From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242
Received for publication, August 15, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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To examine the intracellular trafficking and
translocation of GLUT4 in skeletal muscle, we have generated transgenic
mouse lines that specifically express a GLUT4-EGFP (enhanced green
fluorescent protein) fusion protein under the control of the human
skeletal muscle actin promoter. These transgenic mice displayed EGFP
fluorescence restricted to skeletal muscle and increased glucose
tolerance characteristic of enhanced insulin sensitivity. The
GLUT4-EGFP protein localized to the same intracellular compartment as
the endogenous GLUT4 protein and underwent insulin- and
exercise-stimulated translocation to both the sarcolemma and
transverse-tubule membranes. Consistent with previous studies in
adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform,
but not the related Munc18b isoform, in vivo specifically
inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly,
however, Munc18c inhibited GLUT4 translocation to the transverse-tubule
membrane without affecting translocation to the sarcolemma membrane.
The ability of Munc18c to block GLUT4-EGFP translocation to the
transverse-tubule membrane but not the sarcolemma membrane was
consistent with substantially reduced levels of syntaxin 4 in the
transverse-tubule membrane. Together, these data demonstrate that
Munc18c specifically functions in the compartmentalized translocation
of GLUT4 to the transverse-tubules in skeletal muscle. In addition,
these results underscore the utility of this transgenic model to
directly visualize GLUT4 translocation in skeletal muscle.
The stimulation of glucose uptake in adipose and muscle tissues
primarily occurs through the translocation of the GLUT4 glucose transporter isoform from intracellular storage sites to the cell surface membranes (1-4). Insulin stimulation of this process requires
the tyrosine phosphorylation of the insulin receptor substrate
family of proteins and subsequent activation of the type 1 phosphatidylinositol (PI)1
3-kinase (5-12). Although the precise
signaling steps that occur downstream of the PI 3-kinase ultimately
leading to GLUT4 translocation have remained elusive, recent studies
have begun to resolve the specific trafficking, docking, and fusions
events. In adipocytes, it is well established that GLUT4 storage
compartments contain vesicle-localized proteins (v-SNAREs) that
specifically interact with cognate cell surface target proteins
(t-SNAREs) at the plasma membrane to promote vesicle docking and
fusion (13, 14). Insulin-stimulated GLUT4 translocation is dependent
upon the interaction of the v-SNARE, VAMP2, with the plasma membrane
t-SNAREs, syntaxin 4 and SNAP23 (15-21). Furthermore, the syntaxin
4-binding protein, Munc18c, has been shown to specifically modulate
insulin-sensitive GLUT4 translocation in 3T3L1 adipocytes (22-24).
However, it is important to recognize that the majority of these
studies have been performed in cultured 3T3L1 adipocytes and L6
myotubes due to the inherent technical limitations in the study of
GLUT4 trafficking in adipocytes and skeletal muscle in
vivo.
Skeletal muscle is the primary depot for insulin-stimulated disposal of
blood glucose in vivo and has unique functional and structural characteristics that distinguish it from adipose tissue. Like adipocytes, insulin stimulation of glucose uptake in skeletal muscle also occurs through a PI 3-kinase pathway. But in addition, muscle contraction/exercise also potently stimulates glucose uptake and
GLUT4 translocation independently of the PI 3-kinase pathway (25-29).
Furthermore, unlike the simpler single plasma membrane of adipocytes,
skeletal muscle has two functionally distinct surface membranes, the
sarcolemma and transverse-tubule, both of which function as GLUT4
protein acceptor membranes in the translocation process (30-33).
Currently, there is little functional data with regard to the v-SNARE
and t-SNARE proteins involved in either insulin- or exercise/contraction-stimulated GLUT4 translocation in skeletal muscle.
Thus, to develop a more tractable skeletal muscle system to investigate
GLUT4 translocation, we have generated transgenic mice specifically
expressing a GLUT4-EGFP fusion protein in skeletal muscle. In this
article, we demonstrate that the GLUT4-EGFP fusion protein displays an
identical distribution and trafficking pattern as the endogenous GLUT4
protein. These animals provide a model system to investigate skeletal
muscle GLUT4 translocation in vivo and can be used to
distinguish between sarcolemma and transverse-tubule GLUT4
translocation through the function of the syntaxin 4-binding protein, Munc18c.
Materials--
The rabbit polyclonal GLUT4 and Munc18c
antibodies were obtained as described previously (24). Monoclonal
antibodies 8D5 (specific for Transgenic Mice--
A 2.2-kilobase fragment of the GLUT4
cDNA fused in-frame with the EGFP cDNA was ligated into the
transgenic vector pStec which contains the SV40 polyadenylation and
intron sequences. A 2.2-kilobase fragment of the human skeletal muscle
actin (HSA) promoter was isolated from the pHSAaNeor
plasmid and ligated upstream of the pStec-GLUT4/EGFP vector. The
resulting vector, pStec-HSA-GLUT4/EGFP, was linearized with AflIII, microinjected into the nucleus of pre-implanation
embryos, and transferred to the oviduct of psuedo-pregnant mice as
described previously by the University of Iowa Transgenic Animal
Facility. The resulting pups were screened for the presence of the
transgene by polymerase chain reaction of genomic DNA and the positive
animals bred onto the C57Bl/6 background.
Tissue Extracts--
Various tissues were dissected from carbon
dioxide asphixiated mice and frozen in liquid nitrogen. The tissues
were diced in a lysis buffer containing 25 mM Hepes, pH
7.4, 1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 5 µg/ml leupeptin, and 5 mM
benzamidine and then ground with a handheld pellet pestle (Kontes,
Vineland, NJ) for 20 s. The samples were then transferred to a
Dounce homogenizer and homogenized with 10 hand strokes. After rocking
at 4 °C for 10 min, the samples were centrifuged in a
microcentrifuge for 30 min at 13,000 × g and
the clear lysate subjected to SDS-polyacrylamide gel electrophoresis
and immunoblotting.
Glucose Tolerance Test--
Control and GLUT4-EGFP transgenic
mice were fasted overnight. A basal glucose level was taken with tail
vein blood on a Hemocue Glucometer (Hemocue AB, Angelholm, Sweden). At
time point 0, the mice were given an intraperitoneal injection of 2 grams of glucose per kg of body weight. Glucose measurements were then
taken from tail vein blood at 30, 60, 90, and 120 min post-glucose injection.
Subcellular Fractionation of Skeletal Muscle--
The
subcellular fractionation of skeletal muscle was performed as described
(34). Briefly, following an overnight fast, the transgenic mice were
either left untreated or given an intraperitoneal injection of 21 units
of Humulin R per kg of body weight. After 30 min, the mice were
sacrificed, and the quadriceps and gastrocnemius muscles were dissected
and trimmed of fat and connective tissues. The muscles were put in a
homogenization buffer containing 20 mM Hepes, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml
pepstatin, 5 µg/ml leupeptin, and 5 mM benzamidine, and
homogenized with a Polytron PT-10 homogenizer 3 times in 10-s bursts.
The samples were then centrifuged at 2,000 × g for 5 min and the supernatant was re-centrifuged at 9,000 × g for 20 min at 4 °C. The resulting pellet (P1) was
re-suspended in PBS containing protease inhibitors. The supernatant
from the P1 fraction was then centrifuged at 180,000 × g for 90 min, and the pellet was re-suspended in PBS plus
protease inhibitors. Equal amounts of protein (1 mg) from each pellet
were loaded onto a 4.5-ml 10-30% continuous sucrose gradient and
centrifuged at 216,000 × g for 55 min in an SW50.1
rotor (Beckman-Coulter, Palo Alto, CA). The sucrose gradient was
fractionated into 500-µl aliquots and the pellet (P2) was
re-suspended in PBS plus protease inhibitors. Five µg of protein plus
equal volumes of the fractions were resolved by SDS-polyacrylamide gel
electrophoresis and subjected to immunoblotting.
Immunofluorescence of Muscle--
Transgenic littermates were
fasted overnight and either left untreated or given an intraperitoneal
injection of 21 units of Humulin R per kg of body weight. After 30 min,
the mice were sacrificed, the quadriceps dissected, covered in
TissueTek O.C.T. embedding compound, and snap-frozen in liquid
nitrogen-cooled isopentane. Frozen muscles were stored at
The tissue slices were incubated in blocking solution containing 3%
bovine serum albumin and 5% donkey serum in PBS for 1 h. After
rinsing with PBS, the samples were incubated with a 1:50 dilution of
the primary antibody in the blocking solution for 90 min. The samples
were then washed with PBS three times for 5 min each and then incubated
with a 1:50 dilution of the appropriate secondary antibody. The slides
were washed with PBS three times for 5 min each, dried, mounted with a
drop of Vectashield, and covered with a coverslip. Antibody-labeled
tissues slices were then viewed with a Zeiss LSM 510 Confocal
Fluorescent Microscope.
Due to the high labeling background of the IIID5E1 mouse monoclonal
antibody on mouse tissue, a special antibody labeling protocol was
used. In this particular case, the samples were blocked with avidin and
biotin blocking buffers, and blocked and labeled using the Monoclonal
on Mouse kit according to the manufacturer's instructions.
Exercise Protocol--
Mice were trained to run on a Columbus
Instruments Rodent Treadmill (Columbus, OH) with 3-4 training sessions
consisting of 15 min at a speed of 10-15 m/min at an incline of 10%.
The exercise experiment involved the running of fasted mice for 60 min
at a speed of 15-20 m/min. Trained but not exercised mice were used as
controls for these analyses.
In Vivo Adenoviral Infection of Skeletal Muscle--
Adenovirus
infection of muscle was done according to standard protocols (35, 36).
Briefly, 4-6-week-old mice were anesthetized with a dose of 91 mg/kg
ketamine and 9.1 mg/kg of xylazine. Purified adenovirus (1 × 109 particles) was injected into the right quadriceps of
the mouse just above the kneecap. After 4 days, mice were fasted
overnight and either left untreated or stimulated with insulin (21 units/kg for 30 min) and the muscles were isolated as described above.
Protein Expression and Tissue Specificity of the GLUT4-EGFP
Transgenic Mice--
To develop an in vivo skeletal muscle
model in which the trafficking of GLUT4 could be readily visualized, we
generated several independent transgenic mouse lines expressing the
GLUT4-EGFP fusion protein driven by the HSA promoter. The tissue
specificity of GLUT4-EGFP expression was directly compared with that of
the endogenous GLUT4 protein in the heart, diaphragm, quadriceps,
gastrocnemius, and adipose tissue extracts by immunoblot analysis (Fig.
1). As previously established, control
nontransgenic mice displayed strong GLUT4 immunoreactivity in these
tissues at a molecular mass of ~50 kDa (Fig. 1B).
In contrast, GLUT4 immunoblots of the transgenic mice show the presence
of both the endogenous GLUT4 protein and the GLUT4-EGFP fusion protein
with a molecular mass of ~75 kDa (Fig. 1D). Importantly,
the 75-kDa band was only detected in skeletal muscle tissues
(diaphragm, quadriceps, and gastrocnemius) but was absent in both
cardiac and adipose tissue extracts. As expected, the GFP antibody was
unreactive to the tissues from the nontransgenic mice (Fig.
1A), while a 75-kDa band corresponding to GLUT4-EGFP was
detected in the skeletal muscle extracts from the transgenic mice (Fig.
1C). Again, this GFP immunoreactivity was restricted to the
skeletal muscle tissues and was not observed in cardiac or adipose
tissues. Although the extent of GLUT4-EGFP expression was different
among the 4 transgenic lines isolated, all of these lines displayed an
identical tissue-specific expression pattern (data not shown). These
results demonstrate that GLUT4-EGFP protein expression in these animals
occurs to a similar degree as endogenous GLUT4 protein levels and this
expression is appropriately restricted to skeletal muscle tissue.
GLUT4-EGFP Transgenic Mice Display Increased Glucose
Tolerance--
Previous studies have demonstrated that increased
expression of GLUT4 in skeletal muscle results in relative fasting
hypoglycemia with increased glucose disposal in response to a glucose
challenge (37, 38). Therefore, we fasted GLUT4-EGFP transgenic mice and
nontransgenic littermates and assessed the changes in plasma glucose
levels following an intraperitoneal injection of glucose (Fig.
2). As typically observed, wild-type mice
achieved a peak blood glucose level (~500 mg/dl) at 30 min that
subsequently decrease slowly over next 90 min. Although the GLUT4-EGFP
transgenic mice also reached a peak glucose level at 30 min, the
increase in circulating glucose was markedly reduced averaging only 275 mg/dl. The blood glucose levels were also significantly lower in the
transgenic mice over the entire time frame examined. These data
demonstrate that the addition of EGFP to the COOH-terminal domain of
GLUT4 does not impair its ability to transport glucose and transgenic expression in skeletal muscle results in enhanced glucose
tolerance.
Insulin-stimulated Translocation of GLUT4-EGFP--
The ability of
GLUT4-EGFP to enhance glucose disposal could either result from a basal
increase in the levels of GLUT4-EGFP at the cell surface and/or an
insulin-stimulated translocation of the GLUT4-EGFP from intracellular
storage sites to the cell surface. To investigate the subcellular
distribution of GLUT4-EGFP, we utilized skeletal muscle homogenization
coupled with sucrose velocity sedimentation to separate
sarcolemma/transverse-tubule enriched fractions (P1 and P2) from the
fractions enriched for intracellular membranes (34). As seen in Fig.
3, in the basal state there is a
relatively low level of GLUT4 in the P1 and P2 fractions with the
majority distributed in fractions 4-8 of the continuous sucrose
velocity gradient indicative of an intracellular localization (Fig.
3A). The distribution of the GLUT4-EGFP fusion protein
mirrored the distribution of the endogenous GLUT4 protein. Insulin
stimulation resulted in a decreased amount of endogenous GLUT4
localized to the intracellular compartment with a concomitant increase
in the P1 and P2 fractions (Fig. 3B). Similarly, the GLUT4-EGFP fusion protein also displayed a marked reduction in the
intracellular fractions paralleled with increased levels in the P1 and
P2 fractions in response to insulin. These data demonstrate that the
transgenic GLUT4-EGFP fusion protein localizes to similar intracellular
sites and undergoes an identical pattern of insulin-stimulated translocation as the endogenous skeletal muscle GLUT4 protein.
To distinguish between sarcolemma and transverse-tubule translocation,
we utilized confocal fluorescent microscopy of the GLUT4-EGFP protein
in comparison with the established sarcolemma-localized protein
(
In addition to sarcolemma translocation, several studies have
demonstrated that GLUT4 protein also translocates to the
transverse-tubule membrane (31-33). The transverse-tubules are deep
invaginations of surface membrane that originate from and are
continuous with the sarcolemma membrane. These invaginations are more
difficult to visualize by light microscopy, but can be distinguished by colocalization with an antibody against the transverse-tubule specific
Exercise Also Stimulates the Translocation of GLUT4-EGFP to the
Sarcolemma and Transverse Tubules--
In addition to insulin, a
variety of other stimuli such as exercise, contraction, and hypoxia
also enhance glucose uptake by induction of GLUT4 translocation in
skeletal muscle (3, 39). These "alternative" pathways of GLUT4
translocation are independent of insulin action and occur in the
complete absence of phosphatidylinositol 3-kinase activation (25, 26).
Therefore, we determined whether the GLUT4-EGFP protein was
appropriately distributed into vesicular compartments responsive to an
"alternate" signaling pathway. As shown in Fig.
6, exercise promotes the translocation of
the GLUT4-EGFP protein from intracellular storage sites to the
sarcolemma membrane (Fig. 6, panels a and b). The
sarcolemma translocation was again confirmed by the colocalization of
GLUT4-EGFP with Expression of Munc18c Specifically Inhibits the Insulin-stimulated
GLUT4-EGFP Translocation to Transverse Tubule
Membranes--
Previously, we and others (22-24, 40) have reported
that the syntaxin 4-binding protein, Munc18c, but not the Munc18b
isoform, is an important regulator of insulin-stimulated GLUT4
translocation in adipocytes. To begin to examine the functional role of
Munc18c in skeletal muscle, we compared the level of Munc18c protein
expression among several tissues (Fig.
8). Although it was previously reported that the abundance of Munc18c mRNA was relatively low in skeletal muscle (41), we clearly see expression of Munc18c in skeletal muscle is
comparable to other tissues examined.
Having confirmed the expression of Munc18c in skeletal muscle, we next
assessed the functional role of Munc18c on GLUT4 translocation in
vivo via infection with recombinant adenovirus encoding for Munc18c or Munc18b directly into skeletal muscle (Fig.
9). Injection of the Munc18b adenovirus
into the quadriceps muscle resulted in several fibers overexpressing
the Munc18b protein (Fig. 9, panel a). The overexpression of
Munc18b had no significant effect on the basal state distribution of
GLUT4-EGFP, which remained dispersed throughout the interior of the
fiber (Fig. 9, panels c and e). As typically
observed, insulin stimulation resulted in GLUT4-EGFP translocation to
the sarcolemma and transverse-tubule membranes in both noninfected
fibers as well as those expressing the Munc18b protein (Fig. 9,
panels b, d, and f).
Similarly, adenovirus-mediated expression of Munc18c had no effect on
the distribution of GLUT4-EGFP in the basal state (Fig. 10, panels a, c, and
e). Surprisingly, expression of Munc18c had no effect on the
insulin-stimulated translocation of GLUT4-EGFP to the sarcolemma
membrane compared with the surrounding noninfected fibers (Fig. 10,
panels b and d). In contrast, the infected fibers had a reduced translocation of GLUT4-EGFP to the transverse-tubule membranes (Fig. 10, panels d and f). At low
magnification this is observed as a normal translocation to the outer
surface membrane with an apparent decrease in the interior signal. At
high magnification this is noted by the lack of organized network
appearance within the interior of the infected fibers (compare Fig. 9,
panel f, with Fig. 10, panel f).
Since Munc18c specifically inhibits insulin-stimulated GLUT4
translocation through its binding to syntaxin 4, we next examined the
subcellular distribution of syntaxin 4 in skeletal muscle using the
subcellular fractionation procedure described previously (Fig.
11). Syntaxin 4 primarily is localized
to the P1 membrane fraction, and to a lesser extent the P2 membrane
fraction and the sucrose velocity gradient fractions. In addition,
syntaxin 4 does not display any insulin-induced translocation.
Since this subcellular fractionation protocol does not distinguish
between the two surface membranes found in skeletal muscle, we sought
to determine the relative surface membrane distribution of syntaxin 4 in skeletal muscle (Fig. 12). Confocal
immunofluorescence microscopy indicated that the majority of the
endogenous syntaxin 4 protein was localized at the sarcolemma (Fig. 12,
panels a, c, and e). Syntaxin 4 was also present
in the transverse-tubule membrane compartments as demonstrated by its
colocalization with the The expression of the insulin-responsive glucose transporter GLUT4
is highly restricted to muscle and adipose tissue, the two tissues that
undergo insulin-stimulated GLUT4 translocation (3, 42, 43). However,
the cellular and molecular analysis of GLUT4 translocation has largely
been performed in adipocytes due to the technical limitations
associated with skeletal muscle tissue. Nevertheless,
insulin-stimulated glucose disposal in skeletal muscle is
quantitatively the most important tissue accounting for ~80% of
glucose uptake in the postprandial state (44). In addition, although
the general GLUT4 translocation process in skeletal muscle appears
similar to adipocytes, there are several tissue-specific differences.
Skeletal muscle displays contraction/exercise-stimulated GLUT4
translocation through a pathway independent of but additive with that
of insulin (25, 26, 33). Furthermore, skeletal muscle contains two
distinct surface membranes, the sarcolemma membrane equivalent to the
adipocyte plasma membrane and a unique transverse tubule membrane, both
of which function as acceptor sites for GLUT4 translocation (31, 32,
45). Importantly, the transverse-tubule membrane has 1.5-fold more
surface area than the sarcolemma membrane and therefore quantitatively
accounts for the majority of skeletal muscle glucose uptake (46).
To develop a more tractable system to examine the regulation of GLUT4
translocation in skeletal muscle, we generated transgenic mice
expressing the GLUT4-EGFP fusion protein specifically in skeletal
muscle. This was based upon our previous observations that expression
of the GLUT4-EGFP fusion protein in adipocytes resulted in an identical
distribution of basal and insulin-regulated trafficking compared with
the endogenous GLUT4 protein (24, 47). As expected, the fidelity of the
GLUT4-EGFP transgene was confirmed by its ability to undergo both
insulin- and exercise/contraction-stimulated translocation to the
sarcolemma and transverse-tubule membranes in vivo. This was
confirmed by a more labor intensive subcellular membrane fractionation
method and by fluorescent microscopy which provided an easy and rapid
visual marker to view the movement of GLUT4-EGFP. In addition, the
GLUT4-EGFP protein fluorescence was sensitive enough to distinguish
between translocation to the sarcolemma and the transverse-tubules by
confocal fluorescence microscopy.
Having established the appropriate in vivo regulation of the
GLUT4-EGFP transgene in response to insulin and exercise/contraction, we utilized this system to examine the functional role of Munc18c in
these processes. Munc18c is a ubiquitous homologue of the neuronal n-Sec1/Munc18-1/Munc18a protein (41). This family of syntaxin-binding proteins has been postulated to exert both negative and positive roles
in modulating the docking and fusion of vesicles with target membranes.
For example, overexpression of Munc18c in 3T3L1 adipocytes, or
transgenic overexpression of the Drosophila Rop homologue
decreases GLUT4 translocation and neurotransmitter release,
respectively, consistent with a negative role for Munc18 proteins (24,
48). However, conditional mutants of the yeast homologue Sec1p or null alleles of the Munc18a isoform in mice lead to a complete block of
vesicle release in yeast and neurons, respectively, suggesting an
essential positive role for Munc18 proteins (49, 50). Furthermore, inhibition of Munc18c binding to syntaxin 4 through the use of a
blocking peptide also prevented insulin-stimulated GLUT4 translocation (40).
These apparently contradictory findings can now be partially resolved
based upon the functional and structural analysis of the syntaxin
1A-Munc18a complex (51, 52). Munc18a binding appears to maintain
syntaxin 1 in a closed conformational state in which the juxtamembrane
Hcore domain (directly involved in the SNARE core complex formation) is
in an intramolecular complex with the three amino-terminal coiled-coil
domains, termed the Habc domains. In this state, the syntaxin 1 Hcore
domain is unable to interact with the coiled-coil domains of SNAP25 and
VAMP2 responsible for forming the core complex. However, upon
alteration of Munc18a binding or a conformational change in Munc18a,
syntaxin 1 is able to undergo a transition to an open conformational
state. This releases the Hcore domain from the Habc domain allowing the
Hcore domain of syntaxin 1 to interact with and form a four-helical, coiled-coil bundle with SNAP25 and VAMP2. According to this model, overexpression of Munc18 would inhibit vesicle fusion by maintaining syntaxin in the closed conformational state, whereas complete displacement of Munc18 would prevent the interconversion between the
open and closed conformation, suggesting the necessity of the
transition state for the generation of the syntaxin-SNAP25-VAMP2 core
complex leading to vesicle docking and fusion.
Consistent with this hypothesis, we and others have previously reported
that overexpression of Munc18c in adipocytes inhibit insulin-stimulated
GLUT4 translocation (23, 24). This inhibitory effect was fully reversed
by increased expression of syntaxin 4, indicating a functional
requirement for excess syntaxin 4 over Munc18c (40). Similarly, we have
now observed that increased expression of Munc18c in skeletal muscle
inhibits insulin-stimulated GLUT4 translocation. However, to our
surprise this was specific for the translocation of GLUT4 to the
transverse-tubule membrane but without any effect on the translocation
of GLUT4 to the sarcolemma membrane. This can be reconciled by the fact
that the sarcolemma contains relatively high levels of syntaxin 4 compared with the transverse-tubules. At the level of
adenovirus-mediated skeletal muscle gene expression, sufficient Munc18c
was generated to saturate the transverse-tubule localized syntaxin 4 protein but not the more abundant sarcolemma-localized syntaxin 4. This
would account for the ability of overexpressed Munc18c to specifically
prevent GLUT4 translocation to the transverse tubules but not the sarcolemma.
In summary, the data presented in this article demonstrates that the
expression of the GLUT4-EGFP fusion protein in skeletal muscle of
transgenic mice displays the expected functional properties of the
endogenous GLUT4 protein. This includes the ability to translocate to
both the sarcolemma and transverse-tubule membranes in response to
insulin and exercise/contraction stimulation in vivo.
Furthermore, GLUT4 translocation in skeletal muscle also requires a
functional SNARE complex with the appropriate stoichiometry between the
syntaxin 4 and Munc18c proteins. This mouse model will provide a novel
approach to dissect the regulated and dynamic trafficking events
controlling GLUT4 translocation in skeletal muscle in
vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystroglycan) and IIID5E1 (specific
for the
1 subunit of the L-type calcium channel) were kindly
provided by Dr. Kevin P. Campbell, University of Iowa. A rabbit
polyclonal antibody against recombinant GFP was obtained from
CLONTECH (Palo Alto, CA). A sheep antibody was
prepared against the cytosolic domain of syntaxin 4 was obtained as
described previously (20). A rabbit Munc18 polyclonal antibody that
recognizes Munc18b was purchased from Affinity Bioreagents (Golden,
CO). Vectashield and the Monoclonal on Mouse kit were purchased from
Vector Laboratories (Burlingame, CA). Adenoviral vectors encoding
full-length, Flag-tagged Munc18c and Munc18b were prepared by the
University of Iowa Gene Therapy Vector Core according to standard procedures.
80 °C,
subsequently cut with a Zeiss Cryostat to a thickness of 7 µm and
adhered to glass slides. Similarly, mice were exercised as described
below and the basal and exercised mice were sacrificed and skeletal
muscle tissue prepared as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The human skeletal actin muscle promoter
drives the tissue-specific expression of the GLUT4-EGFP fusion protein
in transgenic mice. Tissues from wild-type (A and
B) and GLUT4-EGFP trangenic (C and D)
mice were isolated and subjected to immunoblotting as described under
"Experimental Procedures." Tissue extracts (5 µg) were prepared
from the heart (H), diaphragm (D), quadriceps
(Q), gastrocnemius (G), and epididymal adipose
depot (F) and subjected to SDS-polyacrylamide gel
electrophoresis. The samples were then immunoblotted with the GFP
antibody (A and C) or the GLUT4 antibody
(B and D). These are representative immunoblots
performed 6 times from four different independent transgenic
mice.
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Fig. 2.
The skeletal muscle specific GLUT4-EGFP
transgenic mice display greater insulin sensitivity than wild-type
mice. Wild-type and GLUT4-EGFP transgenic mice were fasted
overnight and then given an intraperitoneal injection of glucose (2 g/kg). Plasma glucose levels were then determined at the times
indicated as described under "Experimental Procedures." The average
and S.E. of the mean are shown for 7 wild-type (WT) and 10 GLUT4-EGFP
(TG) transgenic mice.
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Fig. 3.
Insulin stimulation results in the skeletal
muscle surface membrane translocation of both the endogenous GLUT4 and
GLUT4-EGFP fusion proteins. GLUT4-EGFP transgenic mice were fasted
overnight and either left untreated (A) or stimulated with
insulin (B) as described under "Experimental
Procedures." The hindquarter skeletal muscle was dissected and
subjected to differential and sucrose velocity centrifugation as
described under "Experimental Procedures." The surface membrane
fractions (P1 and P2) as well as the intracellular membrane fractions
(fractions 1-11) were immunoblotted with the GLUT4 antibody. These are
representative immunoblots performed from four different independent
pairs of transgenic mice.
-dystroglycan) and the transverse-tubule protein marker the L-type
voltage-dependent calcium channel (Figs.
4 and 5). In the basal state, GLUT4-EGFP was primarily distributed throughout the
interior of the muscle fibers with no evidence of sarcolemma localization (Fig. 4, panel a). Following insulin
stimulation, there was a marked redistribution of GLUT4-EGFP resulting
in a strong labeling of the fiber surface coupled with a greater
organization of the fiber interior (Fig. 4, panel b). The
sarcolemma membrane was labeled with the monoclonal
-dystroglycan
antibody and did not change following insulin stimulation (Fig. 4,
panels c and d). Consistent with the results of
the subcellular fractionation, the extent of GLUT4-EGFP and
-dystroglycan colocalization was relatively low in the basal state
but greatly increased following insulin stimulation (Fig. 4,
panels e and f).
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Fig. 4.
Insulin stimulation results in the
translocation of GLUT4-EGFP to the sarcolemma membrane.
GLUT4-EGFP transgenic mice were fasted overnight and either left
untreated (panels a, c, and e) or stimulated with
insulin (panels b, d, and f) as described under
"Experimental Procedures." The quadriceps muscle was frozen,
sliced, and incubated with the -dystroglycan monoclonal antibody and
secondary donkey anti-mouse IgG conjugated to Texas Red (panels
c and d). Sections were visualized by confocal
fluorescent microscopy (×63). These are representative fields from
three independent experiments.
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Fig. 5.
Insulin stimulation results in the
translocation of GLUT4-EGFP to the transverse-tubule membranes.
GLUT4-EGFP transgenic mice were fasted overnight and either left
untreated (panels a, c, and e) or stimulated with
insulin (panels b, d, and f) as described under
"Experimental Procedures." The quadriceps muscle was frozen,
sliced, and incubated with the monoclonal antibody directed against the
1 subunit of the
1-VDCC using the Monoclonal on Mouse kit
according to the manufacturers instructions (panels c and
d). Sections were visualized by confocal fluorescent
microscopy (×63) and then further magnified 11 times. The width of
each panel is ~13 µm. These are representative fields from three
independent experiments.
1 subunit of the L-type voltage-dependent calcium
channel (
1-VDCC) when visualized at high magnification (Fig. 5). In
the basal state, GLUT4-EGFP was randomly distributed within the
interior of the fiber and did not colocalize with the
1-VDCC subunit
(Fig. 5, panels a, c, and e). In contrast,
insulin stimulation resulted in the appearance of GLUT4-EGFP in a
striated pattern consistent with localization of the transverse-tubules
(Fig. 5, panel b). This was confirmed by the
insulin-stimulated colocalization of GLUT4-EGFP with the
1-VDCC
subunit (Fig. 5, panels d and f). Together, these
data demonstrate that in response to insulin, the GLUT4-EGFP fusion
protein translocates to the sarcolemma and transverse-tubule in a
manner similar to that of the endogenous GLUT4 protein.
-dystroglycan (Fig. 6, panels c, d, e,
and f). Similarly, exercise also induced the redistribution
of GLUT4-EGFP to the transverse tubule membranes as detected by
colocalization with the
1-VDCC subunit (Fig.
7, panels a-f). It should also
be noted that the extent of GLUT4-EGFP translocation was weaker than
observed with insulin stimulation, which may reflect the degree of
exercise used in these experiments. Regardless, these data further
demonstrate that the GLUT4-EGFP protein is also stored in the
exercise-responsive population of GLUT4-containing intracellular
vesicles.
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Fig. 6.
Exercise results in the translocation of
GLUT4-EGFP to the sarcolemma membrane. GLUT4-EGFP transgenic mice
were fasted overnight and either left untreated (panels a,
c, and e) or run for 60 min on a treadmill
(panels b, d, and f) as described under
"Experimental Procedures." The quadriceps muscle was frozen,
sliced, and incubated with the -dystroglycan monoclonal antibody and
secondary donkey anti-mouse IgG conjugated to Texas Red (panels
c and d). Sections were visualized by confocal
fluorescent microscopy (×63). These are representative fields from two
independent experiments.
View larger version (83K):
[in a new window]
Fig. 7.
Exercise stimulation results in the
translocation of GLUT4-EGFP to the transverse-tubule membranes.
GLUT4-EGFP transgenic mice were fasted overnight and either left
untreated (panels a, c, and e) or run for 60 min
on a treadmill (panels b, d, and f) as described
under "Experimental Procedures." The quadriceps muscle was frozen,
sliced, and incubated with the monoclonal antibody directed against the
1 subunit of the
1-VDCC using the Monoclonal on Mouse kit
according to manufacturers instructions (panels c and
d). Sections were visualized by confocal fluorescent
microscopy (×63) and then further magnified 11 times. The width of
each panel is ~13 µm. These are representative fields from three
independent experiments.
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[in a new window]
Fig. 8.
Munc18c is expressed in a number of different
tissues, including skeletal muscle. Tissues from a wild-type
C57Bl6 mouse were isolated and subjected to immunoblotting as described
under "Experimental Procedures." Tissue extracts (40 µg) were
prepared from the following tissues: pancreas (P), skeletal
muscle (Sk), heart (H), lung (Lu),
spleen (Sp), brain (B), and liver (Li)
and subjected to SDS-polyacrylamide gel electrophoresis. The samples
were immunoblotted with the Munc18c rabbit polyclonal antibody.
View larger version (60K):
[in a new window]
Fig. 9.
Expression of Munc18b has no effect on the
insulin stimulated translocation of GLUT4-EGFP to either the sarcolemma
or transverse-tubule membranes. The quadricep muscle of GLUT4-EGFP
transgenic mice was injected with a recombinant adenovirus encoding for
the Munc18b protein as described under "Experimental Procedures."
Four days following injection, the mice were fasted overnight and
either left untreated (panels a, c, and e) or
stimulated with insulin (panels b, d, and f). The
quadriceps muscle was frozen, sliced, and incubated with the Munc18b
antibody and secondary donkey anti-rabbit IgG conjugated to Texas Red
(panels a and b). Sections were visualized by
confocal fluorescent microscopy (×63) and then further magnified 11 times (insets). The width of the box is ~13 µm. These
are representative fields from three independent experiments.
View larger version (66K):
[in a new window]
Fig. 10.
Expression of Munc18c has no effect on the
insulin stimulated translocation of GLUT4-EGFP to the sarcolemma
membrane but inhibits GLUT4-EGFP translocation to the
transverse-tubules. The quadricep muscle of GLUT4-EGFP transgenic
mice was injected with a recombinant adenovirus encoding for the
Munc18c protein as described under "Experimental Procedures." Four
days following injection, the mice were fasted overnight and either
left untreated (panels a, c, and e) or stimulated
with insulin (panels b, d, and f). The quadriceps
muscle was frozen, sliced, and incubated with the Munc18c antibody and
secondary donkey anti-rabbit IgG conjugated to Texas Red (panels
a and b). Sections were visualized by confocal
fluorescent microscopy (×63) and then further magnified 11 times
(insets). The width of the box is ~13 µm. These are
representative fields from four independent experiments.
View larger version (31K):
[in a new window]
Fig. 11.
Syntaxin 4 localizes primarily to the P1
fraction, and to a lesser extent to the P2 and sucrose gradient
fractions, and does not translocate in response to insulin. Mice
were treated as described under "Experimental Procedures." 100 µg
of the P1 and P2 fractions and 100 µl of each sucrose gradient
fraction were subjected to SDS-polyacrylamide gel electrophoresis. The
samples were then immunoblotted with a digoxigenin-labeled sheep
Syntaxin 4 antibody and anti-digoxigenin POD Fab fragments (Roche
Molecular Biochemicals, Germany).
1-VDCC subunit (Fig. 12, panels b,
d, and f). However, based upon the relative signal
intensity, the amount of transverse-tubule localized syntaxin 4 protein
was substantially less than that found at the sarcolemma membrane.
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[in a new window]
Fig. 12.
Syntaxin 4 is localized to both the
sarcolemma and transverse-tubule membranes in skeletal muscle. The
quadriceps muscle from wild-type mice was frozen, sliced, and labeled
with the sheep syntaxin 4 antibody (panels a and
b), the -dystroglycan monoclonal antibody (panel
c), or the monoclonal
1-VDCC antibody (panel d) as
described under "Experiment Procedures." Sections were visualized
by confocal fluorescent microscopy (×63). The transverse-tubules
immunofluorescence was magnified 11 times from the confocal image. The
width of each panel is ~13 µm. These are representative fields from
two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Kevin P. Campbell and Valerie Allamand for providing skeletal muscle specific antibodies and assistance with the in vivo adenoviral infections. We also thank Diana Boeglin for assistance with the glucose tolerance tests. We further thank the University of Iowa Transgenic Animal Facility.
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FOOTNOTES |
---|
* This work was supported in part by Research Grants DK33823 and DK25295 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Postdoctoral Fellowship DK09813 from the National
Institutes of Health.
§ Present address: Dept. of Cell Biology, Oklahoma University Health Sciences Center, Oklahoma City, OK 73104.
¶ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, The University of Iowa, Iowa City, IA 52242. Tel.: 319-335-7823; Fax: 319-335-7886; E-mail: Jeffrey-Pessin@uiowa.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M007419200
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ABBREVIATIONS |
---|
The abbreviations used are:
PI, phosphatidylinositol;
SNAP, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor;
GLUT, glucose transporter;
EGFP, enhanced green fluorescent
protein;
PBS, phosphate-buffered saline;
HSA, human skeletal muscle
actin;
1-VDCC, L-type voltage-dependent calcium
channel.
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