(Received for publication, October 18, 1994)
From the
Both the anabolic hormone insulin and contractile activity stimulate the uptake of glucose into mammalian skeletal muscle. In this study, we examined the role of phosphatidylinositol 3-kinase (PI 3-kinase), a putative mediator of insulin actions, in the stimulation of hexose uptake in response to hormone and contraction. Phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate accumulate in skeletal muscle exposed to insulin but not hypoxia, which mimics stimulation of the contractile-dependent pathway of hexose transport activation. The fungal metabolite wortmannin, an inhibitor of PI 3-kinase, completely blocks the appearance of 3`-phospholipids in response to insulin. Moreover, wortmannin entirely prevented the increase in hexose uptake in muscle exposed to insulin but was without effect on muscle stimulated by repetitive contraction or hypoxia. These results support the view that PI 3-kinase is involved in the signaling pathways mediating insulin-responsive glucose transport in skeletal muscle but is not required for stimulation by hypoxia or contraction. Furthermore, these data indicate that there exist at least two signaling pathways leading to activation of glucose transport in skeletal muscle with differential sensitivities to wortmannin.
Insulin, which increases glucose uptake into muscle and adipocytes while decreasing the hepatic production of glucose, is the most important hormone involved in the regulation of hexose metabolism in mammals. Even though 90% of hexose disposal after an oral glucose load occurs in skeletal muscle, the mechanisms by which this is regulated remain poorly understood. In addition to insulin, muscle contraction and hypoxia also increase glucose transport(1, 2) . The acceleration of hexose uptake into skeletal muscle upon exposure to insulin, contractile activity, or hypoxia, which mimics that pathway activated by contraction, is accompanied by a redistribution of the ``insulin-responsive'' facilitated glucose transporter isoform, GLUT4, from an intracellular compartment to the plasma membrane(1, 2) . Presumably, the increase in cell surface transporter in response to any one of these physiological stimuli accounts for much of the augmentation in hexose uptake. Nonetheless, it is unclear if contraction and hypoxia utilize the same signaling pathways as does insulin to activate glucose uptake. Previous studies have implicated some diversity in the means by which glucose uptake is regulated in muscle(1) . The effects of maximal stimulation by contraction and hypoxia are non-additive, whereas activation by insulin and hypoxia are fully or partially additive(2, 3) . This has been interpreted as indicative of alternative mechanisms by which hypoxia or contraction on the one hand and insulin on the other stimulate glucose transport but has not led to significant insight as to the level at which the pathways differ(1) . Thus far, no defined pharmacological agent has been identified that unambiguously abolishes accelerated transport in response to one stimulus while preserving that in response to the other (4, 5) .
Insulin, as well as other growth
factors, rapidly stimulates the enzyme PI
3-kinase()(6, 7, 8, 9, 10) .
The binding of insulin to its receptor leads to tyrosine
phosphorylation of the insulin receptor substrate, IRS-1, which in turn
binds to and activates PI 3-kinase via the SH2 domain of its regulatory
subunit, p85(11) . An increase in the activity of the PI
3-kinase catalytic subunit, p110, results in the accumulation of
phosphatidylinositol derivatives phosphorylated at the D-3
position(6, 12) . Recently, the demonstration that the
fungal metabolite wortmannin inhibits both the activation of PI
3-kinase as well as the insulin-dependent translocation of GLUT4
transfected into Chinese hamster ovary cells and glucose transport in
adipocytes has led to the suggestion that the lipid kinase is an
obligate intermediate in the insulin-signaling
pathway(13, 14, 15, 16) . Moreover,
the insulin-dependent binding of PI 3-kinase to IRS-1 is decreased in
several animal models of insulin resistance(17, 18) .
However, the studies using antagonists of the PI 3-kinase have not
distinguished whether their inhibitory effect was being exerted at the
level of the insulin signal transduction pathway or more directly on
vesicle-mediated redistribution of hexose carriers. In this study, we
explored the role of PI 3-kinase in mediating the stimulation of hexose
uptake into skeletal muscle in response to several distinct,
physiological stimuli.
Figure 1:
Effect of wortmannin
on insulin-stimulated hexose uptake in isolated rat skeletal muscle. A, 2-DG uptake into isolated rat epitrochlearis muscle was
measured as described in the text in the absence (basal) or presence of
300 nM insulin for 30 min, with or without pretreatment with 1
µM wortmannin for 30 min. B, 3-O-MG
uptake of rat epitrochlearis and hemidiaphragm muscles was measured
under the same conditions as in A. The basal uptake into
muscles averaged 0.15 µmol/ml/10 min. Data presented are the
mean ± S.E. of eight muscles for epitrochlearis and seven
muscles for the hemidiaphragm muscles.
Figure 3: The effect of wortmannin on contraction-stimulated 2-DG uptake in muscle. Isolated rat epitrochlearis muscles were treated or not with 1 µM wortmannin, followed by 10 tetanic contractions elicited by stimulating at 50 Hz for 10 s at 1 contraction/min for 10 min. Muscles were stimulated to contract in the absence or presence of wortmannin. 2-DG uptake rate was then measured as described under ``Materials and Methods.'' Data are presented as mean ± S.E. of 5-7 muscles. The difference in the accumulation of hexose with and without wortmannin was not significant (p = 0.65).
Figure 2:
Dose-response curve of wortmannin
inhibition of insulin-stimulated 2-DG uptake in isolated rat
epitrochlearis muscle. Isolated muscles were treated with the indicated
concentrations of wortmannin for 30 min, followed by incubation in the
absence () or presence (
) of 300 nM insulin for 30
min. 2-DG uptake was then measured as described under ``Materials
and Methods.'' Data are the mean ± S.E. of seven muscles
except for wortmannin alone, which is the average of two
muscles.
Figure 4:
The effect of wortmannin on hypoxia and
hypoxia plus insulin stimulation of 2-DG uptake in muscle. Isolated rat
epitrochlearis muscles were treated with or without 1 µM wortmannin for 30 min, followed by incubation in an atmosphere of
5% CO, 95% N
for 60 min (n =
10). In hypoxia plus insulin stimulation experiments (n = 5), insulin was added to 300 nM 30 min after the
start of incubation in 5% CO
, 95% N
. 2-DG
uptake rate was then measured as described in the text and Table 1. Data are presented as the mean ± S.E. Wortmannin
had no effect on hypoxia-stimulated hexose transport (p = 0.52).
Figure 5:
The effect of insulin and hypoxia on the
production of phosphatidylinositol lipids in muscle. Isolated rat
epitrochlearis muscles were incubated in phosphate-free KHB containing
[P]orthophosphate for a total of 4.5 h. When
present, insulin (dottedline) was added to 300
nM at 30 min before the end of the incubation. Hypoxic
conditions (solid line) were induced 60 min before the end of the
incubation by changing the gassing mixture from 5% CO
, 95%
O
to 5% CO
, 95% N
. The lipids were
extracted and analyzed as described under ``Materials and
Methods.'' Shown are the disintegrations/min eluting coincident
with the PtdIns(3,4)P
standard at about 57 min (panelA) and the PtdIns(3,4,5)P
standard at about
88.5 min (panelB). Each tracing was the result of
four muscles.
Figure 6:
The effect of wortmannin on the production
of phosphatidylinositol lipids in muscle. Rat epitrochlearis muscle was
incubated (dottedline) or not (solidline) in the presence of 1 µM wortmannin for
30 min, followed by exposure to insulin for an additional 30 min.
Lipids were extracted and analyzed as in Fig. 5. PanelA, PtdIns(3,4)P; panelB,
PtdIns(3,4,5)P
. Each tracing was the result of four
muscles; note that the data for insulin treatment in the absence of
wortmannin represents the same as that shown in Fig. 5.
In this series of experiments, we have used sensitivity to
the fungal metabolite wortmannin to define two distinct signaling
pathways leading to increased hexose uptake into rat skeletal muscle.
We believe that the ability of wortmannin to inhibit accelerated
transport in response to insulin but not contraction or hypoxia
reflects the obligatory role of PI 3-kinase in the insulin-dependent
pathway but not that activated by the latter stimuli. Several results
support this notion. Insulin, but not hypoxia, stimulated the in
vivo accumulation of the products of PI 3-kinase,
PtdIns(3,4)P and PtdIns(3,4,5)P
(Fig. 5). This is in contrast to the results of a previous
study, in which no labeled PtdIns(3,4)P
and
PtdIns(3,4,5)P
was detected (9) . The reason for
the difference in PtdIns labeling is unclear, but increased efficiency
of labeling in the current series of experiments is most likely. The
inhibition of insulin-activated hexose uptake by 1 µM wortmannin correlated with suppression of the accumulation of
3`-phosphatidylinositol lipids (Fig. 6). Moreover, wortmannin,
which did not affect the phospholipid pattern of hypoxic muscle, also
was completely without effect on the stimulation of hexose uptake in
response to hypoxia or contraction. It should be emphasized, however,
that whereas the accumulation of PtdIns(3,4)P
and
PtdIns(3,4,5)P
probably reflects in vivo PI
3-kinase activity, these experiments do not address whether the lipids
actually represent the critical signaling molecules. It has been
reported that the catalytic subunit of the PI 3-kinase is also capable
of acting as a serine protein kinase(25, 26) , and it
is likely that wortmannin inhibits this activity in parallel in these
experiments.
Several recently published studies have been interpreted as indicative of the presence of multiple independent pathways for the physiological activation of glucose transport in muscle(2, 3, 27) . One of the strongest arguments has been based on the observation that a maximal stimulation of hexose uptake by insulin can be further increased by contraction or hypoxia, whereas maximal activations by hypoxia and contraction are completely non-additive(2) . Efforts to show differential sensitivities of insulin and contraction-stimulated hexose transport to calcium channel blockers have not been successful(4, 5) . In experiments relying on the subcellular fractionation of muscle, Douen et al.(27) showed that in rat hindlimb the amount of GLUT4 on plasma membrane increased significantly and the intracellular membrane GLUT4 decreased correspondingly after insulin stimulation; while the plasma membrane GLUT4 increased after exercise, there was no corresponding decrease in the recovered intracellular membrane pool of GLUT4. Based on these results, they concluded there are two distinct cytoplasmic populations of GLUT4, and, by inference, the biochemical signals initiating translocation of these pools must be different(27) . Our results provide the first direct evidence that there are at least two pathways to activate hexose transport in skeletal muscle, since wortmannin obliterates the response to insulin while leaving the effects of contraction and hypoxia unaffected. The possibility that wortmannin acts at the insulin receptor or IRS-1 to inhibit insulin stimulation has been excluded(16) .
The
potential importance of PI 3-kinase as a mediator of insulin-stimulated
hexose uptake has been suggested from studies in several different
model systems. For example, there have been several demonstrations of
an inverse correlation between insulin resistance and the activation of
PI 3-kinase. In the hyperinsulinemic ob/ob diabetic mouse model, the
amount of PI 3-kinase activity associated with the phosphorylated IRS-1
is decreased in muscle and liver(17) ; insulin-stimulated PI
3-kinase activity is decreased in muscle from mice made obese and
insulin-resistant by injections of gold thioglucose (28) ; and
long term treatment with dexamethasone also reduces the PI
3-kinase-associated IRS-1 in rat muscle(18) . PI 3-kinase has
been implicated more directly in studies on adipocytes using inhibitors
of the lipid kinase. In isolated rat adipocytes, wortmannin blocks both
the stimulation of hexose uptake and the inhibition of lipolysis by
insulin(14, 16) . Similar results have been obtained
in 3T3 L1 adipocytes when another PI 3-kinase inhibitor, LY294002, was
used(15) . However, these studies have failed to distinguish
whether a reduction in PtdIns(3,4)P and
PtdIns(3,4,5)P
affects the insulin-signaling process or
more directly interferes with translocation of GLUT4 glucose
transporters. There are now abundant data implicating a role for PI
3-kinase in vesicular transport events. In the human Hep G2 cell line,
platelet-derived growth factor receptors lacking the high affinity
binding sites for PI 3-kinase could not be internalized after
platelet-derived growth factor stimulation(29) . Even more
strikingly, the yeast PI 3-kinase homologue VPS34p plays a critical
role in delivering newly synthesized proteins to the vacuoles (30) . Exposure of mammalian fibroblasts to wortmannin alters
the steady-state distribution of transferrin receptors in the absence
of exposure of cells to mitogens. (
)Thus, the ability of
wortmannin to inhibit insulin-stimulated but not contraction-activated
hexose uptake in skeletal muscle argues strongly that the site of
action is specific to the insulin-signaling system and not the
apparatus responsible for movement of transporters to the cell surface.
This interpretation, of course, relies on the fact that both
contraction and insulin stimulate hexose uptake predominantly via
translocation of GLUT4 transporters, which, as described above, has
been concluded from sub-cellular fractionation experiments.
In summary, our results support the view that PI 3-kinase is necessary for insulin stimulation of hexose uptake but is not involved in contraction and hypoxia stimulation of hexose uptake.