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Department of Pathology (M.V.E.-F., K.G., R.W.H., A.W.), University of Alabama at Birmingham; and Birmingham Veterans Administration Medical Center (M.V.E.-F., K.G., A.W.), Birmingham, Alabama 35294
Address all correspondence and requests for reprints to: Alan Wells, M.D., D.M.S., Department of Pathology, University of Alabama at Birmingham, Lyons-Harrison Research Building Room 531, Birmingham, Alabama 35294. E-mail: wells{at}lh.path.uab.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dissection of the signaling pathways, activated by the insulin receptor, have suggested that signaling molecules coupled to IRS-1, such as PI3-kinase, are required for stimulation of glucose transport by insulin (reviewed in Ref.3). However, recent reports have shown that GLUT4-mediated glucose transport can be stimulated in the absence of activation of IRS-1 or PI3-kinase (4, 5, 6, 7, 8). Furthermore, studies with rat skeletal muscle suggest differential regulation of GLUT4 translocation by insulin vs. exercise (9). These findings suggest that alternative or redundant signaling pathways may converge on GLUT4 to promote glucose uptake.
To identify these other intracellular mechanisms that can lead to GLUT4-mediated glucose transport in a physiologically relevant cell type, such as adipocytes, we have stably expressed in 3T3-L1 adipocytes, wild-type (WT), and signaling-restricted mutant epidermal growth factor (EGF) receptors (EGFRs). The use of mutant growth factor receptors allows us to selectively activate certain signaling pathways to determine their relevance to stimulation of GLUT4-mediated glucose transport in adipocytes. We have previously demonstrated the ability of EGF to stimulate glucose transport in 3T3-L1 adipocytes in a GLUT4-dependent manner with overexpression of the EGFR (4). High level expression of another receptor with intrinsic tyrosine kinase activity, the platelet-derived growth factor (PDGF) ß receptor, also confers growth factor-dependent GLUT4-mediated glucose uptake (7), suggesting that these receptors stimulate similar signaling pathways to those activated upon insulin binding. This approach has demonstrated the ability to stimulate glucose transport in the absence of activation of either IRS-1 (4) or PI3-kinase (7) and that activation of MAP kinase and Shc was insufficient to stimulate glucose transport (4, 10). Furthermore, we demonstrated that autophosphorylated phosphotyrosine motifs on the EGFR are required for induction of GLUT4-mediated glucose transport but not for stimulation of glucose disposal as glycogen or lipid (4, 10). These findings suggested that specific phosphotyrosine-binding signaling effectors were required to activate the GLUT4-mediated glucose uptake mechanism.
In addition to PI3-kinase and Shc, another protein known to interact
with the EGFR in a phosphotyrosine-dependent manner is phospholipase C
(PLC)-
(11). Recently, in vitro studies, using
signaling-restricted PDGF receptor mutants in CHO cells, have suggested
a PLC-dependent pathway for stimulation of GLUT4 translocation to the
cell surface (12). In addition, in vivo, administration of
lithium [an inhibitor of phosphatases that degrade PLC-generated
inositol phosphates (13, 14)] to both humans and animals resulted in
enhanced insulin stimulation of glucose transport and disposal that is
correlated with an enhanced presence of inositol phosphates generated
by PLC activity (15, 16). Herein, we present evidence that supports the
hypothesis that PLC activity is required for EGF stimulation of glucose
transport in 3T3-L1 adipocytes. Furthermore, we demonstrate that
inhibition of PLC activity diminishes stimulation of glucose transport
by insulin or EGF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PLC assay
PLC activity was assayed by determining the amount of
[3H]-inositol phosphate species generated with hormone
stimulation, as described previously (18). Briefly, 3T3-L1 adipocytes
were incubated overnight in serum-free DMEM that contained 5.0 µCi/ml
[3H]-myo-inositol. Cells were washed twice with PBS and
then incubated in serum-free DMEM containing 10 mM lithium
chloride to enhance accumulation of inositol phosphates generated by
PLC-mediated hydrolysis (19). Cells were then stimulated with either 1
µM insulin or 20 nM EGF for 15 min. Cells
were washed once with PBS and lysed with boiling water. Lysates were
boiled for 5 min and clarified with 5 min centrifugation at 12,000
x g. Subsequent supernatants were loaded onto AG 1-X8 Dowex
anion exchange columns (BioRad Labs, Hercules, CA). Free inositol was
eluted from the column with 20 column volumes of water; glyceroinositol
phosphate was eluted with 20 column volumes of 5 mM sodium
borate-60 mM sodium formate; and inositol phosphates were
eluted with 20 column volumes of 0.1 N formic acid-200 mM
ammonium formate. Eluates were collected and liquid scintillation
counting was performed. The pharmacological agent, U73122
(1-(6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione)
(BIOMOL, Plymouth Meeting, PA) was added to the cells to inhibit PLC
activity. It was introduced into the media 30 min before the addition
of insulin or EGF. U73122 inhibits PLC activities but not PLA2 or PLD
activities (Refs. 20 and 21, and our unpublished observations). The
inactive congener of U73122, U73343
(1-(6((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione),
was added in parallel and served as a control.
2-Deoxyglucose transport assay
2-Deoxyglucose transport assays in 3T3-L1 adipocytes were
performed as previously described (4). Briefly, serum-starved cells
were washed once with Krebs-Ringer phosphate buffer and incubated with
hormone (1 µM insulin or 20 nM EGF) for 20
min at 37 C. 2-Deoxy-D-[1-3H]glucose was
added and the incubation was continued for 10 min. Assays were
terminated with two rapid washes of iced PBS buffer. Cells were
solubilized with NaOH and label detected by scintillation counting.
Pharmacological inhibitors were added to the cells in Krebs-Ringer
phosphate buffer 30 min before hormone stimulation.
Glycogen synthase assay
Glycogen synthase activity was assayed as described (22), with
slight modifications (10). Adipocytes were serum starved for 3 h
in DMEM supplemented with 0.2% BSA and then stimulated with insulin (1
µM) or EGF (20 nM) for 90 min. Cells were
washed once with ice-cold PBS and homogenized with extraction buffer
[100 mM NaF, 10 mM EDTA, 50 mM
Tris-HCl (pH 7.6), 1 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride (PMSF)]. Lysates were then clarified by
centrifugation (12,000 x g for 5 min). Per volume of
supernatant, half-volumes of reaction mix (6.7 mM
[14C)-UDP-glucose [300 µCi/liter], 25 mM
NaF, 20 mmol/liter EDTA, 1% glycogen, 50 mM Tris-HCl, pH
7.6) were added. Incorporation of [14C]-UDP-glucose into
glycogen was determined by precipitation of glycogen on filter paper
with 67% ethanol after 30 min at 30 C, as described above, for the
glucose incorporation into glycogen assay. The label remaining on the
filter paper was detected by scintillation counting. The stimulated
in vitro activity was calculated as the number of counts
incorporated in the presence of 0.25 mmol/liter glucose-6-phosphate
(G6P) divided by the number of counts incorporated in the presence of
10 mmol/liter G6P that represents total glycogen synthase activity.
Pharmacologic inhibitors were added to the cells 30 min before hormone
stimulation.
Lipid synthesis assay
Lipid incorporation of glucose into lipids was measured as
previously described (10). Briefly, cells were serum starved in DMEM
supplemented with 0.2% BSA for 3 h. The medium was then replaced
with Buffer A (25 mM Tris HCl (pH 7.5), 140 mM
NaCl, 1.7 mM KCl, 1 mM CaCl2, 1.47
mM K2HPO4, 0.8 mM
MgSO4, 0.2% BSA) and incubated for 15 min. Cells were
exposed to insulin (1 µM) or EGF (20 nM) for
15 min before addition of [U14C]D-glucose (3
mM glucose, 3 mCi/liter final concentration) and incubated
for 1 h further. The assay was terminated with an ice-cold PBS
wash; cells were scraped in ice-cold methanol and lipids were extracted
as previously described (23). Briefly, the cells were resuspended in a
final mixture of 1:1:0.9:0.0001% chloroform: methanol:
H2O: butylated hydroxytoluene. The lipid-containing phase
was isolated and dried under nitrogen stream before scintillation
counting. Pharmacologic inhibitors were added to the cells 30 min
before hormone stimulation.
Isolation of plasma membrane fractions and immunoblotting of
GLUT4
Subcellular fractionation of 3T3-L1 adipocytes was performed as
previously described (4, 24). Briefly, cells were serum-starved for
3 h in DMEM supplemented with 0.2% BSA and then stimulated with
insulin (1 µM) or EGF (20 nM) for 30 min.
Cells were washed once with ice-cold PBS and homogenized in Buffer A
[250 mM sucrose, 20 mM HEPES (pH 7.4), 1
mM EDTA, 1 mM PMSF] at 4 C. Lysates were
centrifuged at 16,000 x g for 20 min at 4 C. The
resultant pellet was resuspended in Buffer B [20 mM HEPES
(pH 7.4), 1 mM EDTA, 1 mM PMSF] and
fractionated by ultracentrifugation at 100,000 x g
through a 1.12 M sucrose cushion in Buffer B for 1 h
at 4 C. Plasma membranes, isolated from the top of the cushion, were
isolated and pelleted with a 30,000 x g centrifugation
for 30 min at 4 C. Isolated plasma membrane fractions were then
solubilized with Buffer B containing 1% Triton-X-100. Pharmacologic
inhibitors were added to the cells 30 min before hormone
stimulation.
Thirty micrograms of plasma membrane proteins from each treatment condition were separated by SDS-PAGE in a nonreducing, nonheat-treated manner. Separated proteins were then transferred to an Immobilon-P membrane (Millipore, Burlington, MA) and probed with rabbit anti-GLUT4 antibody (East Acres Biologicals, Southbridge, MA) and developed with antirabbit antibodies conjugated to alkaline phosphatase (Promega, Madison, WI).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a known signaling molecule that is
activated efficiently by the EGFR in other cell types and has been
demonstrated to be dependent on phosphotyrosine motifs of the EGFR for
binding to, and subsequent activation by, the receptor (11, 18).
Therefore, we determined whether EGF stimulation of 3T3-L1 adipocytes
overexpressing the EGFR would likewise lead to activation of PLC
activity. Stimulation of cells with 20 nM EGF leads to a
3.37 ± 1.20-fold increase (n = 7) in PLC activity, as
measured by inositol phosphate formation (Table 1
). In parallel, we determined if insulin
stimulation would also activate PLC activity. Insulin (1
µM) effected only a small increase of inositol phosphate
formation (1.06 ± 0.01-fold, n = 2), consistent with a
previous finding in rat adipocytes (25). Finally, the truncated EGFR,
c’973-EGFR, which is devoid of tyrosine autophosphorylation sites,
does not stimulate PLC activity in 3T3-L1 adipocytes (data not shown),
in agreement with findings in fibroblasts (18).
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). Addition of the inactive congener, U73343, at the
same concentration inhibited PLC activity only 16.1 ± 2.0%
(n = 2). At a lower dose, U73122 (17 µM) failed to
inhibit PLC activity (data not shown).
Inhibition of PLC activity diminishes both EGF and
insulin-stimulated glucose transport
To determine whether PLC activation played a role in stimulation
of glucose transport, we used U73122 to inhibit PLC activity while
measuring insulin and EGF-induced glucose transport. In the presence of
U73122, EGF stimulation of glucose transport was inhibited 56.0 ±
12.7% (n = 3, in triplicate), and insulin stimulation of glucose
transport was inhibited 55.9 ± 13.1% (n = 2, in triplicate)
(Fig. 1). U73122 inhibition of
insulin-stimulated glucose transport was confirmed in nontransgenic
parental 3T3-L1 adipocytes. Whereas the concentration of U73122 used
for adipocytes was greater than that used in fibroblasts (18), the
inactive congener of U73122, (U73343) at the same concentration of 50
µM and a lower dose of U73122 (17 µM), both
of which did not inhibit EGF-induced PLC activity, failed to inhibit
EGF and insulin stimulation of glucose transport (data not shown). The
percent inhibition of glucose transport corresponded roughly with the
level of inhibition of PLC achieved.
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). Similarly, inhibition of PLC
activity did not impair either EGF or insulin stimulation of lipid
synthesis. Pretreatment with U73122 yielded hormone-induced lipid
synthesis of 109.2 ± 1.9% and 107.4 ± 12.9% of
non-U73122-treated controls for EGF and insulin, respectively
(4.21 ± 1.42-fold stimulation for insulin, 4.425 ±
0.98-fold for insulin in the presence of U73122; 2.29 ± 0.52-fold
for EGF, 2.50 ± 0.61-fold for EGF/U73122; n = 2, in
triplicate). In addition to supporting our previous hypothesis that
EGF-induced glucose disposal is not dependent on activation of GLUT4 in
3T3-L1 adipocytes (10), these results demonstrate that insulin
signaling of glucose disposal is similarly independent of
GLUT4-mediated glucose transport.
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). By densitometric analysis of the
immunoblots, there was a 56.4% ± 14.8% (P < 0.01)
and a 43.2 ± 11.3% (P < 0.05) decrease in
insulin and EGF-stimulated translocation of GLUT4 to the plasma
membrane, respectively. The decreased membrane association of GLUT4 was
not caused by down-regulation of GLUT4 expression, because total GLUT4
levels were similar in control and U73122-treated cells (data not
shown), as would be expected with only short-term (total 30 min)
treatment of cells.
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). This coincided with a similar
enhancement of GLUT4 translocation to the plasma membrane upon insulin
stimulation (Fig. 5). By densitometric
analysis of the immunoblots, lithium treatment resulted in a 31.1
± 8.6% (P < 0.05) increase in insulin-stimulated
GLUT4 recruitment to the plasma membrane, over insulin treatment
only.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in CHO cells has
suggested that, in addition to PI3-kinase, activation of PLC may
contribute to the translocation of GLUT4 to the plasma membrane (12).
Herein, we demonstrate that in 3T3-L1 adipocytes overexpressing the
full length (WT), but not a truncated (c’973) EGFR, EGF can stimulate
both PLC activity (Table 1) and glucose transport (4). However, insulin
stimulates glucose transport while only minimally increasing PLC
activity (Table 1). These results suggest that despite the low level
stimulation of PLC activity by insulin, adipocytes contain a
significant amount of inducible PLC activity that may be activated by
alternative signaling pathways. The basal levels of PLC activity seem
to decrease slightly with U73122 treatment (Table 1). This would be
consistent with the hypothesis that PLC activity or resultant products
(such as inositol phosphates) are required for GLUT4 translocation in a
permissive manner and would explain why we see the effect of PLC
inhibition on insulin-stimulated glucose transport in the absence of
insulin’s ability to induce PLC activity. However, direct
up-regulation of this signaling pathway, through inhibition of inositol
phosphate degradation [using agents such as lithium (Figs. 4 and 5)],
may provide a stimulatory effect.
Inhibition of PLC activity by the aminosteroid, U73122, was specific
and corresponded to a decrease in both EGF and insulin stimulation of
glucose transport (Table 1). The concentrations used for this inhibitor
were higher in adipocytes than doses needed in other cell types, such
as fibroblasts (18). The reason for the decreased potency of U73122 in
adipocytes is not clear. However, differences in the inhibitory
concentration of U73122 in various cell types have been suggested as
being caused by significant partitioning of the lipophilic drug into
membranes or into fat globules in adipocytes (20). The use of the
inactive congener, U73343, demonstrated no nonspecific aminosteroidal
effects (Table 1). Furthermore, only PLC and glucose transport
activities were impaired with U73122 treatment (Table 1, Fig. 1);
stimulation of glycogen synthase activity or incorporation of glucose
into lipid by insulin or EGF was unaltered in the presence of the PLC
inhibitor (Fig. 2). This extends our previous finding that the glucose
transport and glycogen storage signaling pathways are separate for
EGF signaling (10), to demonstrate a similar divergence for insulin
signaling.
Finally, the role PLC activity may play in the stimulation of glucose
transport is postulated to involve the regulation of GLUT4
translocation to the plasma membrane (Fig. 3). Whereas inhibition of
PLC activity leads to a decrease in hormonal stimulation of GLUT4 to
the plasma membrane, enhancement of inositol phosphate accumulation
secondary to lithium inhibition of inositol phosphatase activity leads
to enhancement of glucose transport (Fig. 4) and an increase in GLUT4
found at the plasma membrane with insulin stimulation (Fig. 5). In sum,
we propose that PLC activity and subsequent generation of inositol
phosphates enhance GLUT4 association with the plasma membrane. This may
normally be a constitutive maintenance function. However, upon
up-regulation by signaling events other than insulin receptor
activation, this signaling pathway may be able to provide enough
stimulus to promote GLUT4 translocation and increased glucose
transport. These studies suggest that PLC activity might represent a
therapeutic target through which enhancement of GLUT4-mediated glucose
transport might be achieved.
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Acknowledgments |
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Footnotes |
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Received May 29, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a substrate for the PDGF and EGF receptor
protein tyrosine kinases in vivo and in vitro.
Cell 57:1109–1122[CrossRef][Medline]
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