Activation of GLUT1 by metabolic and osmotic stress: potential involvement of AMP-activated protein kinase (AMPK)
Kay Barnes1,*,
Jean C. Ingram1,
Omar H. Porras2,
L. Felipe Barros2,
Emma R. Hudson3,
Lee G. D. Fryer4,
Fabienne Foufelle5,
David Carling4,
D. Grahame Hardie3 and
Stephen A. Baldwin1
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
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Fig. 1. Effect of metabolic stress on surface labelling of GLUT1 in Clone 9 cells
by a membrane-impermeant photoaffinity reagent. Clone 9 cells were incubated
with or without 5 mM sodium azide for 30 minutes, as indicated, then
photolabelled with 500 µM Bio-LC-ATB-BMPA in the absence or presence of 400
mM glucose (lane 3) or 20 µM cytochalasin B (lane 4). A control sample
(lane 5) was also UV-irradiated in the absence of Bio-LC-ATB-BMPA. Following
cell lysis with 1% TX-100, samples of total lysate proteins (a) and of
biotinylated cell-surface proteins isolated by adsorption to streptavidin
agarose (b) were subjected to western blotting to detect GLUT1, as described
in the Materials and Methods. The mobilities of the molecular mass markers are
indicated on the left.
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Fig. 2. Effect of AICAR exposure on the kinetic parameters of hexose uptake. Clone
9 cells were treated for 60 minutes with (open symbols) or without (closed
symbols) 500 µM AICAR. Uptake of
[3H]3-O-methyl-D-glucose was then measured at 6°C in
the presence of increasing concentrations of hexose as described in the
Materials and Methods. Transport parameters were estimated by direct fitting
of a rectangular hyperbola to the data using non-linear regression (Sigma
Plot, Jandel). Data shown are means±s.e.m. (3).
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Fig. 3. Effect of AICAR exposure on the abundance of cell-surface GLUT1. Clone 9
cells were exposed for 60 minutes to buffer only (a) or to 500 µM AICAR (b)
or for 24 hours to 250 µM CoCl2 (c). Plasma membrane lawns were
prepared, immunostained and quantified as described in the Materials and
Methods. Data were pooled from two experiments in which eight fields were
measured, each containing an average of 12 cells. The image intensity data
(arbitrary units) shown beneath the images are means±s.e.m. [(8) number
of fields]. *Significantly different (P<0.05) from the
control (ANOVA plus Bonferroni's ad hoc test); NS, not significantly different
from the control. Scale bar, 100 µm.
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Fig. 5. Comparison of the effects of exposure to azide and of infection by an
adenovirus encoding ca-AMPK, or lacking AMPK, on hexose transport. Clone 9
cells in triplicate culture dishes were maintained uninfected (none) or
infected with adenovirus either encoding ca-AMPK (ca) or lacking the AMPK gene
(null) for 48 hours. The cells were washed twice with PBS and treated for 30
minutes with (azide) or without (basal) 5 mM azide. Uptake of
[3H]3-O-methyl-D-glucose was then measured as described in
the Materials and Methods. Results shown in (a) are means±s.e.m. (3).
In parallel, western blots of cell lysates (10 µg protein) were prepared as
described in the Materials and Methods section and stained with mouse
antibodies to c-myc (b). The mobilities of molecular weight markers are
indicated on the left. *This value is significantly different
(P<0.05) from basal uptake rate in non-infected cells and from
basal uptake rate in cells infected with adenovirus lacking the AMPK coding
region.
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Fig. 6. Comparison of the effects of (a) Gö 6976 and (b) Gö 6850 on
metabolic stress-stimulated hexose transport. Clone 9 cells in triplicate
culture dishes were treated with or without the indicated concentrations of
inhibitors for 45 minutes. During the last 30 minutes of this period, they
were treated with (azide) or without (basal) 5 mM sodium azide. Uptake of (a)
[3H]3-O-methyl-D-glucose or (b)
[3H]2-deoxy-D-glucose was then measured as described in the
Materials and Methods. Results shown are the means±s.e.m.;
*this value is significantly different (P<0.05) from
the azide-stimulated uptake rate in the absence of the inhibitor.
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© The Company of Biologists Ltd 2002
subunit isoforms and their phosphorylation in response to
treatment of Clone 9 cells with sodium azide, AICAR and hypertonic sorbitol.
Confluent dishes of cells were untreated (basal), exposed to 5 mM sodium azide
(azide), 500 µM AICAR (AICAR) or 0.4 M sorbitol (sorbitol) for 60 minutes.
Western blots of cell lysates were probed with antibodies against the