in non-stressed cells when compared with those
in cells exposed to sodium azide (Shi et
al., 1995
). Cytochalasin B binds to the endofacial conformation of
the substrate-binding site in facilitative glucose transporters
(Devés and Krupka,
1978
): if the binding sites in Clone 9 cells represent GLUT1
molecules, it follows that both the exofacial and endofacial conformations of
the substrate-binding sites of these transporters are somehow masked in the
catalytically inactive form of the protein proposed to exist in non-stressed
cells. It has been proposed that such masking stems from the binding of an
inhibitory protein, but its identity is not known
(Shi et al., 1995
).
The signal transduction pathways linking metabolic and osmotic stresses to
the activation of cell-surface GLUT1 in Clone 9 cells similarly remain
unclear. However, our findings and those of others
(Abbud et al., 2000) that AICAR
not only activates AMPK but also stimulates glucose transport in a manner akin
to metabolic stress suggested that this enzyme might be involved in the
regulation of transport by stress stimuli in these cells. This hypothesis is
strengthened by two additional findings from the present study: (a) that
expression of constitutively active AMPK mimics the effect of stress on
transport; and (b) that following exposure to sodium azide or hypertonic
sorbitol solutions, AMPK activity is increased in parallel with the
stimulation of hexose uptake. Moreover, as described in the Introduction,
several groups have recently provided evidence for a role for AMPK in
stress-induced translocation of the GLUT4 glucose transporter isoform in
skeletal muscle and heart.
Although our data show that AMPK is likely to be involved in the
stimulation of transport not only by AICAR but also by metabolic and osmotic
stress, the relevant downstream targets of AMPK in Clone 9 cells have not yet
been identified. AMPK is known to phosphorylate and activate endothelial
nitric oxide synthase (eNOS) during ischaemia in rat hearts
(Chen et al., 1999).
Similarly, in mouse H-2Kb muscle cells, activation of AMPK by AICAR
has been shown to stimulate NOS activity, and NOS inhibitors completely block
the stimulation by AICAR of glucose uptake in these cells
(Fryer et al., 2000
). Because
8-Br cGMP also stimulates transport in these cells, and inhibition of
guanylate cyclase by LY83583 blocks stimulation of transport by AICAR, it has
been proposed that activation of AMPK in muscle cells stimulates
GLUT4-mediated glucose transport by activation of NOS coupled to downstream
signalling components, including cGMP
(Fryer et al., 2000
). However,
this mechanism is unlikely to be responsible for stimulation of transport by
stress in Clone 9 cells because transport in these cells is not affected by
8Br-cGMP and is stimulated, rather than inhibited, by LY83583
(Prasad et al., 1999
).
Furthermore, in the present study we have shown that the NOS inhibitors L-NMMA
and L-NAME do not prevent hexose transport stimulation induced by exposure of
cells either to azide or to AICAR.
Conventional PKCs have been reported to be involved in transport
stimulation by mitochondrial uncouplers in other cell types
(Khayat et al., 1998), and the
atypical isoform PKC
is involved in the response to ischaemic hypoxia in
rat cardiomyocytes (Mizukami et al.,
2000
). In an attempt to identify possible downstream components of
stress-regulated signal transduction pathways in Clone 9 cells, we therefore
investigated the role of PKC isoforms in the regulation of glucose transport.
The lack of effect of small molecule inhibitors indicates that conventional
and novel PKC isoforms are unlikely to play a role in the response to stress
in these cells. It was not possible to use a similar approach to investigate
the potential involvement of atypical PKC isoforms because the most widely
used small molecule inhibitor of these enzymes, the bisindolylmaleimide
compound Ro 31-8200 (Standaert et al.,
1997
), has recently been shown to be an equally effective
inhibitor of AMPK in vitro (Davies et al.,
2000
). We found that treatment of Clone 9 cells with this compound
in vivo at a concentration of 10 µM completely prevented both the
activation of AMPK and the stimulation of hexose transport induced by exposure
to AICAR or to sodium azide (data not shown). The membrane-permeable,
myristoylated PKC
pseudosubstrate peptide (myr-SIYRRGARRWRKL) has been
employed as a more specific inhibitor of the atypical PKC isoforms
(Bandyopadhyay et al., 1997b
;
Standaert et al., 1997
;
Bandyopadhyay et al., 1999
).
However, although we found that this peptide, used at a concentration of 40
µM, completely prevented the stimulation of transport by azide and
inhibited that by AICAR by 65%, the peptide was found to weakly inhibit the
activity of purified AMPK in vitro, with an IC50 of approximately
100 µM (data not shown). No definitive conclusions can therefore be drawn
about the involvement of atypical PKC isoforms in the response of Clone 9
cells to stress.
In summary, our findings suggest that metabolic stress stimulates
activation of cell-surface GLUT1 in Clone 9 cells via a pathway that involves
AMPK. The relevant downstream effectors of this enzyme remain unclear, but are
unlikely to include either NOS or conventional and novel PKCs. After
submission of this manuscript, Xi et al. reported that p38 MAP kinase mediates
AICAR-stimulated glucose transport in Clone 9 cells
(Xi et al., 2001). However, we
have previously found that although activation of this enzyme, for example by
anisomycin, can stimulate hexose uptake, the kinase is not involved in the
stimulation of uptake either by azide or by osmotic stress
(Barros et al., 1997
;
Barros et al., 2001
). Further
investigations will therefore be required in order to understand the mechanism
by which transporter activation occurs and to identity additional components
of the signalling pathway involved in the response to stress.
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Acknowledgments |
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