(Received for publication, November 28, 1994)
From the
The precise mechanism by which insulin regulates glucose metabolism is not fully understood. However, it is known that insulin activates two enzymes, phosphatidylinositol 3`-kinase (PI 3`-K) and mitogen-activated protein kinase (MAPK), which may be involved in stimulating the metabolic effects of insulin. The role of these enzymes in glucose metabolism was examined by comparing the effects of insulin, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in 3T3-L1 adipocytes. Treatment of the cells with PDGF or EGF for 5 min increased the MAPK activity 3-5-fold, while insulin treatment produced a 2.5-fold increase. The MAPK activity remained elevated for 1 h after either PDGF or insulin treatment. PDGF and insulin, but not EGF, caused a transient increase in the amount PI 3`-K activity coprecipitated with tyrosine phosphorylated proteins. Although PDGF and insulin caused a similar increase in the activities of these two enzymes, only insulin caused substantial increases in glucose utilization. Insulin increased the transport of glucose and the synthesis of lipid 4- and 17-fold, respectively, while PDGF did not affect these processes significantly. Glycogen synthesis was increased 15-fold in response to insulin and only 3-fold in response to PDGF. Thus, the activation of MAPK and PI 3`-K are not sufficient for the complete stimulation of glucose transport, lipid synthesis, or glycogen synthesis by hormones in 3T3-L1 adipocytes, suggesting a requirement for other signaling mechanisms that may be uniquely responsive to insulin.
While the intracellular events that mediate insulin action are
not fully understood, the regulation of both protein and lipid
phosphorylation are thought to play a prominent role(1) . Upon
binding insulin, the insulin receptor undergoes autophosphorylation on
tyrosine residues, resulting in increased kinase activity that leads to
the phosphorylation of other intracellular
proteins(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
One of these phosphorylated proteins, the insulin receptor substrate-1
(IRS-1), ()is critical for the mitogenic effects of insulin
in some cells(12, 13, 14) . IRS-1 itself does
not contain any catalytic activity, but may be one member of a family
of docking proteins for signaling molecules. Tyrosine phosphorylation
of IRS-1 induces its association with several proteins containing Src
homology 2 (SH2) domains, including
SHPTP
(15, 16) , Nck (17) ,
Grb2(18, 19) , and the p85 regulatory subunit of PI
3`-K(20) . The association of this latter protein with IRS-1
confers an increase in the activity of the 110-kDa catalytic subunit of
the enzyme (21, 22, 23) , resulting in the
production of phosphatidylinositol 3`,4`,5`-trisphosphate in
cells(24) . Similarly, PI 3`-K activity is increased by
virtually all tyrosine kinase receptors, although in most cases
activation involves a direct interaction with the phosphorylated
receptor(25) . While the exact role of this enzyme is not fully
understood, its sequence similarity to the Saccharomyces cerevisiae protein VPS34 suggests that it may participate in membrane
trafficking(26) . Compounds of unknown specificity that inhibit
PI 3`-K activity can attenuate the stimulation of glucose transport and
mitogenesis by insulin(27, 28, 29) .
Additionally, the stimulation of thymidine uptake by insulin can be
inhibited by microinjection of the p85 SH2 domain or anti-p85
antibodies(30) . These data suggest that PI 3`-K activity may
be necessary for certain actions of insulin.
The Shc protooncogene
product also appears to be phosphorylated on tyrosine in response to
insulin or growth factor treatment(31) . The phosphorylation of
Shc induces its association with Grb2(32) , which is an adapter
protein containing both SH2 and SH3 domains(33) . Upon its
activation, Grb2 can target the nucleotide exchange factor Sos, leading
to the binding of GTP to
p21(34, 35) . This activation
of p21
is required for the initiation of a
phosphorylation cascade, leading ultimately to the stimulation of
mitogen-activated protein kinase (MAPK)(34, 36) . This
enzyme is one of the major serine/threonine kinases known to be
activated by insulin in tissue culture cells. Dent et al.(37) have suggested that MAPK is an important intermediate
in the insulin-dependent activation of protein phosphatase-1, an enzyme
that catalyzes the dephosphorylation and subsequent activation of
glycogen synthetase and inhibition of phosphorylase kinase. However,
numerous studies have shown that the activation of MAPK does not always
correlate with the metabolic effects of insulin. For example,
pharmacological agents that activate MAPK, such as phorbol esters and
okadaic acid, can antagonize the metabolic effects of
insulin(38) . Similarly, in cells expressing mutant insulin
receptors, MAPK activation by insulin does not correlate with the
metabolic activities of the hormone(39, 40) .
3T3-L1 adipocytes represent one of the most sensitive tissue culture systems in which to elucidate insulin actions. These cells respond to insulin with increased glucose transport and incorporation of glucose into lipid and glycogen. Moreover, these cells also express receptors for other growth factors which have been shown to activate MAPK and PI 3`-K in other systems. We have exploited these cells to explore the role of MAPK and PI 3`-K in the metabolic effects of insulin.
Figure 1:
MAP kinase is activated in response to
insulin, PDGF, or EGF. A, 3T3-L1 adipocytes were either
untreated or exposed to insulin (INS; 100 nM), PDGF (1, 10 or 100 ng/ml), or EGF (100 ng/ml) for 5 min
prior to preparing extracts. The MAP kinase activity after each
treatment was measured as P incorporated into MAP2 in
vitro. The phosphorylated MAP2 was excised from polyacrylamide
gels and counted. The results shown are the mean ± standard
error for three experiments each done in duplicate. B, the
cells were exposed to 100 nM insulin (squares; solidline) or 10 ng/ml PDGF (circles; brokenline) for the indicated periods prior to
preparing extracts and measuring the MAP kinase activity in those
extracts. The results shown are the mean ± standard error for
two experiments each done in duplicate.
To compare the kinetics of MAPK activation by insulin and PDGF, the time courses were evaluated. Cells were incubated with PDGF (10 ng/ml) or insulin (100 nM) for various times, and the MAPK activity was then measured (Fig. 1B). The MAPK activity reached a 3-fold stimulation 5 min after the addition of insulin, and declined thereafter, although after 1 h of exposure to insulin the enzyme was still modestly elevated. PDGF caused a 5-fold stimulation in enzyme activity that peaked after 10 min and was still elevated 1 h after treatment.
Figure 2:
Phosphatidylinositol 3`-kinase activation
in response to insulin, PDGF, or EGF. A, the cells were
treated as in Fig. 1A. The cell extracts were
immunoprecipitated with anti-phosphotyrosine antibodies. The PI 3`-K
activity in anti-phosphotyrosine immunoprecipitates was assayed in
vitro by P incorporated into phosphatidylinositol.
The resulting phosphatidylinositol 3`-phosphate (PIP) was
resolved by thin layer chromatography. The results shown are
representative of four individual experiments. B, the cells
were treated with insulin (INS; 100 nM), EGF (100 ng/ml), or PDGF (10 ng/ml) for 5 min. Cell
extracts were prepared and immunoprecipitated with antisera against the
p85 subunit of PI 3`-K. The precipitated proteins were separated by
SDS-polyacrylamide gel electrophoresis and visualized by immunoblotting
with anti-phosphotyrosine antibodies. C, the cells were
treated with 100 nM insulin or 10 ng/ml PDGF for the indicated
times prior harvesting the cells and measuring the PI 3`-K activity.
The results shown are representative of three individual
experiments.
Since the phosphoproteins responsible for PI 3`-K activation are different for PDGF and insulin, the time course of activation after addition of the ligands was compared. Both hormones produced a marked increase in activity after only a 5-min exposure, which began to decline by 30 min. After exposure of cells to insulin for 60 min, a fraction of PI 3`-K activity remained associated with anti-phosphotyrosine immunoprecipitates, although PDGF-dependent activity returned to basal (Fig. 2C).
Figure 3:
Stimulation glucose transport, lipid
synthesis or glycogen synthesis in response to insulin or PDGF. A, glucose transport. After a 15-min incubation with
insulin (INS; 100 nM) or PDGF (1, 10, or 100
ng/ml), [C]2-deoxyglucose was added and the
incubation continued an additional 15 min. After extensive washing, the
[
C]2-deoxyglucose that had been internalized by
the cells was determined by scintillation counting. The results are
shown as the mean ± standard error for two experiments each done
in triplicate. B, lipid synthesis. The cells were
treated as in A and incubated with
[
C]glucose for 1 h. After harvesting the cells,
the lipids were extracted and the [
C]glucose
converted to lipid determined by scintillation counting. The results
are the mean ± standard error for two experiments each done in
triplicate. C, glycogen synthesis. The cells were
treated with insulin (INS; 100 nM) or PDGF (100 ng/ml) for 15 min. [
C]Glucose was
added and the incubation continued for an additional hour. Cell lysates
were prepared, and the
C incorporated into glycogen was
determined by scintillation counting. The results are shown as the mean
± standard error for two experiments each done in triplicate. *, p
0.05;**, p
0.025;***, p
0.005.
Insulin can also increase the rate of lipid
synthesis in 3T3-L1 adipocytes. The cells were incubated with either
PDGF or insulin 15 min prior to the addition of
[C-(U)]glucose (5 mM), conditions under
which lipid synthesis is not rate-limited by glucose uptake. After a
1-h exposure, the lipid synthesis from glucose was determined by
scintillation counting. As observed for 2-deoxyglucose transport, PDGF
caused only a 1.5-fold increase in lipogenesis, whereas insulin
produced a 17-fold increase (Fig. 3B).
Insulin also
increases the conversion of glucose into glycogen in the 3T3-L1
adipocytes. Cells were treated with either PDGF or insulin for 15 min
prior to the addition of [C-(U)]glucose.
Following a 60-min incubation, glycogen synthesis was determined by
scintillation counting. Insulin produced a 15-fold increase in glycogen
synthesis. In contrast, glycogen synthesis was increased only 3-fold in
response to PDGF treatment (Fig. 3C).
Although the molecular mechanisms by which insulin regulates
glucose metabolism are not fully understood, changes in protein and
lipid phosphorylation are likely to be involved. While the tyrosine
kinase activity of the insulin receptor initiates the intracellular
signaling cascades responsible for the action of the hormone, the
regulation of key events downstream from the receptor are generally
controlled by changes in serine or threonine phosphorylation. Although
the precise phosphorylation events that regulate these pathways remain
elusive, insulin can stimulate phosphorylation of some proteins and the
dephosphorylation of others(1) . The many cellular responses to
insulin can be easily distinguished by virtue of the dose, the length
of exposure, and the cell type examined(46, 47) ,
suggesting that insulin activates several signaling pathways. It is
likely that the divergence of insulin signaling may occur at the
receptor itself, since differences in insulin action have been observed
in cells expressing mutant receptors. For example, mutation of the two
C-terminal tyrosines of the receptor results in a phenotype, which is
normal with respect to glucose uptake and glycogen
synthesis(48) , but increased in its sensitivity with respect
to mitogenesis(49) , S6 kinase(48) , and MAPK
activation(40) . Also, overexpression of a mutant receptor with
serine substituted at Trp confers normal glucose
transport and glycogen synthesis, but deficient autophosphorylation,
DNA synthesis (50) , p21
(51) , and MAPK
activation(39) .
The ability of insulin to activate both
distinct signaling pathways and diverse metabolic responses in a single
cell has complicated the identification of relevant transduction
mechanisms. Much attention has been focussed on two enzymes, MAPK and
PI 3`-K, which are activated by hormone treatment and lie in distinct
signaling pathways. Dent et al.(37) have suggested
that the insulin-dependent activation of pp90, via
phosphorylation by MAPK, leads to the phosphorylation of site-1 on the
regulatory G subunit of glycogen-associated protein phosphatase-1. This
results in both the dephosphorylation and stimulation of glycogen
synthetase and the inhibition of phosphorylase kinase. Although MAPK is
activated in response to insulin, it is not generally correlated with
the metabolic responsiveness to the hormone in tissue culture models.
Several recent reports have demonstrated that EGF, serum, or thrombin
treatments can activate MAPK in the absence of increased glucose
transport and glycogen
synthesis(52, 53, 54, 55) . The
studies presented here demonstrate that the profound activation of MAPK
by PDGF in a highly responsive cell line does not result in significant
stimulation of glucose transport, lipid synthesis, or glycogen
synthesis, indicating that MAPK activation is not sufficient for
stimulating glucose metabolism. Additional studies have suggested that
MAPK may not be necessary for the regulation of glucose utilization by
insulin. Insulin fails to activate MAPK in PC-12 cells, but
nevertheless stimulates the synthesis of glycogen, lipid and
protein(56) . In addition, a low molecular weight inhibitor
that specifically blocks the stimulation of MAPK by inhibiting its
upstream activator, MEK (MAP kinase kinase), has no effect on the
metabolic actions of insulin in 3T3-L1 cells. (
)
Although MAPK appears to play a limited role in the metabolic actions of insulin, there is evidence that the enzyme PI 3`-K may be important. This enzyme is activated upon the association of its regulatory p85 subunit with insulin receptor substrates such as IRS-1 (21, 22, 23) or pp60 (57) or the insulin receptor itself(24) . PI 3`-K inhibitors, such as wortmannin or LY294002, can block the stimulation of glucose transport and lipogenesis by insulin, suggesting that PI3`-kinase may be necessary for these actions of the hormone(27, 28, 29) . Interestingly, PDGF is also a potent activator of the enzyme in 3T3-L1 adipocytes, presumably through the direct interaction of p85 with the PDGF receptor. However, glucose transport, lipogenesis, and glycogen synthesis remain largely unaffected by exposure to PDGF, suggesting that increased association of PI 3`-K with tyrosine-phosphorylated proteins alone is not sufficient for the stimulation of glucose metabolism. The possibility remains that the subcellular redistribution of PI 3`-K activity in response to PDGF is different than that produced by insulin. Recent reports suggest that in rat adipocytes PI 3`-K activity is localized to low density microsomes in response to insulin (58) . However, whether PDGF causes a similar redistribution is not yet known. Thus, compartmentalization of the enzyme may be the key determinant in signal generation. On the other hand, it is possible that while PI 3`-K activity is involved in membrane vesicle trafficking necessary for metabolic regulation, other intracellular biochemical events that are unique to insulin may provide the important signals regulating glucose metabolism. Moreover, it is likely that the regulation of protein serine/threonine dephosphorylation, which is generally not observed with other growth factors, will provide the critical clue toward understanding metabolic signaling for insulin.