Biochemical Society Transactions (1997) 25 4855
117 Rapid activation of GLUTl by osmotic stress
L. FELIPE BARROS, STEPHEN A. BALDWIN* and MARK
GRIFFITHS*
Departamento de Medicina Experimental, Facultad de Medicina,
Universidad de Chile, Independencia 1027, Casilla 70058,
Santiago 7, Chile and +Department of Biochemistry t
Molecular Biology, University of Leeds, L.eeds LS2 9JT, UK.
The mechanisms and signalling events involved in the
stimulation of glucose uptake by stress in mammalian cells are
largely unknown. However, recent data suggest that the rapid in
situ activation of GLUTl by protein synthesis inhibitors such as
anisomycin is partly mediated by the p38-MAP kinase [1,2]. As
extracellular hyperosmolarity is a known physiologicalactivator
of this serine kinase, we have explored the hypothesis that
osmotic stress results in the activation of GLUTl.
Exposure of rat Clone 9 epithelial cells and mouse 3T3-Ll
adipocytes to 0.4 M sorbitol in Krebs-Ringer-Hepes medium
(0.7 OsM total; 37°C) stimulated the initial rate of 0.2 mM 2deoxyglucose uptake by 2.1 f 0.2(6) and 5.1 O.l(4)-fold,
respectively (mean f s.e.m (number of experiments performed
in duplicate)). Basal uptake rates (pmoVmin/106cells) were 75 f
8(6) for Clone 9 cells and 148 f 29(4) for adipocytes. Half times
of stimulation (min) were 5.6 f 0.9 for Clone 9 cells and 22.7 f
1.5 for adipocytes. The effect was fully reversed upon sorbitol
removal, with half times (min) of 15 f 3.2 for Clone 9 cells and
17 f 3.7 for adipocytes. Control experiments showed a 1.8 f
0.3(3)-fold stimulation, by osmotic stress, of the initial rate of
uptake of 0.2 mM 3-0-methyl-D-glucose at 25°C by Clone 9
cells, which indicates that the stimulation of sugar uptake
observed occurs at the level of transport rather than of hexose
metabolism. Translational arrest by puromycin (100 pg/ml) did
not significantly affect sorbitol-stimulateddeoxyglucoseuptake
(4 f 5(3) % inhibition) indicating that newly synthesised
proteins are not involved in the response.
The stimulation (fold) of deoxyglucose uptake increased with
the severity of the osmotic shock, reaching 2.9 f 0.1(3) (Clone
9) and 9.4 f 1.1(2) (adipocytes) at 0.8 M sorbitol. At higher
osmolarities, sugar uptake decreased steeply, f d l i i well under
control values at 1.2 M. It is noteworthy that the maximum
stimulation achieved by osmotic stress (fold) was similar to that
achieved by 160 nM i n s d i in both 3T3-Ll adipocytes (9.1 f
OS(3)) and Clone 9 cells (2.7 f 0.4(3)[3]). In both cell types,
equiosmolar sodium chloride, sucrose and mannitol caused
similar effects to sorbitol whereas the membrane-permeant
compound# dimethylsulphoxideand urea failed to affect sugar
uptake. Thus it appears that e M v e osmolarity and not solute
concentration itself is the primary stimulus for increased sugar
uptake.
The effect of cell exposure to 0.4 M sorbitol on the apparent
affinity of the transport site for sugars was investigatedin Clone
9 cells by measuring the uptake of deoxyglucose in the presence
of increasing concentrations (2, 4, 8, 16 and 32 mM) of 3 - 0
methyl-D-glucose. From the pooled results of two experimmts,
the concentrations capable of inhibiting sugar uptake by 50%
(ICSO) wen 12 f 1.6 mM for control and 13.3 f 0.9 mM for
sorbitol-treatad cells. In these assays the mean stimulation of
uptake by osmolarity was 2.6-fold. It can therefore be inferred
that the effect of sorbitol is solely due to m increase in the
capacity for sugar uptake (VA.
In order to estimate whether
traasport stimulation by hypcrosmolarity is due to translocation
of intracellular carriers to the plasma membrane (PM) or
*
activation of carriers at the cell,&s
PM lawns pnparedfrom
both cell types [3] were immunoscreened for glucose
transporters. Examination of samples fiom 3 experiments with
3T3-Ll adipocytes suggested that exposure to 0.4 M sorbitol
slightly increased the concentration of GLUT4 at the cell surface
whereas GLUTl was not af€ected. This is in contrast to the large
increase in GLUT4 and GLUTl concentrations in PM lawns
prepared from insulin-stimulated 3T3-Ll adipocytes [3]. In
osmotically-shocked Clone 9 cells no apparent changes in
surface GLUTl concentrationwere detected.
The possible role of p38-MAP kinase in the stimulation of
deoxyglucoseuptake by hyperosmolarity was assessed by using
the selective inhibitor SB203580 (20 @
Sorbitol-stimulated
I).
sugar uptake was not significantly affected by the SB
compound, with inhibitions (%) of 5 f 8(2) and 2 rt 4(2) for
Clone 9 cells and adipocytes, respectively. This finding is in
marked contrast to the complete inhibition by SB203580 of the
stimulation of sugar transport by 300 nM anisomycin in both
cell types [l]. The phosphatidylinositol(PI) 3-kinase inhibitor
wortmantun
*
(250 nM), a potent inhibitor of insulin-stimulated
glucose transport in both cell types [3], failed to a&ct sorbitolstimulated sugar uptake, with inhibitions (%) of -7 f 4(2) and
10 12(2) in Clone 9 cells and adipocytes, respectively.
Finally, by using the equilibrium uptake of 3-o-methyl-Dglucose as an index of cell volume, it was observed that upon
exposure to sorbitol, Clone 9 cells rapidly shrank as perfect
osmometers. This was followed by a slow recovery of cell
volume, reaching control values after two hours of continued
stress This response has been termed Regulatory Volume
Increase (RVI) and requires a continuous supply of metabolic
energy.
We conclude that two mammalian cell types respond to
hyperosmotic shock by rapidly increasing their sugar transport
capacity. The effect appears to be mediated primarily by the in
siru activation of GLUTl carriers at the cell surface. Possible
links between this phenomenon and other stimuli known to
stimulate glucose transport remain to be investigated, as well as
the possible role of glucose transport stimulation in the RVI
response.
*
-
We thank Dr.Jeremy Saklatvala for his kind gift of SB203580
and Mrs Jean Ingram for excellent technical assistance.
Supported by FONDECYT 1961209 and Fundacion Andes
(Chile), and by the BBSRC (UK).
1. Barros, L ,F., Guiton, M. Saklatvala, J. and Baldwin, S.A.
(1997) Submitted for publication.
2. Gould, G.W., Cuenda, A., Thomson, F.J. and &hen (1995)
Biochem. J. 311,735-738.
3. Barros, L.F., Marchant, R.B., and Baldwin, S.A. Biochem.
J.(1995) 309,731-736