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Division of Cell Biology (S.S., T.R., R.S., A.K.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and the Department of Biochemistry, Dartmouth Medical School (S.R.K.), Hanover, New Hampshire 03755
Address all correspondence and requests for reprints to: Amira Klip, Ph.D., Division of Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail amira{at}sickkids.on.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we have characterized vp165 in L6 cells, a rat skeletal muscle cell line that has been used extensively for the investigation of insulin-stimulated GLUT4 translocation (24, 25, 26). We found that the expression of vp165 was up-regulated during differentiation of myoblasts into myotubes. vp165 localized to the same intracellular compartments as GLUT4, and insulin caused translocation of vp165 from the intracellular pool to the cell surface, similar to GLUT4. Finally, we show that the vp165 compartment does not contain the other glucose transporters of L6 cells, GLUT1 and GLUT3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEM, FBS, and other tissue culture reagents were purchased
from Life Technologies (Gaithersburg, MD). 125I-Labeled
protein A, 125I-labeled sheep antimouse IgG, Fc
fragment-specific affinity-purified goat antimouse IgG, and Fc
fragment-specific affinity-purified goat antirabbit IgG were obtained
from ICN (Costa Mesa, CA). Dynabeads M-500 magnetic subcellular beads
were purchased from Dynal (Oslo, Norway). Polyclonal antisera against
the C-terminal sequence of GLUT4 or the C-terminal sequence of GLUT1
were obtained from East Acres Biologicals. Polyclonal antimouse GLUT3
antibody was a gift from Dr. I. Simpson (NIH, Bethesda, MD). Monoclonal
antibody against GLUT4 (1F8) was obtained from Genzyme Diagnostics
(Cambridge, MA). Mouse IgG and rabbit IgG, used as a control for
immunoisolation, were purchased from Sigma Chemical Co. (St. Louis,
MO). An affinity-purified polyclonal antibody against vp165 was raised
against the N-terminal 109 amino acids of rat vp165 (20).
Anti-1-subunit Na+/K+-ATPase
monoclonal antibody (6H) was a gift from Dr. M. Caplan (Yale
University, New Haven, CT). All other reagents were of analytical
grade.
Cell culture
A clonal line of L6 muscle cells selected for high fusion
potential was grown in MEM containing 2% FBS; these cells were
allowed to fuse and differentiate as described previously (27, 28).
Myoblasts were studied just before reaching confluency before any
evidence of differentiation (cell fusion). Fully differentiated
myotubes were studied at more than 90% maximal fusion. For subcellular
fractionation, L6 myotubes were serum deprived for 5 h and then
incubated with or without 100 nM insulin (Humulin R) for 30
min at 37 C and subjected to subcellular fractionation.
Membrane isolation
Subcellular fractionation of L6 myotubes was carried out
according to the method of Piper et al. for 3T3-L1
adipocytes (29) to obtain PM, high density microsome (HDM), and low
density microsome (LDM). Briefly, after pretreatment with or without
insulin, cells were rinsed with cold MEM and scraped with a rubber
policeman. The cells were pelleted, resuspended in homogenization
buffer (250 mM sucrose, 2 mM EGTA, 5
mM NaN3, and 20 mM HEPES, pH 7.4,
with 1 µM leupeptin, 1 µM pepstatin A, 10
µM E-64, and 200 µM
phenylmethylsulfonylfluoride), and homogenized with 10 strokes through
an EMBL Cell Cracker (clearance, 0.0016 in.). The homogenate was
centrifuged at 19,000 x g for 20 min, the pellet was
then resuspended in 5 ml homogenization buffer and layered onto 1.12
M sucrose, 1 mM EDTA, and 20 mM
HEPES, pH 7.4, and centrifuged at 100,000 x g for 60
min. The membranes recovered on top of the 1.12 M sucrose
were resuspended in homogenization buffer and pelleted at 40,000
x g for 20 min. The pelleted membranes were designated PM
based on marker enzyme composition. The supernatant of the 19,000
x g spin was sedimented at 41,000 x g for
20 min to yield HDM as the pelleted material. The supernatant was
centrifuged at 180,000 x g for 75 min to pellet the
LDM. Total membranes of L6 myoblasts and myotubes were prepared as
described previously (30). Subcellular fractionation of rat skeletal
muscle was carried out as described previously for hindquarter muscle
(3, 31). The F35 fraction of rat skeletal muscle was previously shown
to be enriched in GLUT4 (3, 31, 32). Protein concentration was
determined with a BCA kit (Pierce Chemical Co., Rockford, IL) according
to the manufacturer’s instructions.
Immunoisolation of GLUT4- or vp165-containing vesicles
Immunoisolation of GLUT4-containing vesicles was performed using
the monoclonal antibody IF8 immobilized on M-500 magnetic beads.
Coupling of the secondary antibody to the beads was carried out
according to the manufacturer’s instructions. Briefly, 4 x
108 beads were washed twice with PBS, pH 7.4, and then
incubated with 0.4 mg Fc fragment-specific affinity-purified goat
antimouse IgG in 0.1 M borate, pH 9.4, at 37 C for 20
h. The beads were rinsed twice with PBS containing 0.1% BSA, incubated
with 0.2 M Tris-HCl, pH 8.5, with 0.1% BSA for 4 h at
37 C, and washed in PBS-BSA for 5 min at 4 C. The coated beads were
subjected to coupling of primary antibody. Two micrograms of 1F8 or
control mouse IgG were incubated with the coated beads in PBS-BSA at 4
C for 16–18 h under constant rotation and washed four times with
PBS-BSA. L6 myotubes were fractionated to remove nuclei, mitochondria,
and PM, but without sedimenting LDM (33). The supernatant containing
LDM was adjusted to 100 mM potassium phosphate, pH 7.4, and
the protein concentration was measured. An aliquot containing 200 µg
protein, adjusted to 1 ml with PBS, 2 mM EDTA, was added to
the coated beads and incubated for 16–18 h at 4 C under constant
rotation. The supernatant was removed, the beads were washed three
times with ice-cold PBS without BSA, and the resulting supernatants
were combined with the first one. Pooled supernatants were centrifuged
at 200,000 x g for 60 min to sediment membranes.
Immunoisolation of vp165-containing vesicles was performed exactly as
described above, except that affinity-purified antirabbit IgG was
coupled to magnetic beads, and 8 µg affinity-purified vp165 antibody
or control rabbit IgG were used as the primary antibody. The sedimented
membranes were solubilized in 2 x Laemmli’s sample buffer (34)
containing 8 M urea (sample named SN). The material bound
to the beads was equally solubilized in 2 x Laemmli’s sample
buffer containing 8 M urea (sample named Pt).
Immunoblotting
Samples were solubilized in Laemmli’s sample buffer, resolved
by SDS-PAGE, and transferred onto polyvinylidene difluoride membranes.
Immunoblotting was carried out as described previously (31) using
anti-1 Na+/K+-ATPase monoclonal antibody
(1:1000), polyclonal antiserum against GLUT4 (1:1000), GLUT1 (1:2000),
GLUT3 (1:2000), and affinity-purified antibody against vp165 (1
µg/ml). Immunoreactive bands were quantified by laser scanning
densitometry of the autoradiograms.
Statistics
Throughout the study, Student’s paired t test was
applied. This test analyzes the mean of differences between two
conditions, such as control and insulin-treated, even if the control is
assigned a value of 1.0. The test gathers the difference between the
two conditions from each experiment and then analyzes the average of
the differences and determines its significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, immunoblots of vp165
in L6 myoblasts and myotubes are compared to immunoblots of GLUT4 and
1 Na+/K+-ATPase. The F35
fraction of rat skeletal muscle was also probed for positive control of
blotting. Consistent with previous results, the immunoreactive band of
GLUT4 was hardly seen in myoblasts even after long exposure of the
film, but it increased markedly after differentiation into myotubes. In
contrast, a small, but detectable, amount of vp165 protein was present
in myoblasts, and like GLUT4, the amount of vp165 increased
substantially during differentiation into myotubes. Densitometric
analysis of the immunoreactive bands revealed that the amount of vp165
was greater by 4.3 ± 0.7-fold in myotubes than in myoblasts
(P < 0.01; n = 3; Fig. 1B). Consistent with our
previous reports (27, 28), GLUT4 was 9.6 ± 0.8-fold greater in
myotubes than in myoblasts (P < 0.01; n = 3). In
contrast, the expression of 1
Na+/K+-ATPase did not change during
differentiation (1.0 in myoblasts vs. 1.0 ± 0.1 in
myotubes). These results indicate that the expression of vp165 is
up-regulated during differentiation of L6 skeletal muscle cells.
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1
Na+/K+-ATPase in the three distinct membrane
fractions, LDM, HDM, and PM, were determined by immunoblotting.
Densitometric analysis of 10 independent experiments shows that
relative enrichments of vp165 per mg protein in HDM and LDM were
3.71 ± 0.60 and 1.98 ± 0.54, respectively, compared to that
in PM. On the average, the subcellular fractionation of one confluent
10-cm dish of L6 myotubes yielded approximately 107 µg protein from
PM, 47 µg protein from HDM, and 131 µg protein from LDM. When this
yield of protein from PM, HDM, and LDM fractions was considered, the
relative amounts of vp165 in HDM and LDM compared to that in PM were
1.76 ± 0.27 and 2.54 ± 0.60, respectively. By a similar
calculation, the relative recoveries of GLUT4 in the PM, HDM, and LDM
were 1.00, 1.41 ± 0.84, and 3.34 ± 0.80, respectively
(Table 1). In contrast, the relative enrichment of
1-subunit of Na+/K+-ATPase, a PM
protein, per mg protein from PM was approximately 20 times greater than
that from LDM, indicating that the majority of the PM was recovered in
the PM fraction and that the contamination of LDM with PM is low. Thus,
both vp165 and GLUT4 were found mainly in the LDM under basal
conditions.
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). Insulin caused statistically significant decreases
in both proteins in the LDM and statistically significant increases in
the PM. The immunoreactivity of the bands was quantified by laser
scanning densitometry, and the recovery of each protein in PM, HDM, and
LDM was calculated (Fig. 2B). In 10 independent experiments, the
content of vp165 was augmented by 33 ± 13% in PM after insulin
stimulation and was reduced by 51 ± 6% in LDM. Similarly, the
content of GLUT4 protein increased by 36 ± 13% in the PM and
decreased by 27 ± 5% in the LDM. There were not statistically
significant changes in GLUT4 or vp165 in the HDM. Insulin did not
affect the distribution of 1-subunit of
Na+/K+-ATPase in any of the fractions (Figs. 2A, bottom panel, and B).
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shows that a substantial
portion of vp165 was immunoisolated with GLUT4-containing vesicles. The
amounts of immunodetectable GLUT4 protein sedimented nonspecifically by
the nonimmune IgG was subtracted from the amount immunoprecipitated
with 1F8, and this difference was then divided by the amount of GLUT4
protein immunoprecipitated with 1F8. By this calculation, 91 ±
3% (mean ± SE; n = 4) of the GLUT4 in the
pellet was specifically immunoprecipitated with 1F8. The efficiency of
the immunoisolation of vesicles is defined as the ratio of the amount
of antigen immunoprecipitated with 1F8 to the total immunoprecipitable
antigen, which is the sum of the amount left in the SN and the amount
bound to the 1F8. The efficiency of the immunoisolation of
GLUT4-containing vesicles was 83 ± 5%, and that of vp165
coimmunoisolated with GLUT4-containing vesicles was 67 ± 12%
(n = 4). Two other isoforms of glucose transporter, GLUT3 and
GLUT1, are expressed in L6 myotubes (28). Although these isoforms also
translocate to the cell surface in response to insulin (25), it has not
been previously explored whether all glucose transporter isoforms
localize to the same intracellular vesicles. Therefore, we searched for
GLUT1 and GLUT3 on the immunoisolated GLUT4-containing vesicles. As
shown in Fig. 3, the majority of GLUT1 and GLUT3 were excluded from the
immunoisolated GLUT4-containing vesicles.
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). The specificity of the
immunoisolation of vp165-containing vesicles was 90 ± 4%. As can
be appreciated from the figure, vp165 was almost completely
immunoisolated. In four separate experiments, the efficiency of
immunoisolation of vp165-containing vesicles was 93 ± 3%.
Importantly, 90 ± 3% of GLUT4 was simultaneously immunoisolated
with vp165-containing vesicles. In contrast, GLUT1 and GLUT3 were
excluded from these vesicles (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the amount of vp165 also
markedly increased during differentiation into myotubes, but in
contrast to GLUT4, a measurable amount of vp165 was present in
myoblasts. These results are consistent with the observations in mouse
3T3-L1 cells, where the regulation of vp165 expression was also
different from that of GLUT4, and vp165 was also present in the
undifferentiated fibroblasts (23).
Insulin causes translocation of vp165 from the LDM to the PM
concomitantly with GLUT4 in rat adipocytes (17, 18) and in mouse 3T3-L1
adipocytes (23). However, in skeletal muscle, there has been no report
describing the translocation of vp165 to the PM after insulin
stimulation. Coderre et al. demonstrated that after insulin
stimulation in vivo, the amount of vp165 (gp160) is reduced
in an insulin-sensitive intracellular pool of rat skeletal muscle
membranes containing GLUT4, indicating the redistribution of this
molecule (22). However, the increase in vp165 in the PM in response to
insulin was not shown. In the present study we performed subcellular
fractionation of L6 myotubes to demonstrate translocation of vp165 to
the PM after insulin stimulation. This system has been used previously
to study the mechanisms of insulin-induced translocation of GLUT4 in
skeletal muscle cells. We used a subcellular fractionation procedure
that was originally developed for 3T3-L1 adipocytes (29). This method
enabled us to obtain a more pure PM fraction compared to the previously
used method (30). This is supported by the observation that the
relative distribution of 1
Na+/K+-ATPase, a PM marker, determined by
densitometric analysis of the immunoreactive bands on the
autoradiograms is 20 times greater in the PM compared to the LDM. Also,
Fig. 2B shows that the amount of GLUT4 increased by 36 ± 13% in
the PM and decreased by 27 ± 5% in the LDM in response to
insulin. These values are comparable to the increase in cell surface
GLUT4 in response to insulin detected by photolabeling (35),
corroborating the idea that this method for subcellular fractionation
of L6 myotubes is suitable to detect insulin-dependent translocation of
proteins from the LDM to the PM.
In insulin-responsive cells, namely heart, fat, and skeletal muscle, GLUT4 glucose transporters are located in specialized intracellular membrane vesicles that translocate to the cell surface upon insulin stimulation (4, 5, 6). In agreement with observations made in rat adipocytes (17, 18, 21), rat skeletal muscle (21, 22) and mouse 3T3-L1 adipocytes (23), the majority of vp165 in L6 myotubes was colocalized to GLUT4-containing vesicles. Under conditions where 83 ± 5% of GLUT4-containing vesicles were efficiently immunoisolated, 67 ± 12% of vp165 was simultaneously immunoisolated. Although these values are not statistically different, these results suggest that there may be some intracellular membrane vesicles that contain vp165, but are devoid of GLUT4. Conversely, 90 ± 3% of GLUT4 was coimmunoisolated when 93 ± 3% of vp165-containing vesicles were immunoisolated.
Consistent with a previous report that GLUT1 is excluded from
GLUT4-containing vesicles in rat skeletal muscle (21), GLUT1 was not
detected in GLUT4-containing vesicles of L6 myotubes. In addition, we
demonstrated that GLUT3, another glucose transporter isoform expressed
in L6, is also excluded from GLUT4-containing vesicles (Fig. 3).
Exclusion of GLUT1 and GLUT3 from GLUT4-containing vesicles was further
confirmed by the finding that GLUT1 and GLUT3 were not detected on
vp165-containing vesicles when the majority of GLUT4 was
coimmunoprecipitated with vp165-containing vesicles. Taken together,
these findings suggest that vp165 and GLUT4 cosegregate to the same
intracellular compartment, whereas GLUT1 and GLUT3 are excluded from
this compartment. The results also demonstrate that in L6 myotubes
there is a specific compartment of GLUT4 that is distinct from the
compartments populated by the other glucose transporters.
In conclusion, this study shows that in L6 cells, vp165 and GLUT4 colocalize to the same intracellular compartment and are regulated by insulin in a similar fashion. vp165 can, therefore, serve as an excellent marker to study insulin-elicited translocation to the cell surface in these cells. The physiological function of vp165 and its role in established insulin effects are currently not known, but given that vp165 is regulated by insulin like GLUT4 and that vp165 colocalizes with GLUT4, but not with GLUT1 or GLUT3, the consequences of vp165 translocation may be as far reaching as they are for GLUT4 in mediating the anabolic responses to the hormone.
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Acknowledgments |
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1-subunit of
Na+/K+-ATPase antibody, and Dr. I. Simpson for
the GLUT3 antibody. |
Footnotes |
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Received July 18, 1996.
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References |
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2 and ß1
subunits of the Na+/K+-ATPase from
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