(Received for publication, August 1, 1995; and in revised form, September 25, 1995)
From the
Insulin stimulates glucose transport largely by mediating
translocation of the insulin-sensitive glucose transporter (GLUT4) from
an intracellular compartment to the plasma membrane. Using single cell
microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence
detection of GLUT4 proteins, we have determined that inhibition of
endogenous p21 or injection of oncogenic
p21
has no effect on insulin-stimulated GLUT4
translocation. On the other hand, microinjection of
anti-phosphotyrosine antibodies or inhibition of endogenous
phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion
protein markedly inhibits this biologic effect of insulin. These data
suggest that the p21
/mitogen-activated protein
kinase pathway is not involved in this metabolic effect of insulin,
whereas tyrosine phosphorylation and stimulation of
phosphatidylinositol 3-kinase activity are critical components of this
signaling pathway.
Insulin exerts pleiotropic biologic effects. One of insulin's major physiologic effects is the regulation of plasma glucose levels, which is accomplished by suppression of hepatic glucose production and stimulation of glucose uptake into target tissues(1) . The latter effect is primarily due to translocation of insulin-sensitive glucose transporters (GLUT4) from an intracellular vesicular pool to the plasma membrane, where they can lead to glucose uptake(2, 3) . This response is mediated by a metabolic signaling pathway that is divergent from the mitogenic pathway (4) and may, at least in part, utilize distinct signaling molecules. Elucidation of this signaling pathway will yield information rich with implications for a number of human disease states, such as non-insulin-dependent diabetes mellitus, obesity, and polycystic ovarian syndrome. All of these conditions are associated with insulin resistance and feature decreased insulin-stimulated glucose transport as a major manifestation(5) . Thus, the causes of insulin resistance in these pathophysiologic states likely involve defects in the metabolic signaling pathway by which insulin causes an increase in cellular glucose uptake. We have utilized single cell microinjection of 3T3-L1 adipocytes and immunofluorescence microscopy of GLUT4 proteins to assess the transmembrane signaling pathway leading to insulin-stimulated GLUT4 translocation.
Individual, living, differentiated 3T3-L1 adipocytes were microinjected with various reagents, followed by insulin stimulation. Translocation of GLUT4 to the cell surface was then visualized by immunofluorescence microscopy using anti-GLUT4 antibody followed by fluorescein-conjugated second antibody. Fig. 1, A and B, shows a field containing 3T3-L1 adipocytes before (A) and after (B) insulin stimulation. In the basal, unstimulated state, GLUT4 staining is almost exclusively relegated to the intracellular compartment, with typical diffuse punctate and discrete perinuclear staining. After insulin stimulation, a marked difference in the staining pattern occurs, with prominent intense staining uniformly distributed at the cell surface, accompanied by decreased intracellular GLUT4 staining, indicative of translocation of GLUT4 proteins from the intracellular vesicular pool to the plasma membrane. In the basal state, less than 10% of cells display surface GLUT4 staining, whereas after insulin treatment, 60-80% of cells are positive for cell surface GLUT4 localization. Using this technique, translocation occurs with a time course (C) and dose-response curve (D) typical for insulin stimulation of glucose transport, indicating that this visual assay of GLUT4 translocation is representative of the physiologic insulin-stimulated process.
Figure 1: Visualization of insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated without (A) or with (B) 10 ng/ml insulin for 20 min and stained with anti-GLUT4 antibody (1F8) followed by incubation with fluorescein-conjugated anti-mouse IgG antibody. Cells positive for GLUT4 translocation show an increase in plasma membrane-associated fluorescein staining that is visualized as a ring around the cell. C, time course of GLUT4 translocation. 3T3-L1 adipocytes on coverslips were stimulated with 10 ng/ml insulin for various times and stained for GLUT4. D, dose response of GLUT4 translocation. 3T3-L1 adipocytes on coverslips were stimulated with the indicated concentrations of insulin for 20 min and stained for GLUT4. The percentage of cells positive for GLUT4 translocation was calculated by counting at least 100 cells at each point. Mean of two experiments is shown.
We
then used this system coupled with microinjection studies in an attempt
to elucidate the signaling cascade that mediates insulin's effect
on GLUT4 translocation. After insulin binds to its receptor, a series
of events is initiated, including receptor autophosphorylation and
tyrosine phosphorylation of endogenous substrates such as IRS-1 and
Shc(12, 13, 14, 15, 16) .
Through its multiple tyrosine phosphorylation sites, IRS-1 can serve as
a docking protein forming complexes with SH2 domain-containing proteins
such as PI3-kinase(17) . Phospho-Shc forms complexes with
Grb2SOS leading to activation of p21
, which
subsequently stimulates the MAP kinase pathway(18) . While a
great deal is known about insulin's mitogenic actions, relatively
little is known about the signaling processes that mediate GLUT4
translocation. For example, numerous studies have shown that formation
of p21
-GTP and stimulation of the MAP kinase pathway are
essential steps in insulin's mitogenic signaling
pathway(17) . However, whether these molecules are involved in
insulin's metabolic actions remains unclear. For example, it has
been demonstrated that the metabolic and mitogenic signaling pathways
diverge(4) , and it is possible that the point of divergence is
proximal to formation of p21
-GTP. Several studies, using
different experimental approaches, have found results against a role
for p21
in the stimulation of glucose
transport(19, 20, 21, 22, 23) ,
whereas other studies have found evidence in
favor(24, 25) .
Our current technique allows an
opportunity to directly test the importance of intracellular molecules
in a given signaling pathway. Thus, we microinjected 3T3-L1 adipocytes
with a series of Ras-related reagents. As seen in Fig. 2,
microinjection of dominant inhibitory N17 Ras (26) had no
effect on basal or insulin-stimulated GLUT4 localization at maximal (10
ng/ml) insulin concentrations. Identical microinjections of N17 Ras
protein were carried out in cells that were subsequently stimulated
with several submaximal concentrations of insulin. When GLUT4
translocation was quantitated in these injected cells, a dose-response
curve identical to that obtained with uninjected cells (Fig. 1D) was observed (data not shown), indicating
that N17 Ras protein does not shift this dose response. Thus, within
the detection limits of the immunofluorescence assay, it does not
appear that inhibition of endogenous Ras activity affects the extent to
which GLUT4 is translocated or the insulin responsiveness of the
translocation event. A similar lack of effect was observed upon
microinjection of anti-p21 antibody. In contrast, both of
these Ras inhibitory reagents prevented insulin-stimulated DNA
synthesis when they were concurrently injected into Rat 1 fibroblasts
(HIRc cells) overexpressing human insulin receptors. The percentages of
cells positive for BrdUrd staining were 76, 22, 21, and 28% following
injections of control IgG, N17 Ras, anti-p21
antibody,
and anti-Shc antibody, respectively. Oncogenic T24 Ras (27, 28) is a constitutively active protein that will
stimulate downstream effects of p21
-GTP. T24 Ras protein
was microinjected into 3T3-L1 adipocytes, and its effects upon c-Fos
protein expression and translocation of GLUT4 were simultaneously
assessed. As demonstrated in Fig. 3, the T24 Ras protein was
biologically active within the cells and caused a marked induction of
c-Fos protein as visualized by nuclear rhodamine staining with
anti-c-Fos antibody. In contrast, T24 Ras failed to influence GLUT4
localization in the presence or absence of insulin ( Fig. 2and Fig. 3, D and H). Furthermore, concurrent
injections of this preparation of T24 Ras stimulated DNA synthesis in
quiescent HIRc cells to the same extent as insulin. The percentages of
BrdUrd-positive cells following control IgG and T24 Ras injections,
without stimulation by insulin, were 19 and 72%, respectively. Thus,
the signaling pathway leading to c-Fos expression was activated by the
microinjected T24 Ras, while stimulation of this pathway did not
influence GLUT4 translocation. In addition, we have found that
microinjection of anti-Shc antibody (Fig. 2) as well as a Shc
SH2-GST fusion protein (data not shown) did not inhibit
insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, whereas
anti-Shc antibody inhibited insulin-stimulated DNA synthesis by greater
than 80% in HIRc cells, and the Shc SH2-GST protein inhibited epidermal
growth factor-stimulated DNA synthesis. Taken together, these results
argue that activation of p21
is neither necessary nor
sufficient for GLUT4 translocation and indicate that the metabolic
pathway mediating this biologic effect diverges from the mitogenic
signaling pathway proximal to activated p21
.
Figure 2:
Effect of microinjection of N17 Ras,
anti-p21 antibody, T24 Ras, and anti-Shc
antibody on GLUT4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes
on coverslips were microinjected with either control sheep IgG (10
mg/ml), dominant negative mutant p21
protein
(N17) (2.0 mg/ml), monoclonal anti-p21
antibody
(Y13-259) (5 mg/ml), oncogenic p21
(T24
Ras) (2.0 mg/ml), or anti-Shc antibody (5 mg/ml). One hour after
microinjection, cells were stimulated in the absence (open
bars) or presence (hatched bars) of 10 ng/ml insulin for
20 min. The cells were fixed, permeabilized, and stained for GLUT4 and
injected IgG. The percentage of cells positive for GLUT4 translocation
was determined in each experiment by analyzing at least 100 cells
positive for injected IgG. Error bars represent S.E. for three
experiments.
Figure 3:
Microinjection of oncogenic T24 Ras
induces c-Fos protein expression but not GLUT4 translocation. 3T3-L1
adipocytes on coverslips were microinjected with either control sheep
IgG (10 mg/ml) (A-D) or oncogenic p21 (T24 Ras) (2 mg/ml) combined with sheep IgG (10 mg/ml) (E-H). The cells were stained for GLUT4 and injected IgG
followed by staining for c-Fos. A and E, phase
contrast. B and F, IgG staining, demonstrating
injected cells. C and G, c-Fos staining. D and H, GLUT4 staining. Arrows indicate injected
cells. 70% of T24 Ras-injected cells demonstrated c-Fos nuclear
staining, while none of the control IgG injected or uninjected cells
showed this staining pattern.
There is evidence to suggest that PI3-kinase may be an intermediary molecule facilitating insulin-stimulated GLUT4 translocation(29, 30, 31, 32) . When cells are treated with the PI3-kinase inhibitor Wortmannin, insulin stimulation of glucose transport is inhibited(31, 32) . Although this is suggestive of a role for PI3-kinase in this effect of insulin, the specificity of Wortmannin as an inhibitor has recently been questioned(33) . To directly assess the relevance of PI3-kinase in the metabolic effects of insulin, we utilized a GST fusion protein comprising the N-terminal SH2 domain of the p85 subunit of PI3-kinase. In previous studies, we have shown that microinjection of this SH2-GST fusion protein into HIRc cells completely inhibited insulin, insulin-like growth factor-I, and epidermal growth factor-induced DNA synthesis(9) . As seen in Fig. 4, microinjection of the p85 SH2-GST protein inhibited insulin-stimulated GLUT4 translocation by 75%. The specificity of this effect is verified by the fact that a GST fusion protein containing the Shc SH2 domain was without effect on GLUT4 translocation (data not shown). In support of this, we also found that preincubation of cells with Wortmannin (1 µM) completely inhibited GLUT4 translocation (data not shown).
Figure 4: Inhibition of GLUT4 translocation by microinjection of GST-p85 SH2 fusion protein or anti-phosphotyrosine antibody. Either control sheep IgG (10 mg/ml), a GST fusion protein containing the N-terminal SH2 domain of p85 (12 mg/ml) combined with sheep IgG (10 mg/ml), or a monoclonal anti-phosphotyrosine antibody (pY 20) (5 mg/ml) were microinjected into 3T3-L1 adipocytes. One hour after injection, the cells were stimulated without (open bars) or with (hatched bars) 10 ng/ml insulin for 20 min and then stained for GLUT4.
Insulin leads to autophosphorylation of the insulin receptor with tyrosine phosphorylation of IRS-1, which then binds to the SH2 domains of PI3-kinase leading to activation of this enzyme(17) . To evaluate the role of tyrosine phosphorylation in this pathway, we also microinjected anti-phosphotyrosine antibodies into 3T3-L1 cells. The monoclonal anti-phosphotyrosine antibody markedly inhibited GLUT4 translocation indicating the necessity for tyrosine phosphorylation as well as PI3-kinase activity in GLUT4 translocation (Fig. 4).
Previous
studies examining whether molecules in the p21/MAP kinase
pathway are involved in insulin stimulation of glucose transport have
yielded conflicting
results(19, 20, 21, 22, 23, 24, 25) .
Some of these studies have not specifically measured GLUT4
translocation or have utilized cell lines that do not express GLUT4.
Others have used transfection to generate cell lines overexpressing
Ras-related molecules. With this latter approach, the possibility
always exists that the selected cell lines will have adapted to the
expressed transgene in such a way as to make the results
non-representative of the physiologic cell context. Single cell
microinjection avoids many of these problems, since our studies can be
done in relevant insulin target cells (adipocytes), with direct
assessment of the biologic event in question (GLUT4 translocation).
Furthermore, the acute introduction of test molecules into the cell
interior followed by rapid assay of insulin action does not allow cells
enough time to undergo adaptive changes to the perturbation.
Single living cell microinjection has been a powerful technique that has proven useful in identifying components of the signaling pathway for growth factor-mediated mitogenesis(8, 9, 11, 34, 35, 36, 37) . The current system provides the means to identify the intracellular molecular components that transduce insulin's major metabolic effect, i.e. stimulation of GLUT4 translocation. In the current report our data have shown that the Ras pathway, which is a critical component of mitogenic signaling, is unnecessary for insulin stimulation of GLUT4 translocation. As such, this provides clear evidence for the divergence of metabolic and mitogenic signaling events. We also find that PI3-kinase is a necessary molecule coupling the tyrosine phosphorylation signal generated by the insulin receptor to glucose transport stimulation. Although our studies do not elucidate the mechanism whereby PI3-kinase facilitates GLUT4 translocation, our results are consistent with recent studies (38) showing that following insulin stimulation, PI3-kinase can be localized to the intracellular GLUT4-containing vesicles, consistent with a role for this enzyme in trafficking of GLUT4 to the cell surface.
Using the approaches contained in this report, we anticipate that further molecules in this key signaling pathway will be identified in a timely manner. As the details of this signaling pathway unfold, new insights into the mechanisms of insulin-resistant glucose transport stimulation in human disease states are likely to emerge.