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(Received for publication, June 19, 1995; and in revised form, September
11, 1995) From the
Augmentation of glucose transport into skeletal muscle by GLUT4
translocation to the plasma and T-tubule membranes can be mediated
independently by insulin and by contraction/exercise. Available data
suggest that separable pools of intracellular GLUT4 respond to these
two stimuli. To identify and characterize these pools, we fractionated
skeletal muscle membranes in a discontinuous sucrose density gradient.
Fractions of 32 and 36% sucrose exhibited the highest enrichment of
GLUT4 and were independently responsive to insulin and exercise,
respectively. The combination of the two stimuli depleted both GLUT4
fractions simultaneously. Both vesicle populations contained the gp160
aminopeptidase, whose expression had previously been shown to be
specific to muscle and fat and restricted to GLUT4 vesicles in the
latter tissue. In muscle, gp160 translocates exactly as does GLUT4 in
response to insulin and exercise. The contraction- and
insulin-sensitive GLUT4 pools also contained secretory
component-associated membrane protein/glucose transporter vesicle
triplet but not GLUT1 and caveolin. Immunoadsorption of the two pools
followed by silver staining did not reveal any obvious difference in
their major protein components. On the other hand, sedimentational
analysis in sucrose velocity gradients revealed that the
insulin-sensitive GLUT4 vesicles had a larger sedimentation coefficient
than the exercise-sensitive vesicles. Thus, the separation of the two
intracellular GLUT4 pools should be useful in dissecting what are
likely to be different signal transduction pathways that mediate their
translocation to the cell surface.
In skeletal muscle, stimulation of glucose uptake by insulin is
achieved by recruiting GLUT4 vesicles from their intracellular site to
the plasma membrane and to the T-tubules (Hirshman et al.,
1990; Douen et al., 1990; Goodyear et al., 1991;
Marette et al., 1992). A similar recruitment mechanism is
thought to be responsible for the increased glucose uptake seen in
response to exercise (Douen et al., 1990; Goodyear et
al., 1991) and hypoxia (Cartee and Holloszy, 1990; Cartee et
al., 1991). However, there are likely to be mechanistic
differences between the stimulation of glucose transport by insulin and
exercise. Thus, it has been recently demonstrated that the fungal
metabolite wortmannin blocks insulin-stimulated glucose transport in
muscle but has no effect on contraction or hypoxia-stimulated glucose
uptake (Yeh et al., 1995). Furthermore, it has been known for
some time that the effects of insulin and exercise are additive with
regard to glucose transport in muscle (Zorzano et al., 1986;
Constable et al., 1988; Wallberg-Henriksson et al.,
1988; Ploug et al., 1990, 1992) as are their effects on GLUT4
appearance at the plasma membrane (Gao et al., 1994). Finally,
it has been demonstrated in denervated muscle (Turinsky, 1987) and in
muscles of obese Zucker rats (Brozinick et al., 1992; Dolan et al., 1993; King et al., 1993; Brozinick et
al., 1994) that muscle contraction can stimulate glucose transport
even when the ability of insulin to do so is impaired. Taken together,
these data suggest that muscle may possess two different pools of GLUT4
vesicles, one insulin-sensitive and one exercise-sensitive. Although
some evidence has been presented supporting the existence of two such
pools (Douen et al., 1990), others have not obtained data
consistent with this postulate (Goodyear et al., 1991), and
the pools have heretofore not been isolated or characterized. The
likely reason for this is the difficulty in isolating relatively pure
membrane fractions from muscle, a tissue consisting of multiple fiber
types and a complex subcellular membrane structure. The cell surface
includes T-tubules as well as sarcolemma, and the abundant myofibrils
hinder facile membrane purification. Nevertheless, many investigators
have fractionated skeletal muscle membranes to study GLUT4
translocation, and a comparison of some of the different methods for
membrane isolation from this source has been performed by Fushiki et al.(1989). They concluded that the method of Grimditch et al.(1985) gives a good preparation for studying the
insulin-dependent GLUT4 enrichment in the plasma membrane, whereas the
procedure of Klip et al.(1987) is preferable for the analysis
of GLUT4-containing microsomal membranes. However, the latter
procedure, while providing results supportive of two transporter pools
translocating to the plasma membrane (Douen et al., 1990),
employs only 25, 30, and 35% sucrose fractions, and it did not result
in identification of an intracellular GLUT4 fraction that responded to
exercise/contraction. Given this background, we determined that adding
32 and 38% sucrose layers to this published protocol (Klip et
al., 1987) allowed us to clearly separate intracellular GLUT4
vesicles that responded independently to exercise and insulin. Here we
present a biochemical characterization of these pools.
Figure 1:
Distribution of GLUT1, GLUT4, and gp160
aminopeptidase in muscle membrane fractions subjected to discontinuous
sucrose density gradient centrifugation. Subfractionation of skeletal
muscle membranes was performed as described under ``Experimental
Procedures.'' Membrane protein (15 µg) was then subjected to
SDS-polyacrylamide gel electrophoresis. Immunoblotting (A) was
performed with a polyclonal antibody against GLUT1, a monoclonal
anti-GLUT4 antibody (1F8), and an anti-gp160 anti-peptide antibody as
described under ``Experimental Procedures,'' and detection of
antigen was by appropriate secondary antibody and enhanced
chemiluminescence (GLUT4 and gp160) or iodinated protein A (GLUT1).
Quantitation (B) was performed as described under
``Experimental Procedures.'' The total membranes applied to
the gradient are called the homogenate (H), and the pellet (P) is the bottom of the gradient. The positions of GLUT4 and
gp160 are indicated by the closed symbols, GLUT1 by is
indicated by open circles, and protein is indicated by open triangles.
Figure 3:
Insulin- and exercise-dependent
translocation of gp160 aminopeptidase. An experiment was performed that
was similar to that of Fig. 2except that anti-gp160
aminopeptidase antibody was used to show translocation of this protein
in response to insulin and exercise. Ins refers to membranes
from insulin treated animals, and Exe refers to membranes from
exercised animals. C, control.
Figure 5:
gp160 aminopeptidase and SCAMPs/GTV3
proteins are present in GLUT4-containing vesicles from exercise- and
insulin-recruitable pools. Western blotting of a portion of the same
preparation used in the previous figure was performed with the
appropriate antibodies as described in the previous figure legends and
under ``Experimental
Procedures.''
Figure 2:
Skeletal muscle contains two separate
GLUT4 pools: insulin-sensitive and exercise-sensitive. Rats were left
untreated(-) or were injected in vivo with 1.5 units of
insulin (top, +), subjected to a 30-min swimming period (middle, +), or a combination of both swimming and
insulin (see ``Experimental Procedures'') (bottom,
+). Membrane protein (50 µg) from the 32 and 36% sucrose
fractions was resolved by SDS-polyacrylamide gel electrophoresis and
analyzed by Western blotting. Detection of GLUT4 was accomplished with
monoclonal antibody (1F8) and iodinated secondary antibody. A
representative blot is shown in A. B shows the
quantitative analysis of autoradiographs from several experiments as
determined by excising the appropriate region of the blot and counting
it in a
Figure 4:
Silver staining of vesicles from 32 and
36% sucrose fractions. Membrane protein (0.5 mg) from the fractions of
interest were adsorbed with 100 µl of 1F8 or nonspecific IgG beads
as described under ``Experimental Procedures.'' The figure is
a silver stained 10% polyacrylamide gel with the proteins identified by
Western blot indicated by the arrows.
Figure 6:
Fractionation of muscle membranes by
sucrose velocity gradient centrifugation. Fractionation of
GLUT4-containing vesicles and detection of antigen was performed as
described under ``Experimental Procedures.'' A shows
the autoradiogram, and B shows the densitometric analysis of
the blot (closed symbols) expressed as arbitrary units. The open circles are the protein values from the 32% sucrose
fraction, and the open triangles represent protein from the
36% fraction. Ins and Exe are as defined in the
legend to Fig. 3. Shown is a representative experiment that was
perfomed on three independent occasions.
As noted above, GLUT4-enriched vesicles
contain a muscle- and fat-specific aminopeptidase, gp160 (Kandror and
Pilch, 1994; Kandror et al., 1994), and they also possess
three antigenically related integral membrane proteins of unknown
function, called SCAMPs/GTV3 for secretory component-associated
membrane protein/glucose transporter vesicle triplet (Brand et
al., 1991; Thoidis et al., 1993; Laurie et al.,
1993). Fig. 5shows a Western blot of immunoisolated vesicles
from the 32 and 36% sucrose fraction using three antibodies to these
vesicle constituents. Again, there are no striking differences between
the two fractions, although the slowest migrating form of SCAMPs/GTV3
appears somewhat less abundant in the insulin-sensitive vesicles. As is
the case in fat cells, GLUT1 (Zorzano et al., 1989; Kandror et al., 1995a) and caveolin (Kandror et al., 1995b)
are completely excluded from the GLUT4-containing vesicles in both
sucrose fractions (data not shown).
Previous work by Douen et al.(1990) in skeletal
muscle had demonstrated that both insulin and exercise increased GLUT4
in a fraction enriched in plasma membrane markers, whereas only insulin
provoked a statistically significant, concomitant decrease in the
internal pool that they measured, a 35% sucrose fraction. We modified
their fractionation protocol by eliminating the 35% fraction and
replacing it with 32 and 36% sucrose fractions, and we also added a 38%
sucrose fraction. These relatively minor additions allowed a sufficient
increase in the resolution of membrane separation as compared with the
prior study (Douen et al., 1990), and this allowed us to
isolate two fractions of 32 and 36% sucrose that were highly enriched
in GLUT4 (Fig. 1). The former responded to insulin and the
latter to exercise with regard to translocation to the cell surface (Fig. 2). We compared the composition of the insulin-sensitive
vesicles to the exercise-sensitive pool by silver staining and by
Western blotting with antibodies to known vesicular components ( Fig. 4and Fig. 5), but we saw no obvious differences in
overall protein composition by either method. We did observe
differential sedimentational behavior, however, in the vesicles from
the two pools (Fig. 6). What then is the molecular basis for
the different behavior of the insulin- and exercise-sensitive pools?
Previous studies from our lab have compared the composition and
sedimentational properties of GLUT4-containing vesicles obtained from
unstimulated and insulin-stimulated adipocytes with those from muscle
exposed to insulin or not (Kandror et al., 1995a). Somewhat
surprisingly and despite different methods of tissue homogenization and
vesicle isolation, we found no obvious differences in protein
composition determined by silver staining, nor did the vesicles from
the two tissues behave differently in sucrose velocity and density
gradients (Kandror et al., 1995a). We take this as evidence
that GLUT4 vesicles are a unique type of endosomal compartment that is
the same in muscle and fat. With regard to changes in sedimentation
behavior observed here (Fig. 6) and in the previous paper
(Kandror et al., 1995a), there are two possible explanations.
First, the protein composition may be the same in vesicles isolated
from different fractions under different conditions, but the protein to
lipid ratio may vary. The presence or absence of specific phospholipids
may be an important parameter in GLUT4 vesicle trafficking, and we are
currently examining this possibility. With regard to the apparent
constant protein composition in GLUT4 vesicles as determined by silver
staining, in order to obtain a clean result with this highly sensitive
technique, vesicles adsorbed to beads are thoroughly washed, and this
washing is very likely to remove peripheral membrane proteins that may
or may not be different in insulin versus exercise responsive
GLUT4 vesicles. Moreover, GLUT4 vesicles are not abundant and possibly
important differences in minor protein components from differentially
responsive vesicles will not be detected until specific antibodies to
these are available. Thus, we are also working to identify additional
protein components in GLUT4 vesicles by the immunological approach
previously used to identify GLUT4 (James et al., 1988) and
SCAMPs/GTV3 (Thoidis et al., 1993). Regardless of the
reason, GLUT4 vesicles are able to respond differentially to exercise
and insulin, most likely by different mechanisms (Yeh et al.,
1995). We suggest that this may be, in part, as a result of the
association of GLUT4 vesicles with glycogen particles in a saturable
manner. That is, this association is governed by the amount of glycogen
in the muscle, and a variety of studies provide indirect evidence for
this hypothesis. For example, exercise depletes glycogen concomitant
with GLUT4 translocation (Fig. 2). Shortly after exercise, rats
are more sensitive with respect to insulin-stimulated glucose transport
(Zorzano et al., 1986), and we suggest that this is due to the
larger available pool of free GLUT4 vesicles resulting from glycogen
depletion. Additionally, just after denervation of muscle and before
changes in GLUT4 gene expression, glycogen is depleted, and glucose
transport is enhanced (Coderre et al., 1992). Finally,
transgenic mice overexpressing GLUT1 in muscle have dramatically
increased glycogen levels and are insulin-resistant (Gulve et
al., 1994). It should be noted that in our current protocol, the
homogenization and fractionation conditions (Fig. 1) remove all
measurable glycogen prior to this analysis. Therefore, we are in the
process of trying other methodology to experimentally verify this
possible association of GLUT4 vesicles with glycogen particles.
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27584-27588
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Animals
Male Sprague-Dawley rats
(175-200 g) were purchased from the Charles River Breeding
Laboratory (Wilmington, MA). The animals were fasted overnight and
divided into three groups: untreated controls, those injected with
insulin (1.5 units/rat) via the portal vein 8 min prior to being
sacrificed, and those exercised. The latter consisted of a 30-min
swimming period in a water bath maintained at 37 °C. For all three
groups, the rats were anesthetized with sodium pentobarbital (60 mg/kg
of body weight by intraperitoneal injection), insulin was administered
or not, hind limb muscles were removed, and membranes were immediately
isolated. These surgical procedures were approved by the Institutional
Animal Care and Use Committee of Boston University School of Medicine.Preparation of Membranes from Skeletal
Muscle
Membranes from mixed skeletal muscle were prepared
essentially according to Klip et al.(1987). Red and white
gastrocnemius and soleus muscles from rat hind limb were rapidly
removed and trimmed of connective tissue, fat, and nerves. The muscles
(2.5-3.0 gm/rat) were then minced and homogenized for 5 s by
Polytron in a buffer containing 20 mM HEPES, 250 mM sucrose, 5 mM EDTA, 1 µM leupeptin, 1
µM pepstatin, 1 µM aprotinin A, pH 7.2, at 4
°C. The homogenate was centrifuged at 1,200
g for
10 min at 4 °C. The supernatant was saved, and the pellet was
resuspended, homogenized, and centrifuged again at 1,200 g for 10 min. The two supernatants were then combined and
centrifuged at 9,000 g for 10 min at 4 °C. The
resulting supernatant was then centrifuged at 200,000 g for 140 min at 4 °C. The pellet was then resuspended in 7 ml
of 38% sucrose (w/v) and 5 ml each of 25, 30, 32, and 36% sucrose
solutions were layered on the top. The 30% sucrose layer was adjusted
to 7 ml in order to minimize plasma membrane contamination with
intracellular membranes. The gradient was centrifuged at 68,000 g for 14 h. Membranes from the gradient were then collected,
diluted with homogenization buffer, and centrifuged again at 200,000
g for 140 min. The resulting pellets were resuspended
in the homogenization buffer, and total membrane protein content was
determined by the BCA protein assay (Pierce).Fractionation of Microsomes in Sucrose Velocity
Gradient
Microsomes from the 32 and 36% sucrose fractions
were resuspended in phosphate-buffered saline, and an equal amount of
protein from each fraction was loaded on a 4.6-ml 10-30%
continuous sucrose gradient containing 20 mM HEPES, pH 7.5,
100 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol. Membranes were centrifuged at 48,000 rpm for 55
min at 4 °C. Membranes from the gradient were then collected into
200-µl fractions(25, 26, 27, 28, 29, 30) starting
from the bottom of the tube. Protein content and refractive index were
measured. The position of GLUT4 was determined by Western blotting.Immunoadsorption of GLUT4
Vesicles
Purified anti-GLUT4 antibody 1F8 (James et
al., 1988) as well as nonspecific IgG were coupled to acrylamide
beads (Reacti-gel GF2000, Pierce) at a concentration of 1 mg of
antibody/ml of resin according to the manufacturer's
instructions. Microsomes from the 32 and 36% sucrose fractions were
incubated with each antibody for 16 h at 4 °C. The beads were
collected by centrifugation, washed three times with phosphate-buffered
saline, and eluted with sample buffer (Laemmli, 1970). Portions of the
eluate were used for silver stain and for Western blotting.Gel Electrophoresis and
Immunoblotting
Membranes were subjected to electrophoresis
in 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate
(Laemmli, 1970), transferred to an Immobilon-P membrane (Millipore,
Bedford, MA), which was treated with 5% nonfat dry milk to block
nonspecific interactions of antibodies. The membranes were then
incubated with anti-GLUT4 antibody (James et al., 1988), a
monoclonal antibody directed against SCAMPs/GTV3 (
)(Thoidis et al., 1993), and a monoclonal antibody for the
dihydropyridine receptor (a gift of Dr. Kevin Campbell, University of
Iowa). Rabbit polyclonal antibody recognizing gp160 aminopeptidase
(Kandror and Pilch, 1994) and GLUT-1 (a gift of Dr. C. Carter-Su,
University of Michigan) were also used. The antigen-antibody complexes
were detected with either I-goat anti-mouse antibody,
I-protein A, or horseradish peroxidase-conjugated
antibodies and an enhanced chemiluminescence detection system (DuPont
NEN). When horseradish peroxidase-conjugated secondary antibodies were
used, the autoradiograph was scanned in a computing densitometer
(Molecular Dynamics) and expressed in arbitrary units. For some
experiments both approaches were utilized with virtually identical
results.
Materials
Electrophoresis chemicals were
obtained from National Diagnostics (Atlanta, GA), and collagenase,
aprotinin, leupeptin, and pepstatin were purchased from Boehringer
Mannheim. I-goat anti-mouse antibody and
I-protein A were obtained from DuPont NEN. Other reagents
were from Sigma.
Statistical Analysis
The statistical
analysis of the immunoblots was performed using an unpaired
Student's t test for comparing the experimental group
with the control.
Muscle Membrane
Fractionation
Fig. 1A shows the
distribution of GLUT4, GLUT1, and gp160 aminopeptidase in the
discontinuous sucrose density gradient where detection is by Western
blotting in all cases. Fig. 1B is the quantitative
analysis of the data together with the protein values. The 32 and 36%
sucrose fractions are specifically enriched in GLUT4 to the greatest
extent of any fractions, and this is also true for gp160 aminopeptidase
(Kandror et al., 1994), the latter being co-localized exactly
as is GLUT4 in fat cells (Kandror and Pilch, 1994) (see also Fig. 3and Fig. 5). A readily detectable amount of GLUT4
is present in the 25 and 30% sucrose fractions, but this represents
only 18% of the total in fractions 25 through 36. Previous studies have
shown that the 25% sucrose fraction is enriched in plasma membrane
markers (Douen et al., 1990) and that the 30% sucrose fraction
may also be slightly enriched in plasma membrane (Douen et
al., 1989). We observe that GLUT1 is most abundant in the 25%
sucrose fraction consistent with this fraction being enriched in plasma
membrane, although much of muscle GLUT1 may derive from the perineural
sheath (Handberg et al., 1992). In any case, our interests in
this study are focused on the intracellular GLUT4 pools mobilized by
insulin and exercise, and therefore, we did not characterize the 25 and
30% sucrose fractions further. We also used antibodies for the T-tubule
marker, the dihydropyridine receptor, which overlaps somewhat in
distribution with GLUT4 but is most enriched in the 30% sucrose
fraction (data not shown). Finally, most (70%) of the protein
applied to the gradient appears in the 38% sucrose fraction including
60% of the GLUT4. As shown in Fig. 1B, there is no
enrichment of GLUT4 in 38% sucrose fraction as compared with the
homogenate, and thus, this fraction very likely represents
heterogeneous and impure membrane structures from all parts of the
cell. The remaining 30% of GLUT4 distributed in the 32 and 36% sucrose
fractions represents the intracellular GLUT4 pools. This value compares
with previous studies of intracellular GLUT4 vesicles obtained in
50% yield from muscles frozen in liquid nitrogen, pulverized,
homogenized, and fractionated by differential centrifugation (Rodnick et al., 1992).
counter. The data are expressed in arbitrary units where
each fraction was compared with the 36% sucrose fraction, which was set
at a value of 1. Each experiment was performed three times on separate
occasions, and the results are expressed as the means ± S.E. The
asterisks indicate p < 0.05 compared with
control.
Selective Response of Intracellular Fractions to
Insulin and Exercise
We tested the 32 and 36% sucrose
fractions for the ability to respond to insulin and exercise (swimming)
as described under ``Experimental Procedures.'' Fig. 2A depicts a representative experiment showing the
Western blot analysis from these fractions. It can be seen that GLUT4
is depleted from the 32% sucrose fraction in response to insulin,
whereas the 36% sucrose fraction is depleted by exercise, and these
responses are specific to each fraction. The combination of both
insulin and exercise depletes both fractions simultaneously. The
quantitative analysis of three such experiments is shown in Fig. 2B, only for the treatments separately. However,
the combination of insulin and exercise resulted in 50% GLUT4
depletion from both fractions (data not shown). Because gp160
aminopeptidase is co-localized with GLUT4 in fat (Kandror and Pilch,
1994) and muscle (Kandror et al., 1995a), we also determined
its response to the two stimuli, and as expected, it behaves like GLUT4 (Fig. 3). That is, gp160 aminopeptidase is depleted from the 32%
sucrose fraction in response to insulin and from the 36% sucrose
fraction in response to exercise. We did not perform the complete
analysis of this translocation as we show in Fig. 2for GLUT4.
Immunoadsorption and Silver Staining of GLUT4
Vesicles
In attempts to determine possible biochemical
differences that might underlie the differential response of the two
vesicle populations, we used immobilized anti-GLUT4 antibody to
immunoabsorb transporter-containing vesicles from the 32 and 36%
sucrose fractions, and we analyzed their protein composition by silver
staining (Fig. 4) and by Western blot (Fig. 5). As shown
in Fig. 4, we observed no obvious differences in the composition
of the major proteins in vesicles derived from the two fractions, and
GLUT4 is clearly visible as a diffuse band. The background (IgG)
staining for the 36% sucrose fraction is always higher than that for
the 32% fraction, presumably due to its higher overall protein content (Fig. 1B). It is not entirely obvious why the protein
composition of the immunoadsorbed vesicles is so similar in the
exercise- and insulin-mobilizable pools (see ``Discussion'').
In order to obtain clean silver staining, we must thoroughly wash
vesicles bound to antibody immobilized on acrylamide beads, and it is
possible that this procedure removes a peripheral membrane protein or
proteins that regulate insulin versus exercise responsiveness.
Alternatively, because it is difficult to obtain GLUT4 vesicles in
large quantities and silver staining is not uniform for every protein,
we may simply have a detection problem. In fact, the vesicles from the
32% pool differ in their sedimentation properties from those in the 36%
fraction (Fig. 6).
Sucrose Velocity Gradient
To further
characterize GLUT4 vesicles from the 32 and 36% sucrose fraction,
membranes from the two fractions were analyzed by sucrose velocity
gradient centrifugation, and their sedimentation coefficients were
compared. Fig. 6A shows the raw Western blotting data,
and Fig. 6B shows the quantitative analysis of these
data along with the protein distribution (closed symbols).
GLUT4 vesicles from these fractions have a narrow distribution, and a
comparison of the insulin-responsive and exercise-responsive pools
reveal close but nonetheless distinct sedimentation coefficients (Fig. 6B). The overall sedimentation properties of each
fraction (open symbols) are distinct with shoulders that
correspond to the GLUT4 vesicles. Thus, these two GLUT4 vesicle
populations can be distinguished on the basis of their densities and
their sedimentation coefficients.
)
We thank Neil Ruderman for many helpful discussions
and for careful reading of this manuscript and Dr. Kevin Campbell for
the gift of anti-dihydropyridine receptor antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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J. O. Holloszy A forty-year memoir of research on the regulation of glucose transport into muscle Am J Physiol Endocrinol Metab, March 1, 2003; 284(3): E453 - E467. [Abstract] [Full Text] [PDF]
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L. L. Tortorella and P. F. Pilch C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E514 - E524. [Abstract] [Full Text] [PDF]
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E. Tomas, A. Zorzano, and N. B. Ruderman Exercise Effects on Muscle Insulin Signaling and Action: Exercise and insulin signaling: a historical perspective J Appl Physiol, August 1, 2002; 93(2): 765 - 772. [Abstract] [Full Text] [PDF]
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 H. C. Chen, G. Bandyopadhyay, M. P. Sajan, Y. Kanoh, M. Standaert, R. V. Farese Jr., and R. V. Farese Activation of the ERK Pathway and Atypical Protein Kinase C Isoforms in Exercise- and Aminoimidazole-4-carboxamide- 1-beta -D-riboside (AICAR)-stimulated Glucose Transport J. Biol. Chem., June 21, 2002; 277(26): 23554 - 23562. [Abstract] [Full Text] [PDF]
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H. Ai, J. Ihlemann, Y. Hellsten, H. P. M. M. Lauritzen, D. G. Hardie, H. Galbo, and T. Ploug Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle Am J Physiol Endocrinol Metab, June 1, 2002; 282(6): E1291 - E1300. [Abstract] [Full Text] [PDF]
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E. B. Marliss and M. Vranic Intense Exercise Has Unique Effects on Both Insulin Release and Its Roles in Glucoregulation: Implications for Diabetes Diabetes, February 1, 2002; 51(90001): S271 - 283. [Abstract] [Full Text] [PDF]
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J. S. Fisher, J. Gao, D.-H. Han, J. O. Holloszy, and L. A. Nolte Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E18 - E23. [Abstract] [Full Text] [PDF]
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R. Aslesen, E. M. L. Engebretsen, J. Franch, and J. Jensen Glucose uptake and metabolic stress in rat muscles stimulated electrically with different protocols J Appl Physiol, September 1, 2001; 91(3): 1237 - 1244. [Abstract] [Full Text] [PDF]
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 C. Yang, S. Mora, J. W. Ryder, K. J. Coker, P. Hansen, L.-A. Allen, and J. E. Pessin VAMP3 Null Mice Display Normal Constitutive, Insulin- and Exercise-Regulated Vesicle Trafficking Mol. Cell. Biol., March 1, 2001; 21(5): 1573 - 1580. [Abstract] [Full Text]
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K. Kawanaka, L. A. Nolte, D.-H. Han, P. A. Hansen, and J. O. Holloszy Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats Am J Physiol Endocrinol Metab, December 1, 2000; 279(6): E1311 - E1318. [Abstract] [Full Text] [PDF]
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F. W. Booth, S. E. Gordon, C. J. Carlson, and M. T. Hamilton Waging war on modern chronic diseases: primary prevention through exercise biology J Appl Physiol, February 1, 2000; 88(2): 774 - 787. [Abstract] [Full Text] [PDF]
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A. V. Chibalin, M. Yu, J. W. Ryder, X. M. Song, D. Galuska, A. Krook, H. Wallberg-Henriksson, and J. R. Zierath Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2 PNAS, January 4, 2000; 97(1): 38 - 43. [Abstract] [Full Text] [PDF]
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 C. A. Millar, A. Shewan, G. R. X. Hickson, D. E. James, and G. W. Gould Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes Mol. Biol. Cell, November 1, 1999; 10(11): 3675 - 3688. [Abstract] [Full Text]
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B. Urso, D. L. Cope, H. E. Kalloo-Hosein, A. C. Hayward, J. P. Whitehead, S. O'Rahilly, and K. Siddle Differences in Signaling Properties of the Cytoplasmic Domains of the Insulin Receptor and Insulin-like Growth Factor Receptor in 3T3-L1 Adipocytes J. Biol. Chem., October 22, 1999; 274(43): 30864 - 30873. [Abstract] [Full Text] [PDF]
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