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Mediates Insulin-Induced Glucose Transport in Primary Cultures of Rat Skeletal Muscle
Faculty of Life Sciences (L.B., A.A., A.B., T.T., S.R.S.)
Gonda-Goldschmied Center Bar-Ilan University Ramat-Gan 52900,
Israel
Institute of Molecular Oncology (T.K.) and Department
of Microbiology (M.O.) Showa University Tokyo
142-8555, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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by insulin participates in the mediation of insulin effects on
glucose transport in skeletal muscle. Studies were performed on primary
cultures of rat skeletal myotubes. The role of PKC in
insulin-induced glucose uptake was evaluated both by selective
pharmacological blockade and by overexpression of wild-type and
point-mutated inactive PKC isoforms in skeletal myotubes. We found
that insulin induces tyrosine phosphorylation and translocation of
PKC to the plasma membrane and increases the activity of this
isoform. Insulin-induced effects on translocation and phosphorylation
of PKC were blocked by a low concentration of rottlerin, whereas the
effects of insulin on other PKC isoforms were not. This selective
blockade of PKC by rottlerin also inhibited insulin-induced
translocation of glucose transporter 4 (GLUT4), but not glucose
transporter 3 (GLUT3), and significantly reduced the stimulation of
glucose uptake by insulin. When overexpressed in skeletal muscle,
PKC and PKC
were both active. Overexpression of PKC induced
the translocation of GLUT4 to the plasma membrane and increased basal
glucose uptake to levels attained by insulin. Moreover, insulin did not
increase glucose uptake further in cells overexpressing PKC.
Overexpression of PKC did not affect basal glucose uptake or GLUT4
location. Stimulation of glucose uptake by insulin in cells
overexpressing PKC was similar to that in untransfected cells.
Transfection of skeletal myotubes with dominant negative mutant PKC
did not alter basal glucose uptake but blocked insulin-induced
GLUT4 translocation and glucose transport. These results demonstrate
that insulin activates PKC and that activated PKC is a major
signaling molecule in insulin-induced glucose transport. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit of the insulin receptor
(IR) activates the IR tyrosine kinase, inducing a cascade of
events leading to stimulation of glucose uptake into several tissues
(1). Glucose uptake is preceded by translocation of the GLUT3 and GLUT4
glucose transporters (GLUTs) from internal membranes to the plasma
membrane (2). While several of the key molecules participating in this
cascade have been identified, the precise steps between IR activation
and GLUT translocation have not been entirely delineated. Activation of
the IR leads to phosphorylation of the IR substrate family of proteins.
This leads to activation of a number of downstream signaling pathways
including mitogen-activated protein (MAP) kinase and
phosphatidylinositol 3-kinase (PI3K) (1). One component of this
cascade is protein kinase C (PKC), which has been shown to be activated
by insulin in some systems (3, 4, 5). PKC comprises a family of
serine-threonine kinases that play an important regulatory role in a
variety of biological phenomena (6, 7). The family is composed of a
number of individual isoforms that are categorized according to their
mechanisms of activation. It is generally believed that the enzymes,
when quiescent, are located in the cytoplasm and that upon activation
they are translocated to the plasma membrane (7). The pattern of PKC
isoform distribution varies among different tissue (8).
Although PKC isoforms are activated by substances released
intracellularly by PI3K activity, the involvement of specific
PKC isoforms in insulin-induced glucose uptake has not been
definitively established. This is primarily because of the failure of
phorbol ester-induced down-regulation of certain PKC isoforms to alter
either basal or insulin-stimulated glucose transport (9, 10). However,
recent studies implicate certain PKC isoforms, including , ß2,
, and 
, in the insulin-signaling cascade (11, 12, 13). We recently
reported that rat skeletal muscle in primary culture expresses six
isoforms, , 
, 
, ß2, , and , and that
insulin-stimulated glucose uptake involves activation of PKCs ß2,
, and (14). Although PKC has been shown to participate in
insulin signaling in some systems (15), this isoform was barely
detectable in skeletal muscle in primary culture. The activation of
PKCs ß2, , and was associated with a specific increase in
tyrosine phosphorylation and translocation of the three isoforms. The
insulin-induced effects on glucose transport, and on PKCß2 and ,
were blocked by selective inhibitors of phosphatidylinositol-3-kinase
(PI3K), whereas those on PKC were not. These findings raise a
question as to whether or not PKC is likely to be involved in
insulin-induced glucose uptake.
Although several roles have been ascribed to PKC in a variety of
systems (16, 17, 18, 19, 20), the possible involvement of this isoform in
insulin-induced glucose transport has not been reported. The potential
participation of this isoform in insulin signaling is suggested by the
in vitro findings that coincubation of PKC with IR
resulted in tyrosine phosphorylation of purified PKC, accompanied by
an increase in its activity (21). Similarly, it was recently reported
that insulin could induce coprecipitation of PKCs , , and
with IR in NIH-3T3 cells expressing the human IR (22).
Skeletal muscle is the major target organ for insulin regulation of blood glucose levels. The preparation of primary skeletal muscle cultures obtained from neonatal rat pups is a useful model for the study of regulation of glucose uptake by insulin. These cells, plated initially as individual myoblasts, align and fuse into multinucleated muscle fibers by day 3–4 in vitro. The mature fibers display resting membrane and action potentials that are nearly identical to those seen in vivo, and the physiological expression of a number of membrane proteins in this preparation, in contrast to muscle cell lines such as L6, resembles closely that observed in vivo (23, 24, 25). An earlier report from this laboratory suggested that activation of PKC might be a fundamental early signal in stimulation of the Na+/K+ pump by insulin in cultured skeletal muscle, but the specific isoforms involved were not considered (26).
In this study, we have used the primary muscle culture system to
specifically investigate the possibility that insulin activation of
endogenous PKC may play a role in insulin stimulation of glucose
transport in skeletal muscle. We have found that selective blockade of
PKC resulted in elimination of insulin-induced stimulation of
glucose transport and GLUT4 translocation. In addition, overexpression
of PKC increased glucose transport and translocated GLUT4. Taken
together, these findings indicate that PKC is a major isoform
mediating insulin-induced effects on glucose transport in skeletal
muscle.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and PKC and . As shown in Fig. 1
, insulin
treatment resulted in translocation and tyrosine phosphorylation of
PKC but not PKC. Translocation of PKC to the plasma membrane
was detectable within 10 min after addition of insulin to the cultures,
and membrane levels remained elevated for least 30 min (Fig. 1A).
Insulin-induced tyrosine phosphorylation of PKC (Fig. 1B) could be
detected as early as 1 min after insulin stimulation, remained elevated
for 10 min, and decreased to near control levels by 30 min. The
insulin-induced translocation and tyrosine phosphorylation of PKC
was accompanied by a significant increase in its kinase activity, which
peaked by 1 min and gradually returned to control levels (Fig. 1C).
Insulin had no effect on PKC activity.
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in a manner similar to that by
which insulin activates PKCs ß2 and (14). To evaluate the
possibility that this activation of PKC may be important to
insulin-induced glucose transport, we studied effects of a selective
inhibitor of PKC, rottlerin, on insulin-induced activation of
PKC, glucose uptake, and GLUT4 translocation. Rottlerin prevents the
binding of ATP to its binding site, thus preventing PKC
phosphorylation and activation; it reportedly blocks PKC at a
concentration of 3–6 µM, one-tenth of that required to
block the other PKC isoforms (50–100 µM) in cultured
skeletal muscle (27). Figure 2A presents
a Western blot showing effects of rottlerin (5 and 100
µM) on insulin-induced translocation of PKCs ß2, ,
and . As expected, 5 µM rottlerin significantly
reduced insulin-induced translocation of PKC while translocation of
PKC ß2 and was essentially unaltered. At a concentration of 100
µM, the translocation of PKC ß2 and was also
significantly reduced, thus demonstrating the selectivity of low
concentrations of rottlerin for PKC. Figure 2B shows that tyrosine
phosphorylation of the three PKC isoforms was affected in a similar
dose-effect relation. We next examined the effects of rottlerin on
insulin-induced glucose uptake and translocation of GLUTs 3 and 4. The
graph in Fig. 3A shows that
rottlerin, when added to the cultures to a final concentration of 5
µM, selective for inhibition of PKC, nearly eliminated
the effect of insulin to increase glucose uptake. Finally, as seen in
Fig. 3B, this concentration of rottlerin also blocked insulin-induced
translocation of GLUT4 but not that of GLUT3.
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severely impairs the ability of
insulin to increase glucose transport and to translocate the GLUT4
glucose transporter. They further indicate that this PKC isoform may
play an important role in this phenomenon. However, the pharmacological
approach is not specific. Therefore, to test the role of PKC more
directly, we used an adenovirus expression system to overexpress
specific PKC isoforms in skeletal myotubes. As can be seen in Fig. 4A, myotubes overexpressing PKC isoforms
and show high protein expression of each isoform compared with
endogenous protein levels. Overexpression of each isoform resulted in
an increase in activity of that isoform without altering the activity
of other isoforms. Thus, overexpression of PKC did affect the
activity of PKCs , , and ß2 (data not shown). The
overexpression of a point-mutated dominant negative PKC resulted in
a high level of expression of this protein (Fig. 4C), which, however,
was inactive. The basal activity and insulin-induced stimulation of
PKCs ß2 and were not altered by the overexpression of dominant
negative PKC (Fig. 4D).
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and
PKC. Figure 5C shows that basal glucose uptake in cells
overexpressing PKC was elevated to levels similar to those attained
by addition of insulin to control myotubes. In addition, insulin failed
to further increase glucose uptake in cells overexpressing PKC. In
contrast, overexpression of PKC in myotubes did not alter either
basal or insulin-induced glucose uptake. To validate the contribution
of endogenous PKC to insulin-induced glucose uptake in myotubes,
cells were infected with a dominant negative PKC adenovirus
construct, which down-regulates endogenous PKC activity.
Overexpression of this dominant negative PKC did not alter basal
glucose uptake. However, whereas insulin increased glucose uptake
by 250% in control myotubes, it increased glucose uptake by only
50–60% in cells overexpressing dominant negative PKC.
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). As further shown in Fig. 5A, concomitant with the increase in glucose uptake, overexpression of
PKC caused an increase in plasma membrane GLUT4 to levels achieved
by insulin in control cells. There was no change in translocation of
GLUT 3 by PKC overexpression. Moreover, addition of insulin to cells
overexpressing PKC did not cause a further translocation of
additional GLUT4 to the plasma membrane. Indeed, stimulation by insulin
of cells overexpressing PKC induced a decrease in plasma membrane
GLUT4 and an increase in the amount of GLUT4 in the internal membrane
fraction. In contrast, overexpression of PKC neither translocated
GLUT4 nor altered the effect of insulin to translocate this transporter
to the plasma membrane (Fig. 5A). As shown in Fig. 5B, overexpression
of the dominant negative PKC completely abrogated insulin-induced
translocation of GLUT4. No change in expression of GLUT4 in internal
membrane and plasma membrane components could be detected.
Overexpression of dominant negative PKC altered neither basal
distribution nor insulin-induced translocation of GLUT3 (not
illustrated). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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in primary skeletal muscle cells and that
the activated PKC is associated with selective translocation of the
insulin-sensitive glucose transporter GLUT4 to the plasma membrane and
increased glucose uptake. These results were validated by both
pharmacological intervention and overexpression of wild-type and
dominant negative PKC. Pharmacological blockade of PKC was
accomplished by utilizing rottlerin, which preferentially inhibits
PKC at low concentrations, presumably by competing with ATP for its
binding site (27). At a concentration reportedly selective for PKC
(5 µM), we were able to selectively block insulin-induced
translocation and tyrosine phosphorylation of this PKC isoform without
altering insulin-induced translocation and tyrosine phosphorylation of
PKCs ß2 and . We have recently shown that insulin-induced
activation of PKCs ß2 and similarly involves tyrosine
phosphorylation and translocation, and that this occurs via a
PI3K-dependent pathway (14). The kinase responsible for phosphorylation
of PKC has not yet been identified. It is known, however, that
phosphorylation can occur by the Src family of kinases as well as by
autophosphorylation (28). Similar inhibition of insulin-induced
activation of PKC was obtained with overexpression of dominant
negative PKC. Of special interest is the finding that this selective
inhibition of PKC was associated with blockade of insulin-induced
translocation of the GLUT4 transporter and a significant reduction in
insulin-induced glucose uptake. Insulin-induced translocation of GLUT3,
on the other hand, was not affected by rottlerin. These results
strongly suggest that PKC is likely to play a cardinal role
in insulin-induced translocation of GLUT4 and accompanying increase in
glucose transport in skeletal muscle. Moreover, these findings,
together with those of a recent study from our laboratory (14),
indicate that insulin-induced translocation of GLUT3 does not involve
PKC but may involve the participation of PKCs ß2 and , which
are also activated by insulin.
The notion that PKC is involved in mediation of insulin effects (not
necessarily exclusively) on glucose transport is strengthened further
by the results of experiments on overexpression of this isoform. Thus,
overexpression of PKC translocated GLUT4 to the plasma membrane,
increased basal glucose uptake, and actually induced PKC to leave
the plasma membrane and increase in the internal membrane fraction. The
mechanism of this reverse translocation is not clear and requires
further study. One possible explanation may involve mechanisms similar
to those that are responsible for glucose-induced down-regulation of
plasma membrane GLUT4 in skeletal muscle (29, 30). Clearly, however,
overexpression of PKC interfered with the ability of insulin to
translocate GLUT4 to the plasma membrane and to further increase
glucose uptake. The latter finding indicates that PKC and insulin
share a common pathway. In contrast, overexpression of PKC was
without effect on either GLUT4 translocation, basal glucose uptake, or
on insulin-induced translocation of GLUT4 and glucose transport.
These findings attest not only to the involvement of PKC in
mediation of insulin-induced effects on glucose uptake, they also
conclusively demonstrate that PKC is not at all involved in either
basal or insulin-stimulated glucose transport in skeletal muscle,
either directly or indirectly via activation of other signaling
proteins.
Our findings are not in accord with the conclusion that diacylglycerol
(DAG)-sensitive PKC isoforms are unlikely to participate in
insulin-induced glucose transport (10). This conclusion is based on the
failure of phorbol ester-induced down-regulation of certain PKC
isoforms to alter either basal or insulin-stimulated glucose transport
(9, 10). This down-regulation assumes that PKC activation depends
entirely on translocation of a given isoform to the plasma membrane. As
recently pointed out, however, there are certain limitations to this
notion (7, 21). Many PKC isoforms can be detected in the particulate
fraction of cells independent of the activation state, and PKC
stimulation need not occur exclusively by translocation to the plasma
membrane (31, 32). In this regard, translocation to the nucleus, as
well as association with cytoskeletal components on activation, is well
documented. In addition, products of lipid hydrolysis, such as free
cis-unsaturated fatty acids, may activate PKC directly in
the cytosol (33). Hence, down-regulation of DAG-sensitive isoforms by
chronic phorbol ester stimulation would not appear to be a reliable
approach for definitive studies implicating or ruling out the
participation of these isoforms in insulin signaling. Using specific
blockade of PKC by pharmacological inhibition and by overexpression
of kinase-inactive PKC, we have shown that this isoform is important
in insulin-induced glucose transport. Moreover, other studies have also
shown that DAG-sensitive PKCß2 is also an important participant in
insulin effects on glucose uptake (11, 14).
Our results do not agree with a previous report that PKC was not
involved in basal or insulin-induced glucose uptake in rat adipocytes
(10). One possible explanation for this discrepancy might be related to
the difference in tissues studied; adipocytes and skeletal muscle may
indeed utilize different PKC isoforms in the signaling of
insulin-induced glucose transport. Another possibility for the failure
to find a significant role for PKC in insulin signaling might be
related to the different methods used for overexpression. Using
adenovirus constructs, we obtained very high efficiency of protein
expression. More than 90% of the cultured cells demonstrated increased
protein levels, and cells overexpressing the and PKC isoforms
displayed an increase of more than 10-fold in specific PKC activity as
compared with wild-type controls. In contrast, a study using
electroporation for transfecting the primary adipocyte cultures (10)
achieved only an approximately 2-fold increase in PKC activity;
activity levels for and ß2 were not reported.
The results of our studies on overexpression of PKC indicate that
this isoform is essential for insulin-induced translocation of GLUT4.
Thus, in cells overexpressing the dominant negative PKC, not only
was insulin unable to increase activity of this enzyme, insulin had no
detectable effect on GLUT4 translocation. The stimulation of glucose
transport that remains in cells expressing dominant negative PKC can
be attributed to the effect of insulin to translocate GLUT3. These
findings were similar to those we obtained in pharmacological studies
utilizing rottlerin; this agent, in concentrations that selectively
inhibit insulin effects on PKC, blocked translocation of GLUT4 but
not of GLUT3.
Whereas this is the first report implicating PKC in insulin
signaling, PKC has been shown to be important in a number of cell
functions. Thus, PKC has been shown to be involved in stimulation of
the Na+/K+ antiproton in C6 glioma
cells, in hydrolysis of phosphoinositide and formation of
PGE2, and in keratinocyte and murine erythroleukemia cell
differentiation (26, 34, 35, 36). PKC has also been implicated in
Sis-induced transformation of NIH 3T3 cells as well as activation of
Na-K-2Cl cotransport (36, 37, 38). In addition, PKC has been
demonstrated to participate in carbachol-induced secretion in parotid
acinar cells (40). Finally, changes in activation state of PKC are
associated with actions of various growth factors and other agents
including platelet-derived growth factor, transforming growth
factor-, substance P, ligand of IgE receptor, and extracellular ATP
or UTP (41, 42, 43).
Our findings of PKC involvement in insulin-induced glucose uptake
should not be taken as a negation of a role for other PKC isoforms, or
other protein kinases such as protein kinase B (44) or MAP
kinase (45) in this phenomenon. Indeed, regarding other PKC isoforms,
we also reported that insulin stimulation of PKCs ß2 and play an
important role in the activation of glucose uptake by insulin. We would
suggest that several of the isoforms participate at different steps in
the pathway. Ample evidence has been presented in studies on other cell
types for the involvement of DAG-sensitive PKC isoforms, especially
PKCß2 (as well as the non-DAG-sensitive PKC) in insulin-induced
glucose transport (5, 11, 12, 13). In addition, we have recently reported
that insulin activates PKCs ß2 and (associated with tyrosine
phosphorylation and translocation) in primary cultures of rat skeletal
muscle (14). Thus, in primary cultures of skeletal muscle, the
DAG-sensitive PKCs ß2 and , in addition to , are activated by
insulin and appear to be involved in mediation of glucose uptake
stimulated by insulin. The determination of the precise steps
at which each of these isoforms acts in the insulin-signaling pathway
remains to be investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Preparation of Rat Muscle Cell Cultures
Skeletal muscle cultures were prepared from thigh muscles
obtained from 1- to 2-day neonatal rats as described previously
(23, 24, 25). The muscles were removed from the limbs, washed in PBS to
remove excess blood cells, and then transferred to a
Ca2+-free, 0.25% trypsin solution containing EDTA (1
mM) for incubation with continuous stirring at 37 C. Cells
were collected after serial trypsinization (successive 10-min periods
until all tissue was dispersed), centrifuged for 5 min at 500 x
g, and resuspended in growth medium (83% DMEM-high glucose,
15% horse serum, 2% chick embryo extract), to a concentration of
0.8 x 106 cells/ml for plating in collagen-coated
10-cm plastic tissue culture (10 ml/dish) or 24-well plates (400
µl/well). Cultures were grown in water-saturated atmosphere of 95%
air-5% CO2 at 37 C. On day 5 in culture, myotubes were
transferred to low glucose (4.5 mM), serum-free DMEM
containing 1% BSA for 24 h before study.
Preparation of Cell Lysates for Immunoprecipitation
Culture dishes (90 mm; Nunc, Roskilde, Denmark)
containing the muscle cells were washed with Ca
2+/Mg2+-free PBS and then mechanically detached
in RIPA buffer (Tris HCl, pH 7.4, 50 mM; NaCl, 150
mM; EDTA, 1 mM; NaF, 10 mM; Triton
X-100, 1%; SDS, 0.1%; Na deoxycholate, 1%) containing a cocktail of
protease inhibitors (leupeptin, 20 µg/ml; aprotinin, 10 µg/ml;
PMSF, 0.1 mM; DTT, 1 mM) and phosphatase
inhibitors (orthovanadate, 200 µM; pepstatin, 2 µg/ml).
After scraping, the preparation was centrifuged at 20,000 x
g for 20 min at 4 C. The supernatant was used for
immunoprecipitation.
Immunoprecipitation
To 0.3 ml of cell lysate, 25 µl of Protein A/G Sepharose were
added and the suspension was rotated continuously for 30 min at 4 C.
The preparation was then centrifuged at 20,000 x g at
4 C for 10 min, and 30 µl of A/G Sepharose were added to the
supernatant along with specific monoclonal antibodies to the individual
PKC isoforms (dilution 1:100). This was rotated overnight at 4 C. The
suspension was then centrifuged at 20,000 x g for 10
min at 4 C, and the pellet was washed twice as above with RIPA buffer.
The beads were eluted with 25 µl of sample buffer (0.5 M
Tris HCl, pH 6.8; 10% SDS; 10% glycerol; 4% 2-ß-mercaptoethanol;
0.05% bromophenol blue). The suspension was again centrifuged at
15,000 x g (4 C for 10 min) and washed four times in
TBST. Sample buffer was added and the samples were boiled for 5 min and
then subjected to SDS-PAGE.
Cell Fractionation
Crude membrane preparations were isolated from muscle cell
cultures according to a modification of the method described by Klip
and Ramlal (9). Culture dishes (90 mm; Nunc) containing the muscle
cells were washed with Ca2+/Mg2+-free PBS and
then mechanically detached in Ca2+/Mg2+-free
PBS containing 2 mM EDTA with a rubber policeman. The cells
were pelleted by centrifugation at 500 x g for 10 min
at 4 C. The pelleted cells were resuspended in sonication buffer (Tris
HCl, pH 7.4, 50 mM; NaCl, 150 mM; EDTA, 2
mM; EGTA, 1 mM; sucrose, 250 mM)
containing leupeptin, 20 µg/ml; aprotinin, 10 µg/ml; PMSF, 0.1
mM; DTT, 1 mM; orthovanadate, 200
µM; and pepstatin, 2 µg/ml. The suspension was
homogenized in a Dounce glass homogenizer (30 strokes) and centrifuged
at 1100 x g for 5 min. The supernatant was centrifuged
at 31,000 x g for 60 min. The supernatant from this
centrifugation was centrifuged at 190,000 x g for 60
min to collect the light microsome fraction. The 31,000 x
g pellet was resuspended in homogenization buffer to a final
volume of 500 µl and placed on a discontinuous sucrose gradient of
500 µl each of 32% (wt/wt), 40% (wt/wt), and 50% (wt/wt) sucrose
solution in 5 mM Tris, pH 7.5. This gradient was
centrifuged at 210,000 x g for 50 min. The plasma
membranes banded above the 32% layer, and the 32/40% and 40/50%
interfaces were collected by puncture with a syringe. These fractions
were diluted in homogenization buffer containing 1% Triton X-100,
freeze-thawed four times, and centrifuged at 30,000 x
g for 30 min, and the supernatant was designated the
membrane protein. All membrane fractions were stored at -70 C until
use.
Western Blot Analysis
Protein (20–25 µg) was electrophoresed through
SDS-polyacrylamide gels (7.5 or 10%) and electrophoretically
transferred onto Immobilon-P (Millipore Corp., Bedford,
MA) membranes. After transfer, the membranes were subjected to standard
blocking and incubation procedures and were incubated with monoclonal
antibodies to specific PKC isoforms and phosphotyrosine, and polyclonal
antibodies to glucose transporters. The membranes were washed four
times for 15 min in Tris-buffered saline-Tween 20 [TBST] and then
further incubated for 20 min at room temperature with horseradish
peroxidase-labeled secondary antibody (goat antirabbit or mouse
IgG) diluted 1:10,000 in blocking buffer. After three washes (1 x
15 min and 2 x 5 min) in TBST, the membranes were treated with
ECL reagent for 1 min, and then exposed on (Eastman Kodak Co., Rochester, NY) x-ray film for the required times (5–30
sec) and developed.
Adenovirus Constructs
The recombinant adenoviruses were constructed in three steps.
Initially the coding sequence for PKC and in the form of cDNA
was inserted into the cassette cosmid. The cassette cosmid for
constructing recombinant Ad of the E1-substitution type, pAdex1, was an
11-kb charomid vector bearing an Ad5 genome spanning 0–99.3 map units
(mu) with deletions of E1 (mu 1.3–9.3) and E3 (mu 79.6–84.8), in
which a unique SwaI site was created by linker insertion at
the E1 deletion. The expression unit was excised with the appropriate
restriction enzymes, blunt ended with Klenow fragment of DNA polymerase
I, and purified by gel electrophoresis. Thereafter the fragment was
ligated with SwaI-linearized pAxCAwt (46).
After overnight ligation, the DNA sample was digested with
SwaI, to exclude empty re-ligated cosmids lacking a coding
sequence, and an aliquot was packaged in vitro using
Gigapack (Stratagene, La Jolla, CA). Colonies were
obtained after plating the transduced Escherichia coli
DH5, and the majority of the clones contained the desired insert.
Ad5-dIX, which has an E3 deletion (mu 79.6–84.8) was used
as the parent virus for recombinant Ad construction. The DNA-terminal
protein complex (DNA-TPC) of the parent Ad was prepared and
purified utilizing CsCl density gradient with guanidine hydrochloride.
The DNA-TPC was digested with EcoT22I and was gel filtered through a
Sephadex G-50 spin column. The EcoT22I-digested adenovirus DNA-TPC was
mixed with the cassette cosmid bearing the desired expression unit, and
human embryonic kidney 293 cells were transfected with the mixed DNA by
the calcium phosphate method using CellPhect Transfection kit
(Pharmacia Biotech). One day later, the cells were
dispensed in 96-well plates in 10-fold serial dilutions and mixed with
untransfected 293 cells. After being maintained in culture for 10–15
days, virus-containing supernatants were isolated and propagated
further to assess restriction analysis and expression of inserted
genes.
Mutated PKC Adenovirus Construct
The dominant negative mutant of mouse PKC was generated by
substitution of the lysine residue at the ATP binding site with alanine
(47). The mutant cDNA was cut from SRD expression vector with
EcoRI and ligated into the pAxCA1w cosmid cassette to
construct Ax vector. Its kinase-negative nature was demonstrated by
abrogation of autophosphorylation activity (48).
PKC Isoform Viral Infection
After differentiation of cultured rat muscle into myotubes, the
culture medium was aspirated and cultures were infected with the viral
medium containing PKC or recombinant adenoviruses for 1 h.
The cultures were then washed twice with DMEM and refed. Cells 10
h postinfection were transferred to serum-free DMEM containing 4.5
mM for 24 h. Control and insulin-treated cultures were
used for glucose uptake experiments, or extracted and fractionated into
cytosolic and membrane fractions. The fractions were
electrophoretically separated and blotted with appropriate
antibodies.
PKC Activity
Specific PKC activity was determined in freshly prepared
immunoprecipitates from mature muscle cultures after appropriate
treatments. These lysates were prepared in RIPA buffer without NaF.
Activity was measured with the use of the SignaTECT Protein Kinase C
Assay System (Promega Corp., Madison, WI) according to the
manufacturer’s instruction. PKC biotinylated pseudosubstrate was used
as the substrate in these studies.
Glucose Uptake
The total and nonspecific rates of glucose transport were
measured in triplicate samples in 24-well plates with the use of
[3H]2-DG (13). After appropriate treatment, cells were
washed three times with 0.5 ml PBS, the final wash being replaced
immediately with 0.5 ml PBS containing 1 µCi/ml of
[3H]2-DG in glucose at a concentration of 2
mM. Cells were then incubated for 15 min at 37 C, after
which time they were washed four times with 0.5 ml cold (4–6 C) PBS
and then lysed by addition of 300 ml Triton X-100 (1%) and incubation
for 30 min. The contents of each well were transferred to counting
vials and 3.5 ml scintillation fluid were added to each vial. Samples
were counted in the 3H window of a Tricarb scintillation
counter. Nonspecific uptake was determined in the presence of excess
(100 mM) D-glucose. Net specific uptake was
then calculated as the difference between the total and nonspecific
values. Baseline glucose uptake values under control conditions ranged
from 14–20 nmol/min/mg protein.
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FOOTNOTES |
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Supported in part by the Sorrell Foundation, the Ben and Effie Raber Research Fund, and The Harvett-Aviv Neuroscience Research Fund. S.R.S. is the incumbent of the Louis Fisher Chair in Cellular Pathology.
Received for publication June 17, 1999. Revision received August 24, 1999. Accepted for publication September 1, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß in rat adipocytes and protein kinase C , ß and in rat
skeletal muscle. Biochem J 308:181–187
, ß and ) by insulin in 3T3/L1 adipocytes. J
Biol Chem 272:2551–2558 and
noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated
glucose transport in L6 myotubes. Endocrinology 138:4721–4731 as a downstream effector of
phosphatidylinositol 3-kinase during insulin stimulation in rat
adipocytes. Potential role in glucose transport. J Biol Chem 272:30075–30082, ),
conventional (, ß) and novel (, ) protein kinase C isoforms
on insulin-stimulated translocation of epitope-tagged GLUT4 glucose
transporters in rat adipocytes: specific interchangeable effects of
protein kinases C- and C-. Biochem J 337:461–470
. Mol Cell
Biol 14:6727–6735 inhibits sis-induced transformation of NIH3T3 cells. Oncogene 13:731–737[Medline]
and in NIH
3T3 cells. Cytokine 9:577–581[CrossRef][Medline]
. Mol Cell
Biol 17:3418–3428[Abstract]
phosphorylation site. J Biol Chem 271:26404–26409. Eur J Biochem 259:555–564[Medline]
in
response to its activation. J Biol Chem 269:2349–2352 is involved
in the stimulation of the Na+-H+ exchanger in
C6 glioma cells. Mol Pharmacol 48:995–1003[Abstract]
in arachidonic acid
cascade, superoxide formation and phosphoinositide hydrolysis. Biochem
J 292:203–207
blocks 1- adrenergic activation of Na- K- 2Cl cotransport.
Am J Physiol 273:C1632–C1640
in
salivary gland epithelial cells. J Biol Chem 270:13490–13495 from late phase
down-regulation, despite synergy in diacylglycerol accumulation.
Biochem J 318:519–525
abolishes the kinase activity and renders it
down-regulation-insensitive. A molecular link between
autophosphorylation and down-regulation. J Biol Chem 265:6296–6300This article has been cited by other articles:
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