(Received for publication, July 27, 1995; and in revised form, August 21, 1995)
From the
Insulin binding results in rapid phosphorylation of insulin
receptor substrate-1 to activate p21 and
mitogen-activated protein kinase. Insulin also activates the ribosomal
protein S6 kinase (pp70 S6 kinase) independently of the Ras pathway.
Chronic (18 h) treatment of L6 muscle cells with insulin increases
glucose transport activity severalfold due to biosynthetic elevation of
the GLUT1 and GLUT3 but not the GLUT4 glucose transporters. Here we
investigate the roles of p21
and pp70 S6 kinase
in the insulin-mediated increases in GLUT1 and GLUT3 expression. L6
cells were transfected with the dominant negative Ras(S17N) under the
control of a dexamethasone-inducible promoter. Induction of Ras(S17N)
failed to block the insulin-mediated increase in GLUT1 glucose
transporter protein and mRNA; however, it abrogated the
insulin-mediated increase in GLUT3 glucose transporter protein and
mRNA. Inhibition of pp70 S6 kinase by rapamycin, on the other hand,
eliminated the insulin-mediated increase in GLUT1 but had no effect on
that of GLUT3 in both parental and Ras(S17N) transfected L6 cells.
These results suggest that the biosynthetic regulation of glucose
transporters is differentially determined, with pp70 S6 kinase and
p21
playing active roles in the
insulin-stimulated increases in GLUT1 and GLUT3, respectively.
Insulin mediates a wide spectrum of biological responses
including stimulation of glucose influx and metabolism in muscle and
adipocytes, transport of amino acids, transcription of specific genes
and mitogenesis(1, 2) . These are determined by
signals initiated by insulin binding, leading to rapid
autophosphorylation of receptor tyrosine residues (3) and
tyrosine phosphorylation of Shc (4) and IRS-1. ()IRS-1 serves as a docking protein for Src homology 2 (SH2)
domain proteins including phosphatidylinositol 3-kinase, and GRB2, an
adaptor protein linked to Sos, a guanine nucleotide exchange
factor(3, 5) . Association of IRS-1 or Shc with
GRB2-Sos results in the release of GDP from p21
and a consequent increase in the amount of
GTP-p21
(5) . Like all GTP-binding
proteins, p21
cycles between inactive GDP-bound
and active GTP-bound conformations(6) . Ras binds directly to
the serine/threonine kinase Raf-1, which in turn phosphorylates and
activates MAPK/ERK kinase to phosphorylate and activate MAPK
(ERK)(7, 8, 9, 10) . Insulin also
stimulates the ribosomal protein S6 kinases pp70 S6 kinase and pp90 S6
kinase(3, 11) . The two S6 kinases, originally
identified by their ability to phosphorylate the 40 S ribosomal protein
S6, are regulated by distinct mechanisms, as MAPK directly
phosphorylates and activates pp90 S6 kinase but has no effect on pp70
S6 kinase(3, 5) , whereas phosphatidylinositol
3-kinase is required for activation of pp70 S6 kinase but not pp90 S6
kinase or MAPK(12) .
The L6 cell line is derived from neonatal rat thigh skeletal muscle and retains several properties of skeletal muscle(13, 14) . During all stages of their development, L6 cells express the GLUT1 glucose transporter, a ubiquitous isoform that is widely distributed and is believed to provide cells with basal glucose requirements(15) . They also express the GLUT3 glucose transporter, which is expressed in fetal (16) and regenerating muscle (17) and in neuronal cells of the brain (18) . In contrast, the fat/muscle-specific GLUT4 glucose transporter is not expressed until alignment and onset of cell fusion into myotubes (15, 19, 20) . Our laboratory has previously demonstrated that in response to chronic treatment with insulin (for several hours), glucose transport activity increases severalfold due to an increase in GLUT1 mRNA and protein levels(2, 21) . We also observed that sustained insulin-like growth factor-1 treatment leads to an increase in GLUT3 mRNA and protein levels(22) . By contrast, prolonged insulin treatment was associated with a small decrease in the levels of GLUT4 protein and mRNA(21) . Similar observations on GLUT1 and GLUT4 have been made in 3T3-F442A adipocytes(23) .
Despite long
standing recognition of the signaling cascades that link the insulin
receptor to the nucleus, it remains unknown whether they participate in
the insulin-induced regulation of expression of specific genes with the
exception of those encoding for transcription factors (24) .
Therefore, the objective of this study was to investigate the role of
the signaling cascade, in particular the role of p21 and pp70 S6 kinase, in the insulin-induced increases in
GLUT1 and GLUT3 expression after prolonged (18 h) treatment with the
hormone. Using a constructed L6 cell line transfected with a dominant
negative Ras, Ras(S17N) under the control of a dexamethasone-inducible
promoter(25) , and using rapamycin, a specific inhibitor of
pp70 S6 kinase(11, 26) , we investigated the roles of
p21
and pp70 S6 kinase in the insulin-mediated
regulation of expression of GLUT1 and GLUT3 protein and mRNA.
Figure 1:
Inhibition of insulin-induced elevation
in GLUT1 protein by rapamycin but not by expression of Ras dominant
negative mutant. A, L6 cells overexpressing Ras(S17N) were
treated with or without 1.5 µM dexamethasone (DEX) for 24 h prior to treatment without (basal (B))
or with 100 nM insulin (I) in the presence or absence
of 30 ng/ml rapamycin (R) for 18 h. Total membranes were
prepared and immunoblotted as described under ``Experimental
Procedures.'' The content of the immunoreactive 1
Na
/K
-ATPase subunit is illustrated
for assessment of equality of protein loading. This is a representative
blot of four independent experiments. B, the results of four
independent experiments were densitometrically scanned. The content of
GLUT1 protein in basal cells in the absence of dexamethasone was
assigned a value of 1.0, and other values were expressed in relative
units. Values represent means ± S.E. of four independent
experiments. *, significance at 95% compared with basal cells in the
absence of dexamethasone.**, significance at 95% compared with basal
cells in the presence of dexamethasone.
On the other hand, rapamycin, a
specific inhibitor of pp70 S6 kinase, almost completely eliminated the
increase in GLUT1 glucose transporter protein in response to insulin
(shown in Fig. 1A and quantified in Fig. 1B). This was observed under control conditions
(in the absence of dexamethasone) as well as when the dominant negative
p21 was induced (in the presence of dexamethasone).
Therefore, the insulin-stimulated increase in GLUT1 protein is a pp70
S6 kinase-dependent event.
Parallel results were obtained with GLUT1
mRNA; chronic treatment with insulin resulted in a 101% increase in
GLUT1 mRNA above the basal value (mean of four independent
experiments). The induction of dominant negative Ras by dexamethasone
failed to block the insulin-mediated increase in GLUT1 mRNA. Insulin
still caused a 128% increase in GLUT1 mRNA above the value in the
presence of dexamethasone (mean of four independent experiments). These
results indicate that insulin action on GLUT1 gene expression occurs
independently of p21.
Figure 2:
Inhibition of insulin-induced elevation in
GLUT3 protein by expression of Ras dominant negative but not by
rapamycin. A, L6 cells overexpressing Ras(S17N) were treated
with or without 1.5 µM dexamethasone (DEX) for 24
h prior to treatment without (basal (B)) or with 100 nM insulin (I) in the presence or absence of 30 ng/ml
rapamycin (R) for 18 h. Total membranes were prepared and
subjected to immunoblot analysis as described under ``Experimental
Procedures.'' The content of immunoreactive 1
Na
/K
-ATPase subunit is illustrated
for assessment of equality of protein loading. This is a representative
blot of four independent experiments. B, the results of four
independent experiments were densitometrically scanned. The content of
GLUT3 protein in basal cells in the absence of dexamethasone was
assigned a value of 1.0, and other values were expressed in relative
units. Values represent means ± S.E. of four independent
experiments. *, significance at 95% compared with the basal cells in
the absence of dexamethasone.**, significance at 95% compared with
rapamycin alone.
Rapamycin, on the other hand, was without effect on the insulin-stimulated increase in GLUT3 protein. In the absence of dexamethasone, rapamycin did not block the ability of insulin to increase GLUT3 (Fig. 2). Furthermore, rapamycin did not interfere with the ability of Ras(S17N), when induced by dexamethasone, to eliminate the insulin-mediated increase in GLUT3 glucose transporter as shown in Fig. 2. Thus, the insulin-mediated increase in GLUT3 is a pp70 S6 kinase-independent event.
Parallel results were obtained for GLUT3 mRNA. GLUT3 mRNA
rose by 104% above the basal levels in response to chronic treatment
with insulin (mean of four independent experiments). Unlike the effects
on GLUT1 mRNA, however, the induction of dominant negative Ras was
associated with an insulin-mediated increase in GLUT3 mRNA of only 9%
above basal levels (mean of four independent experiments). These
results indicate that insulin action on GLUT3 gene expression is a
p21-dependent event.
Figure 3: Inhibition of insulin-induced phosphorylation of MAPK (ERK) in cells overexpressing Ras(S17N) but not in parental L6 cells. Parental (A) or Ras(S17N)-transfected (B) cells were treated with or without 1.5 µM dexamethasone (DEX) for 24 h, then stimulated with or without 100 nM insulin for 5 min, and lysed, and MAPK phosphorylation was detected in the whole lysates as described under ``Experimental Procedures.'' This is a representative blot of two independent experiments.
Figure 4: Effects of rapamycin and dexamethasone on the insulin-mediated increases in GLUT1 and GLUT3 in parental L6 cells. Parental L6 cells were treated with or without 1.5 µM dexamethasone (DEX) for 24 h prior to treatment without (basal (B)) or with 100 nM insulin (I) in the presence or absence of 30 ng/ml rapamycin (R) for 18 h. Total membranes were prepared and subjected to immunoblot analysis using specific anti-GLUT1 (A) or anti-GLUT3 (B) antibodies as described under ``Experimental Procedures.''
To confirm that the inhibition of the
insulin-mediated increase in GLUT3 seen earlier after induction of
Ras(S17N) by dexamethasone (Fig. 2) is not due to an inhibitory
effect of dexamethasone itself, the elevation in GLUT3 in response to
insulin was examined in the presence and absence of dexamethasone in
parental L6 cells. As shown in Fig. 4B, dexamethasone
had no effect on the insulin-mediated increase in GLUT3 (compare lanes2 and 6 of panelB).
Furthermore, rapamycin did not affect the increase in GLUT3 in response
to insulin (lane4 of panelB).
Hence, insulin action on GLUT3 is indeed a p21-dependent
and pp70 S6 kinase-independent phenomenon.
Exposure of L6 muscle cells to insulin for several hours
increases total levels of both GLUT1 and GLUT3 glucose transporters but
has no effect on GLUT4 expression(2, 21) . Here we
examined the roles of p21 and pp70 S6 kinase as mediators
in the pathway(s) by which insulin elevates GLUT1 and GLUT3 expression,
since both p21
and pp70 S6 kinase appear to mediate
communication between the insulin receptor and nuclear events.
Ras(S17N) expressed in L6 muscle cells has reduced affinity for GTP
and inhibits the activity of endogenous p21 by
interfering with Sos needed for Ras activation(25) . Induction
of Ras(S17N) by dexamethasone was also shown to diminish the ability of
insulin to stimulate the tyrosine phosphorylation of MAPK ( (25) and Fig. 3B). Moreover, dexamethasone
itself had no inhibitory effect on the insulin-induced MAPK
phosphorylation in parental L6 (Fig. 3A). Hence, L6
cells expressing Ras(S17N) provide a useful system for studying the
role of p21
in insulin signaling.
In agreement with
the work of others(30) , the use of the glucocorticoid
dexamethasone was associated with an increase in total GLUT1 protein in
parental (Fig. 4A) and in Ras(S17N)-transfected L6
cells (Fig. 1). However, induction of Ras(S17N) by dexamethasone
failed to eliminate the insulin-mediated increase in total GLUT1
protein or mRNA, suggesting that insulin action on GLUT1 gene
expression occurs independently of p21. The failure of
Ras(S17N) to block the insulin-mediated increase in GLUT1 is not a
result of a dysfunctional mutation since in the same cells GLUT3
expression was prevented (Fig. 2) and MAPK phosphorylation was
diminished (Fig. 3B). In another study, microinjection
of dominant inhibitory forms of p21
or neutralizing
antibodies directed against p21
in 3T3-L1 adipocytes
blocked the insulin-induced increase in GLUT1 at the cell
surface(31) . However, that study did not examine if GLUT1
expression was prevented. Therefore, it is possible that the dominant
inhibitory forms of p21
or the neutralizing antibodies
directed against p21
interfered with the machinery
sorting GLUT1, thus preventing it from reaching the cell surface. The
disparity between the results of Hausdorff et al.(31) and those reported here could also be due to the
differences between a stable expression and microinjection or to cell
type-specific regulation of GLUT1. Indeed, dominant inhibitory mutants
of p21
interfere with the insulin-induced activation of
the collagenase promoter in CHO and HeLa cells but not in A14 cells (24) .
Supporting the idea of Ras-independent signaling
pathways for insulin-induced nuclear events, insulin provoked normal
DNA synthesis in CHO cells transfected with mutant mSos protein devoid
of the guanine nucleotide exchange activity (CHO-IR/Sos cells),
yet the insulin-stimulated formation of GTP-bound p21
and
the phosphorylation of MAPK were markedly inhibited(32) .
Moreover, consistent with our results, Kozma et al.(33) observed that the total amount of GLUT1 protein was
similar in parental 3T3-L1 cells and in those expressing an activated
mutant N-Ras
protein. The possible involvement of
p21
in the acute regulation of glucose transport
mediated by rapid translocation of glucose transporters to the cell
surface remains controversial. Microinjection of activating or
inhibitory forms of p21
had no effect on the rapid action
of insulin in 3T3-L1 adipocytes(31) , but introducing
p21
neutralizing antibodies attenuated the rapid effects
of the hormone in cardiac myocytes(34) .
Considering the importance of pp70 S6 kinase in the regulation of protein synthesis(26) , we asked whether insulin increases GLUT1 protein level in L6 muscle cells by activation of this kinase. The drug rapamycin blocks the activation of pp70 S6 kinase(11, 26) , and its effect is selective insofar as it does not block the activation of other kinases such as Raf-1, MAPK, or pp90 S6 kinase(11, 26) . Indeed, inhibition of pp70 S6 kinase by rapamycin blocked the ability of insulin to increase GLUT1 protein in parental cells (Fig. 4A) and in cells transfected with Ras(S17N) (Fig. 1). Moreover, rapamycin also blocked the dexamethasone-induced elevation in GLUT1 protein in both parental and transfected L6 cells. pp70 S6 kinase has been dissociated from playing a role in the acute translocation of GLUT4 to the cell surface by insulin(11) . The data presented herein demonstrate that pp70 S6 kinase might be a mediator of some of the chronic effects of insulin, such as the stimulation of GLUT1 expression but not of GLUT3. It is also likely that the dexamethasone-induced increase in GLUT1 is a pp70 S6 kinase-dependent event.
Information on the
regulation of the GLUT3 gene and its protein product remains scarce
possibly due to the absence of this glucose transporter from adult
human or rodent muscle. In this study we demonstrated for the first
time that chronic treatment with insulin, like insulin-like growth
factor-1, leads to an increase in GLUT3 protein and mRNA in L6 muscle
cells. We also addressed for the first time the mechanism by which the
GLUT3 glucose transporter protein increases in response to prolonged
exposure to insulin. In contrast to GLUT1, the insulin-induced
increases in GLUT3 protein and mRNA were found to be
p21-dependent. The inhibition of insulin action on GLUT3
after induction of Ras(S17N) by dexamethasone was not due to any
inhibitory effects of dexamethasone itself. Dexamethasone did not
affect the insulin-induced increase in GLUT3 in parental L6 (Fig. 4B). In addition, rapamycin did not interfere
with the ability of insulin to stimulate GLUT3 protein expression in
parental (Fig. 4B) or transfected cells (Fig. 2), thus ruling out a role for pp70 S6 kinase in the
mechanism by which GLUT3 glucose transporter protein is induced by
insulin.
In conclusion, signaling for the insulin-dependent
synthesis of GLUT1 and GLUT3 glucose transporters occurs through
different pathways; expression of the former is governed by pp70 S6
kinase whereas expression of the latter is determined via
p21.