(Received for publication, July 10, 1995)
From the
Insulin causes the activation of phosphatidylinositol 3-kinase
(PI 3-kinase) through complexation of tyrosine-phosphorylated
YMXM motifs on insulin receptor substrate 1 with the Src
homology 2 domains of PI 3-kinase. Previous studies with inhibitors
have indicated that activation of PI 3-kinase is necessary for the
stimulation of glucose transport in adipocytes. Here, we investigate
whether this activation is sufficient for this effect. Short peptides
containing two tyrosine-phosphorylated or thiophosphorylated
YMXM motifs potently activated PI 3-kinase in the cytosol from
3T3-L1 adipocytes. Introduction of the phosphatase-resistant
thiophosphorylated peptide into 3T3-L1 adipocytes through
permeabilization with Staphylococcus aureus -toxin
stimulated PI 3-kinase as strongly as insulin. However, under the same
conditions the peptide increased glucose transport into the
permeabilized cells only 20% as well as insulin. Determination of the
distribution of the glucose transporter isotype GLUT4 by confocal
immunofluorescence showed that GLUT4 translocation to the plasma
membrane can account for the effect of the peptide. These results
suggest that one or more other insulin-triggered signaling pathways,
besides the PI 3-kinase one, participate in the stimulation of glucose
transport.
A major metabolic effect of insulin is the stimulation of glucose transport into muscle and adipose cells. The immediate basis of this effect is the insulin-induced increase in the amount of the glucose transporter isotype GLUT4 in the plasma membrane of these cells(1) . This increase results from an enhanced rate of trafficking of GLUT4 to the plasma membrane and possibly also some slowing of the rate of endocytosis of GLUT4 from the plasma membrane(2) . The trafficking of GLUT4 to the cell surface may proceed by the docking and fusion of specialized secretory vesicles enriched in GLUT4(1, 2, 3) .
An important
unsolved issue is the identity of the signaling pathway(s) from the
insulin receptor that triggers this translocation of GLUT4 to the
plasma membrane. The insulin receptor is a tyrosine kinase. Binding of
insulin to the extracellular domain activates the kinase function in
the intracellular domain, and the receptor both autophosphorylates and
phosphorylates substrate proteins. Among the latter is insulin receptor
substrate 1 (IRS-1). ()IRS-1 is phosphorylated on multiple
tyrosine residues, and through these, binds and so regulates at least
three Src homology 2 (SH2) domain-containing
proteins(4, 5) . Phosphatidylinositol 3-kinase (PI
3-kinase) is one of the proteins that associates with IRS-1. It
consists of an 85-kDa regulatory subunit with two SH2 domains and a
110-kDa catalytic subunit. The binding of two tyrosine-phosphorylated
YMXM motifs present in IRS-1 to the two SH2 domains markedly
stimulates PI 3-kinase activity(6, 7) . This signal
transduction pathway from the insulin receptor to PI 3-kinase thus
accounts for the rapid elevation of PI 3,4-bisphosphate and
3,4,5-trisphosphate seen in vivo in response to
insulin(8, 9) . The downstream components of the
pathway have not yet been elucidated. The finding that PI
3,4,5-trisphosphate stimulates some isotypes of protein kinase C in
vitro suggests a role for these kinases(10) .
Several lines of evidence have indicated that the activation of PI 3-kinase by insulin is required for GLUT4 translocation to the plasma membrane. First, wortmannin, a potent inhibitor of the enzyme, blocks insulin stimulation of glucose transport in rat and 3T3-L1 adipocytes and in muscle tissue(11, 12, 13) . Second, the compound LY294002, another inhibitor that is structurally unrelated to wortmannin, has also been found to prevent the stimulation of transport in 3T3-L1 adipocytes(9) . In each study, except for the one with muscle, it was directly shown that GLUT4 translocation was blocked. Lastly, the microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase into 3T3-L1 adipocytes prevented GLUT4 translocation in response to insulin (14) . The mutant form of this subunit is one that is unable to bind to the catalytic subunit of PI 3-kinase but can still bind to IRS-1, since it retains both SH2 domains.
Although these studies indicate that activation of PI 3-kinase is required for insulin stimulation of glucose transport via GLUT4 translocation, they do not address the question of whether it is sufficient. The present study examines this issue. Previously, we have shown that simple peptides containing two tyrosine-phosphorylated YMXM motifs bind to and activate PI 3-kinase as well as IRS-1 itself does(7) . Here, these peptides have been used to activate PI 3-kinase selectively in permeabilized 3T3-L1 adipocytes, and the effect on glucose transport has been determined.
Figure 1:
Binding of
peptides to the SH2 domains of PI 3-kinase. The relative affinities of
the peptides for binding to the glutathione S-transferase
fusion protein containing both SH2 domains of PI 3-kinase were measured
in the Biacore assay described under ``Experimental
Procedures.'' Results are expressed as percent of the binding of
the PI 3-kinase SH2 domain protein to the immobilized peptide in the
absence of competing peptide. ,
YZPZSGS
YZPZS;
,
YZPZSGS
YZPZS;
,
YZPZSGSYZPZS.
Figure 2:
Stimulation of PI 3-kinase activity by
Tyr(P) and Tyr(S) peptides. The cytosolic fraction from basal 3T3-L1
adipocytes was incubated with peptide in 100 µl at room temperature
for 15 min and then assayed for PI 3-kinase by the addition of a
mixture of [P]ATP and PI (60 µl).
,
YZPZSGS
YZPZS;
,
YZPZSGS
YZPZS. The concentrations given are the
ones in the initial 100-µl incubation mixture. The values are the
means ± S.D. for triplicate measurements in a representative
experiment of three that gave similar results. The activity in the
cytosol prepared at the same time from 3T3-L1 adipocytes treated with
100 nM insulin for 3 min and in cytosol to which 200
µM GTP
S was added are also
shown.
Previous studies have found
that glucose transport can be measured in adipocytes permeabilized by
electric discharge (25) or with the detergent streptolysin
O(12) , because the rate of hexose uptake catalyzed by the
glucose transporter is much greater than the rate of either its uptake
or of the loss of the 2-deoxyglucose 6-phosphate product through the
pores. This also proved to be the case in the 3T3-L1 adipocytes
permeabilized with -toxin. Cytochalasin B, a specific inhibitor of
glucose transport(18) , at 25 µM, reduced the
2-deoxyglucose uptake into permeabilized cells in the basal and
insulin-treated states to 24 and 9% of the uninhibited rates,
respectively, such that residual 2-deoxyglucose uptake was the same for
the cells in the two states.
The effects of insulin, GTPS, and
the Tyr(S) peptide on the uptake of 2-deoxyglucose by the
-toxin
permeabilized 3T3-L1 adipocytes are shown in Fig. 3. Both
insulin and GTP
S stimulated transport approximately 3-fold. These
results are consistent with the previous finding that insulin
stimulation of glucose transport is preserved in streptolysin
O-permeabilized 3T3-L1 adipocytes and that GTP
S also enhances
transport in these permeabilized cells(12) . Earlier studies
have also shown that both insulin and GTP
S elicit the
translocation of GLUT4 to the plasma membrane in
-toxin
permeabilized rat adipocytes (22) and streptolysin
O-permeabilized 3T3-L1 adipocytes(26) . The stimulation of
transport by GTP
S that we observed provides further evidence that
the 3T3-L1 adipocytes were permeabilized by
-toxin, since
GTP
S did not enhance transport in the absence of
-toxin
treatment.
Figure 3:
Glucose transport in permeabilized
adipocytes. 3T3-L1 adipocytes were permeabilized with -toxin and
then left untreated (basal) or exposed to 100 nM insulin, 200
µM GTP
S, or peptide for 15 min, followed by the assay
of 2-deoxyglucose (dGlc) uptake for 5 min as described under
``Experimental Procedures.'' In the case of treatment with
the peptide plus insulin, the peptide was added 5 min before insulin,
and exposure to insulin was for 10 min before the transport assay. Panel A, 10 µM
YZPZSGS
YZPZS; panel B, 10 and 100
µM YZPZSGSYZPZS. The values are the means ± the
range for measurements on uptake by two 35-mm wells of cells in a
representative experiment. Similar results were obtained with the
thiophosphorylated peptide in four other separate experiments of this
type. *, p
0.05.
The Tyr(S) peptide, at 10 µM, consistently caused a small (approximately 1.5-fold) but statistically significant increase in glucose transport, whereas the nonphosphorylated peptide had no effect (Fig. 3, panels A and B, respectively). In other experiments where the Tyr(S) peptide was tested at higher concentrations (30 and 100 µM), the stimulation of transport was no greater; also, lower concentrations of the peptide (1 and 0.1 µM) did not enhance the transport significantly. Moreover, the combination of the Tyr(S) peptide (10 µM) and insulin (100 nM) gave the same rate of transport as insulin alone (panel A). Thus, the peptide did not inhibit the effect of insulin.
In one experiment of an analog of the product of the PI 3-kinase, reaction was examined for its effect on glucose transport. Permeabilized cells were treated with a water-soluble PI 3,4,5-trisphosphate (the dioctanoyl compound(10) ; kindly provided by Drs. J. R. Falck and L. C. Cantley) at 20 µM. No stimulation of glucose transport occurred (data not shown).
Figure 4:
PI 3-kinase activity in the cytosol from
permeabilized 3T3-L1 adipocytes. Cells were permeabilized with
-toxin and treated with no addition (basal), 100 µM insulin, 10 µM
YZPZSGS
YZPZS,
or 200 µM GTP
S for 15 min, and then the cytosolic
fraction was prepared and assayed for PI 3-kinase activity as described
under ``Experimental Procedures.'' Cells exposed to Tyr(S)
peptide in the absence of toxin (intact cells) were also assayed. The
values are the means ± S.D. for triplicate measurements in a
representative experiment. Two other separate experiments gave similar
results.
Figure 5:
GLUT4 distribution in permeabilized 3T3-L1
adipocytes by immunofluorescence. 3T3-L1 adipocytes on coverslips were
permeabilized with -toxin, treated with various agents, and fixed
with formaldehyde; GLUT4 was then detected by confocal
immunofluorescence, as described under ``Experimental
Procedures.'' Shown are representative images of basal cells (A) and cells treated with 100 nM insulin (B), 200 µM GTP
S (C), and 10
µM
YZPZSGS
YZPZS (D). The large clear circles within the cells are fat droplets. Similar
results were obtained in four repetitions of this entire
experiment.
The main finding of this study is that activation of PI 3-kinase is not sufficient to stimulate glucose transport in 3T3-L1 adipocytes to the same extent as insulin. This finding suggests that one or more additional signaling pathways are involved. While this study was in progress, Wiese et al.(28) reached the same conclusion by a different approach. These authors found that treatment of 3T3-L1 adipocytes with PDGF caused no significant stimulation of glucose transport, even though it led to an increase in PI 3-kinase activity similar to that elicited by insulin. Despite the fact that these two lines of investigation point to the same conclusion, it should be noted that alternative explanations for the results remain possible. For example, the stimulation of glucose transport may require that PI 3-kinase be activated at a specific subcellular location, and insulin may, to some extent, activate PI 3-kinase in different subcellular locations than do the Tyr(S) peptide and PDGF. Also, PDGF activates some signaling pathways not activated by insulin(4, 5, 29) , and one of these may have inhibited the stimulation of glucose transport.
Our observations that the PI 3-kinase-activating peptides did increase glucose transport to a modest extent and did cause redistribution of GLUT4 to the cell surface, as assessed by immunofluorescence, are consistent with the conclusion, derived from the use of inhibitors (see the Introduction), that activation of PI 3-kinase is involved in GLUT4 translocation and transport stimulation. There are, however, complicating factors that should be considered in the interpretation of these observations. With regard to the stimulation of transport, it is possible that translocation of the transporter isotype GLUT1 contributes to part or all of this effect. 3T3-L1 adipocytes contain considerable intracellular GLUT1, part of which is in vesicles with GLUT4 and part of which is in separate vesicles, and a portion of this isotype also translocates to the cell surface in response to insulin (18, 30) . Studies with inhibitors indicate that the activation of PI 3-kinase is also necessary for the smaller increases in cell surface GLUT1 and its associated transport activity caused by insulin(12, 31) . Moreover, we have previously shown that microinjection of the Tyr(P) peptide into Xenopus oocytes stimulates glucose transport to the same extent as does insulin-like growth factor I(32) . The immediate basis of this effect on transport in oocytes has not been determined but may be GLUT1 translocation to the plasma membrane.
In regard to the redistribution of GLUT4 elicited by the Tyr(S) peptide, the qualitative nature of the immunofluorescence methodology precludes a definitive interpretation. It is possible that modest translocation of GLUT4 to the plasma membrane, consistent with the observed increase in transport (about 20% of the insulin effect), appears as a distinct increase in staining at the cell border. Alternatively, the peptide may cause GLUT4 vesicles to migrate to the plasma membrane as effectively as insulin does but not trigger their fusion with the membrane. This situation would probably not be distinguishable from completed translocation by immunofluorescence. Another possibility is that the peptide causes an increase in GLUT4 in the plasma membrane as large as insulin does but that an additional process is required for stimulation of transport. The determination of GLUT4 subcellular distribution by other methods, such as quantitative immunoelectron microscopy(27) , will be required to decide which of these possibilities is in fact occurring.
Although our data suggest that a signaling pathway, in addition to the activation of PI 3-kinase, is necessary for the stimulation of GLUT4 translocation and glucose transport, there is at present evidence against any of the other known signaling pathways from the insulin receptor being required for this effect (reviewed in (33) ). Most relevant here are studies in which specific inhibitors of signaling pathways were used, since if two signaling mechanisms are necessary, specific inhibition of only one will block the insulin stimulation of transport, whereas specific activation of only one will not elicit it. Insulin treatment of 3T3-L1 adipocytes also causes the stimulation of the 70-kDa ribosomal S6 kinase, the activation of the SH2 domain-containing tyrosine phosphatase PTP2C, the elevation of the GTP form of Ras, and the activation of the mitogen-activated protein (MAP) kinase cascade. Specific inhibition of the activation of the 70-kDa S6 kinase by rapamycin does not block insulin-stimulated GLUT4 translocation or glucose transport(34) . Similarly, inhibition of the activation of PTP2C through the microinjection of antibodies or isolated SH2 domains does not prevent insulin-stimulated GLUT4 translocation(35) . In the case of Ras, inhibition of GTP loading by microinjection of a dominant negative mutant or a neutralizing antibody or by prevention of Ras isoprenilation does not impair GLUT4 translocation or the increase in glucose transport(36, 37) . Although no complete reports have yet appeared regarding specific inhibitors of the MAP kinase cascade, a recent abstract presents data showing that a specific inhibitor of MAP kinase kinase does not inhibit insulin stimulation of glucose transport(38) . Moreover, neither PDGF nor epidermal growth factor, both of which activate the MAP kinase cascade in 3T3-L1 adipocytes, increases glucose transport significantly(28, 39, 40) . Taken together, these results thus imply that there may be an as yet undiscovered signaling pathway specific to insulin that is necessary for GLUT4 translocation and transport stimulation.