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and Noninvolvement of Diacylglycerol-Sensitive PKCs in Insulin-Stimulated Glucose Transport in L6 Myotubes1
J. A. Haley Veterans Hospital and the Departments of Internal Medicine and Biochemistry/Molecular Biology (G.B., M.L.S., L.G., R.V.F.), University of South Florida College of Medicine, Tampa, Florida 33612; and Centro de Biologia Molecular "Severo Ochoa" (J.M.), Universidad Autonoma, Canto Blanco, 28049 Madrid, Spain
Address all correspondence and requests for reprints to: Robert V. Farese, M.D., Research Service (VAR 151), J. A. Haley Veterans Hospital, 13000 Bruce Downs Boulevard, Tampa, Florida 33612.
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Abstract |
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in L6 myotubes, and the dependence of this activation on
phosphatidylinositol (PI) 3-kinase. We also evaluated a number of
issues that are relevant to the question of whether diacylglycerol
(DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-, are more
likely to play a role in insulin-stimulated glucose transport in L6
myotubes and other insulin-sensitive cell types. We found that insulin
increased the enzyme activity of immunoprecipitable PKC- in L6
myotubes, and this effect was blocked by PI 3-kinase inhibitors,
wortmannin and LY294002; this suggested that PKC- operates
downstream of PI 3-kinase during insulin action. We also found that
treatment of L6 myotubes with 5 µM tetradecanoyl
phorbol-13-acetate (TPA) for 24 h led to 80–100% losses of all
DAG-dependent PKCs (
, ß1, ß2, 
, 
)
and TPA-stimulated glucose transport (2-deoxyglucose uptake); in
contrast, there was full retention of PKC-, as well as
insulin-stimulated glucose transport and translocation of GLUT4 and
GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1
myocytes, TPA treatment did not elicit increases in PKCß2 messenger
RNA or protein in L6 myotubes, and selective retention of this PKC
isoform could not explain the retention of insulin effects on glucose
transport after prolonged TPA treatment. Of further interest, TPA
acutely activated membrane-associated PI 3-kinase in L6 myotubes, and
acute effects of TPA on glucose transport were inhibited, not only by
the PKC inhibitor, LY379196, but also by both wortmannin and LY294002;
this suggested that DAG-sensitive PKCs activate glucose transport
through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than
directly through PKC. Also, the cell-permeable, myristoylated PKC-
pseudosubstrate inhibited insulin-stimulated glucose transport both in
non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus,
the PKC- pseudosubstrate appeared to inhibit a protein kinase that
is required for insulin-stimulated glucose transport but is distinct
from DAG-sensitive PKCs. In keeping with the latter dissociation of
DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196,
which inhibits PKC-ß (preferentially) and other DAG-sensitive PKCs at
relatively low concentrations, inhibited insulin-stimulated glucose
transport only at much higher concentrations, not only in L6 myotubes,
but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat
soleus muscles. Finally, stable and transient expression of a
kinase-inactive PKC- inhibited basal and insulin-stimulated glucose
transport in L6 myotubes. Collectively, our findings suggest that,
whereas PKC- is a reasonable candidate to participate in insulin
stimulation of glucose transport, DAG-sensitive PKCs are unlikely
participants. |
Introduction |
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Several other recent findings are germane to the conundrum described
above. On the one hand, in support of the argument that DAG-sensitive
PKCs are required for insulin action, phorbol ester-induced
down-regulation of PKC has been reported to be attended by an
alteration in PKC-ß messenger RNA (mRNA) splicing, thus causing a
decrease in PKC-ß1 and a paradoxical increase in
PKC-ß2 in BC3H-1 myocytes; moreover, this mechanism has
been suggested to account for retention of glucose transport effects of
insulin in this experimental paradigm (6). On the other hand, several
other recent findings militate against the involvement of DAG-sensitive
PKCs, and speak for the involvement of the DAG-insensitive PKC, viz.,
PKC-, in insulin-stimulated glucose transport. First, phorbol esters
have been found to activate a membrane-associated form of
phosphatidylinositol (PI) 3-kinase in rat adipocytes (7) and 3T3/L1
cells (8), and the relatively small effects of phorbol esters on
glucose transport in 3T3/L1 cells [although not in rat adipocytes
(7)] are blocked by the PI 3-kinase inhibitor, wortmannin (8): thus,
DAG-sensitive PKCs do not directly activate glucose transport and are
unlikely to serve as downstream effectors for PI 3-kinase-dependent
increases in glucose transport, at least in some cell types. Second, we
have recently found that insulin provokes an increase in the enzymatic
activity of immunoprecipitable PKC- in 3T3/L1 cells (9). Third, in
transfection studies in 3T3/L1 fibroblasts and adipocytes, we have
found that: 1) stable expression of wild-type PKC-, but not PKC-
or PKC-ß2, provokes increases in basal and
insulin-stimulated glucose transport; and 2) stable expression of a
kinase-inactive mutant form of PKC- inhibits basal and
insulin-stimulated glucose transport (9).
Presently, we have attempted to gain further insight into the question
of whether DAG-sensitive or DAG-insensitive PKCs are more likely to
play a role in insulin-stimulated glucose transport in L6 myotubes. To
this end, we have studied in L6 myotubes: 1) the activation of
immunoprecipitable PKC- by insulin and its dependence on PI
3-kinase; 2) the completeness of depletion of specific PKC isoforms
during prolonged, high-dosage phorbol ester treatment; 3) effects of
phorbol ester on PKC-ß1 and PKC-ß2 mRNA; 4)
effects of phorbol esters on PI 3-kinase; 5) effects of inhibitors of
PI 3-kinase on acute, phorbol ester-induced increases in glucose
transport; and 6) effects of stable and transient transfection of
wild-type and kinase-inactive PKC- on glucose transport. We also
studied the effects of selective inhibitors of PKC-ß and other
DAG-sensitive PKCs on insulin-stimulation of glucose transport both in
L6 myotubes, and several other commonly used insulin-sensitive cell
types, i.e. rat adipocytes, 3T3/L1 adipocytes, BC3H-1
myocytes, and rat soleus muscles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pseudosubstrate (Quality Controlled Biochemicals,
Inc., Hopkinton, MA), wortmannin (Sigma), LY294002 (BioMol Research
Laboratories, Inc., Plymouth Meeting, PA), LY379196 (a selective
PKC-ß inhibitor, kindly provided by Drs. Michael Jirousek and Kirk
Ways, Eli Lilly Co.), CG53353 (a selective PKC-ß2
inhibitor, kindly provided by Drs. Anna Suter and Doriano Fabbro, Ciba
Geigy Corp.), and other substances were added to the medium, and
incubations were conducted as described in the text.
PKC isoforms in total cell lysates were immunoblotted as described (1, 10, 11, 12), except that quantitation of chemiluminescence was accomplished
with a BioRad 32P/Chemiluminescence Molecular Analyst
Imaging System, which provides linear responses for both
32P and chemiluminescence. Polyclonal antisera for PKC-,
, and were obtained from Life Technologies (Grand Island,
NY). Polyclonal antisera for PKC-ß1 and
PKC-ß2 were obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Antisera specificities were confirmed by 1)
immunoabsorption (i.e. loss of immunoreactivity) with the
immunizing peptide and 2) by blotting against recombinant PKCs obtained
from Sf9 insect cells infected with baculovirus containing
isoform-specific PKC complementary DNA (cDNA), and/or NIH3T3, 3T3/L1 or
L6 cells transfected with cDNAs encoding respective PKCs (see Refs. 9,
10, 12, 13, and Santa Cruz catalog). We presently could not detect
PKC-
in L6 myotubes using methods successfully applied to mature rat
skeletal muscle (12). GLUT4 and GLUT1 glucose transporter levels were
measured by Western analysis using antisera obtained from Charles
River.
PKC- enzyme activity was measured in specific immunoprecipitates as
described (9). Cells were lysed by sonication in Buffer I containing 20
mM Tris-HCl (pH 7.5), 0.25 M sucrose, 1.2
mM EGTA, 20 mM ß-mercaptoethanol, 1
mM phenylmethylsulfonylfluoride (PMSF), 20 µg/ml
leupeptin, 20 µg/ml aprotinin, 1 mM Na pyrophosphate, 1
mM NaF, 1% Triton X-100, 0.5% Nonidet and 150
mM NaCl. Lysates were first cleared with rabbit preimmune
serum, followed by treatment with second antibody (goat antirabbit IGG
antiserum) and protein AG-sepharose beads. PKC- was then
immunoprecipitated by overnight treatment at 4 C with anti-PKC-
antiserum obtained from either Life Technologies or Santa Cruz
Biotechnology, followed by addition of second antibody. Precipitates
were collected on protein AG-sepharose beads, washed and suspended in
Buffer II containing 50 mM Tris/HCl (pH, 7.5), 5
mM MgCl2, 100 µM
Na3VO4, 100 µM
Na3P2O7, 1 mM NaF and
100 µM PMSF. The suspension was then incubated for 8 min
at 30 C in 100 µl Buffer II containing 3–5 µCi
[
-32P]ATP (New England Nuclear, Boston, MA), 50
µM ATP, 4 µg phosphatidylserine (PS), and 40
µM[159Ser]-PKC-(AA153–164)-NH2
(Upstate Biotechnology, Inc., Lake Placid, NY) [this PKC-
pseudosubstrate analog is an excellent substrate for PKC- (14) and
we have found that this substrate is phosphorylated very well by
PKC- and PKC- (both set at 100%), and poorly by PKC- (35%),
PKC-ß1 (24%), PKC-ß2 (10%) and PKC-
(5%) using PKCs purified from Sf9 insect cells infected with cDNAs
encoding these PKCs (kindly supplied by Dr. Larry Ballas, Sphinx
Division, Eli Lilly Co.). PKC- pseudosubstrate (as a relatively
specific PKC- inhibitor) was used in the assay to determine blank
values, i.e. cpm incorporated independently of PKC-
(
15% of total cpm), which were subtracted from total cpm. After
incubation, an aliquot of the reaction mixture was spotted on p81
paper, washed in 5% acetic acid, and counted. As reported elsewhere
(9), PKC- immunoprecipitates contained no detectable PKC-, ß,
or ; additionally, recovery of PKC- was approximately 50%
and was not influenced by insulin treatment or improved by adding a
2-fold excess of antibody.
PKC-ß1 mRNA and PKC-ß2 mRNA were measured by ribonuclease protection assay (RPA) as described (9, 13) (original cDNAs were kindly provided by Dr. Yoshitaka Ono, Kobe, Japan). In these RPAs, the protected RNA fragments spanned the splice sites and extended well into both adjacent exons of fully processed PKC-ß1 mRNA (base 1792 to 2058) and PKC-ß2 mRNA (base 1792 to 1962); accordingly PKC-ß1 and PKC-ß2 mRNAs cannot be confused with prespliced (smaller) precursors (note - the splice site is at base 1865 of the coding region of rat PKC-ß). The validity of this method has been confirmed by observing specific increases in PKC-ß2 (but not PKC-ß1) mRNA in 3T3/L1 cells transfected with cDNA encoding PKC-ß2 mRNA (9). Quantitation was accomplished with the Biorad Phosphorimager Molecular Analyst Imaging System.
Wild-type PKC- and PKC-ß2 and kinase-inactive PKC-
(i.e. point-mutated in the ATP-binding site; see Ref.15),
each contained in a pCDNA3 eukaryotic expression vector (Invitrogen,
Carlsbad, CA), were transfected into L6 cells using Lipofectamine
according to instructions of the manufacturer (Life Technologies). In
stable transfection experiments, colonies were selected by resistance
to G418 (500 µg/ml), cultured to confluence in 24-well plates (for
glucose transport studies) or 100-mm plates (for immunoblot studies),
and then differentiated to myotubes as described above. Growth rates
and differentiation did not appear to be influenced by the transfection
of vectors or vectors containing inserted PKC- cDNAs. In transient
transfection experiments, fully differentiated L6 myotubes were
transfected directly in 24-well plates and then allowed to incubate for
2 days in fresh medium before experimental use. Transfection with
ß-galactosidase (ß-gal)-containing constructs (also in pCDNA3) were
used to judge transient transfection rates, which were at least
approximately 25%.
[3H]2-Deoxyglucose (2-DOG) uptake in L6 myotubes was measured as described (1). In these assays, cells were incubated in glucose-free KRP with or without inhibitors as described in the figure legends and then stimulated for 30 min with or without agonist (insulin or TPA), followed by determination of uptake during a 5-min incubation in medium containing 0.1 mM [3H]2-DOG (New England Nuclear). In a more limited number of experiments, 2-DOG uptake was also measured in rat adipocytes, 3T3/L1 adipocytes, BC3H-1 myocytes and rat soleus muscles, as described previously (7, 9, 16, 17, respectively).
Membrane-associated PI 3-kinase activity was measured as described
previously (7). In brief, total membranes were obtained by
centrifugation of postnuclear homogenates at 100,000 x
g for 60 min, and 100 µg membrane protein were incubated
for 10 min with 50 µM [32P]ATP, 10
mM MgCl2, 20 µg PI, and other components of the assay
system in a final volume of 75 µl. After incubation,
PI-3-PO4 was extracted, resolved by TLC, and counted in the
Biorad Phosphorimager Molecular Analyst Imaging System.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation
in L6 myotubes is specifically activated in these cells.
Presently, we found that 20 nM insulin provoked, on the
average, 80% increases in the enzyme activity of immunoprecipitable
PKC- in L6 myotubes (Fig. 1
).
Moreover, PI 3-kinase appeared to be required for the activation of
PKC-, as this effect of insulin was not observed in myotubes
incubated in the presence of either 100 nM wortmannin or
100 µM LY294002 (Fig. 1).
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, treatment of L6
myotubes with 5 µM TPA for 24 h was attended by
large (80–100%) losses of immunoreactive PKC-, ß1,
ß2, and ; concomitantly, acute effects of TPA on
glucose transport were completely lost in down-regulated myotubes. The
DAG-insensitive PKC-, on the other hand, was fully retained in
TPA-treated myotubes, and this correlated well with full retention of
insulin-stimulated (a) 2-DOG uptake (Fig. 2) and (b) translocation of
both GLUT4 and GLUT1 to the plasma membrane (Fig. 3). Unlike what was reported in studies
of BC3H-1 myocytes (see Refs. 6 and 18), neither PKC-ß2
protein (Fig. 2) nor PKC-ß2 mRNA (Fig. 4) was induced by 24-h TPA treatment in
L6 myotubes. (It may be noted that acute TPA treatment, like insulin,
provoked increases in plasma membrane levels of both GLUT4 and
GLUT1-see Fig. 3).
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) and
LY294002 (Fig. 6) inhibited TPA-, as well
as insulin-stimulated, 2-DOG uptake in L6 myotubes: maximal inhibition
was observed at 50 nM wortmannin and 50 µM
LY294002, concentrations that are well within the range of those
reported to inhibit PI 3-kinase (19, 20). These findings suggested
that: 1) phorbol esters activate glucose transport by cross-talking of
PKC with PI 3-kinase; and 2) DAG-sensitive PKCs cannot be viewed simply
as serving as downstream effectors for PI 3-kinase-dependent activation
of glucose transport in L6 myotubes. It may also be noted that basal
2-DOG uptake was mildly inhibited by wortmannin and LY294002,
suggesting the presence of significant (although variable; see below)
basal PI 3-kinase activity in control myotubes. (This was also noted
previously; see Ref.19).
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, both
insulin and TPA provoked increases in membrane-associated PI 3-kinase
activity. This finding is similar to those observed in rat adipocytes
(7) and 3T3/L1 adipocytes (8), and, moreover, provides an explanation
for the inhibitory effects of wortmannin and LY294002 on acute effects
of TPA on glucose transport in L6 myotubes.
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pseudosubstrate on insulin and
TPA-stimulated glucose transport in L6 myotubes, and/or another protein
kinase, distinct from DAG-sensitive PKC family members, may operate
downstream of PI 3-kinase and participate in both insulin-stimulated
and TPA-stimulated glucose transport. Accordingly, it was of interest
to find that the cell-permeable, myristoylated PKC- pseudosubstrate
(myr-SIRRGARRWRKL-NH2) inhibited insulin-stimulated, and to
a variable extent, basal 2-DOG uptake, both in non-down-regulated L6
myotubes and in L6 myotubes down-regulated by 24-h treatment with 5
µM TPA (Figs. 8 and 9). Inhibition of insulin effects was
complete at 10–30 µM PKC- pseudosubstrate,
concentrations of myristoylated pseudosubstrate that are well within
the range of those reported to inhibit PKC in other cellular systems
(see Ref.21). The partial, but variable, inhibition of basal 2-DOG
uptake by the PKC- pseudosubstrate, like that occurring with
wortmannin and LY294002, probably reflects significant activation PI
3-kinase and a distal protein kinase(s) in control L6 myotubes. Also,
it may be noted that, despite significant differences in amino acid
sequences of various PKC pseudosubstrates (see Ref.22), the isoform
specificity of the PKC- pseudosubstrate is presently uncertain, and
it is possible, if not likely, that it may also inhibit PKCs other than
PKC-: nevertheless, the inhibitory effects of the PKC-
pseudosubstrate in L6 myotubes in which all DAG-sensitive PKCs
were effectively depleted by 24-h TPA treatment, suggested that a
TPA-resistant PKC, possibly PKC-, may be required for
insulin-stimulated glucose transport.
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pseudosubstrate inhibited TPA-stimulated 2-DOG
uptake in L6 myotubes (Fig. 9
). This inhibition, however, appeared to
be slightly less complete than that observed with insulin, suggesting
that a small part of the TPA effect may be mediated by a protein kinase
or other factor that is relatively resistant to the PKC-
pseudosubstrate. It may also be noted that a comparably sized,
myristoylated peptide containing the amino acid sequence,
myr-ITRARRAPSVAN-NH2, did not inhibit insulin-stimulated
2-DOG uptake (Fig. 9), suggesting at least some degree of specificity
of the sequence of the PKC- pseudosubstrate.
Effects of expression of transfected PKC- on insulin-stimulated
glucose transport in L6 myotubes
Stable expression of wild-type PKC- and PKC-ß2
provoked 2-fold increases in immunoreactive levels of these PKCs but
failed to affect insulin effects on glucose transport (data not shown);
it may therefore be surmised that these PKC isoforms, at least in their
inactive forms, are present in amounts that are not rate-limiting for
glucose transport. On the other hand, stable expression of
kinase-inactive PKC- provoked a 50% decrease in basal and
insulin-stimulated 2-DOG uptake (Fig. 10). Concomitantly, immunoreactive
PKC- was increased approximately 2-fold [i.e. an
increase of 127 ± 11% (mean ± SE; n =
7)] above control] in cells stably transfected with kinase-inactive
PKC- (Fig. 10): this correlated well with the observed 50%
inhibition of glucose transport, if it is assumed that transfected,
catalytically inactive PKC- competes equally with native,
catalytically active PKC- for either activating substances and/or
substrates. Along these lines, it may be noted that in previous studies
of 3T3/L1 adipocytes(9), we observed that, as in the present study,
stable transfection of the same kinase-inactive PKC- led to a 2-fold
increase in immunoreactive PKC-, with no change in total cellular
PKC- enzyme activity (9) (this is the expected result, if the
transfected PKC- is catalytically inactive, but does not inhibit
endogenous PKC- enzyme): this suggested that the specific enzyme
activity of total cellular PKC- was decreased by 50% in these
transfected 3T3/L1 cells. We presume that the same situation prevailed
in L6 myotubes stably transfected with kinase-inactive PKC-.
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was not unexpected because basal transport was
also frequently inhibited (albeit to a variable extent) by wortmannin,
LY294002 and myristoylated PKC- pseudosubstrate (this inhibition of
basal activity probably reflects variable stimulatory effects of
residual serum or endogenous paracrine or autocrine activators that may
have survived the washings and preincubations, or continue to be
released from the myocytes, thus raising the basal transport level). It
may also be noted that the inhibition of glucose transport by
kinase-inactive PKC- could not be explained by changes in levels of
GLUT4 and/or GLUT1 glucose transporters, as these were increased by
80 ± 17 (n = 5; P < 0.01) and 88 ± 29
(n = 6; P < 0.05) percent, respectively.
Interestingly, increases in Glut4 and/or Glut1 glucose transporters
were also observed in 3T3/L1 cells stably transfected with
kinase-inactive PKC- (9); it is therefore possible that decreases in
basal and insulin-stimulated glucose transport may have resulted in
compensatory increases in Glut4 and/or Glut1 glucose transporters.
Whatever the explanation, the decrease in basal and insulin-stimulated
glucose transport in these transfected cells, in the face of increases
in levels of Glut4 and Glut1, suggested that 1) the levels of these
glucose transporters were not rate-limiting for basal glucose
transport; and 2) the inhibitory effects of kinase-inactive PKC- may
have been partly offset by increases in Glut4 and Glut1 glucose
transporters.
Similar to changes observed in stable transfectants, transient
transfection of kinase-inactive PKC- led to a modest (40%) but
significant inhibition of insulin-stimulated glucose transport (Fig. 10). This inhibition was associated with approximately 2-fold increases
in immunoreactive PKC-, which, based upon transfection rates with
ß-gal, appeared to reside in at least 25% of the cells. Inasmuch as
the latter may have been underestimated by microscopic analysis, it may
be surmised that there were approximately 4- to 8-fold increases in
PKC- in these transiently transfected cells, and this may explain
the apparently greater relative (perhaps full) inhibitory effect of
kinase-inactive PKC- in transient transfection experiments.
Effects of selective PKC-ß inhibitors (LY379196 and CG53353) on
insulin-stimulated glucose transport in L6 myotubes and other cell
types
As our results suggested that PKC-ß and other DAG-sensitive PKCs
are not likely to be important for insulin effects on glucose
transport, it was of interest to use a PKC inhibitor that provided a
wide difference between inhibitory effects on PKC-ß and other PKCs.
For this purpose, we used LY379196, which (as determined with
recombinant, baculovirus-derived PKCs, purified from Sf9 insect cells
by Dr. Michael Jirousek, Eli Lilly Co.) provides a difference of nearly
three orders of magnitude between the inhibition of
PKC-ß1 (IC50, 50 nM) and
PKC-ß2 (IC50, 30 nM), on the one
hand, and PKC- [IC50, 48 µM], on the
other hand. [Note: IC50s for PKC-, PKC-, PKC-,
PKC-
and PKC- = 0.6, 0.6, 0.7, 0.3, and 5 µM,
respectively; also note that these IC50s are similar in
relative distribution to those of another, structurally similar,
selective, PKC-ß inhibitor, LY333531; see Ref. 23]. As shown in Fig. 11, LY379196, in concentrations up to
10 µM, had no effect on control or insulin-stimulated
2-DOG uptake, and caused inhibition only at higher concentrations in
both L6 myotubes and rat adipocytes. In contrast, LY379196, in
relatively low concentrations, fully inhibited TPA-stimulated DOG
uptake in L6 myotubes (Fig. 11); this confirmed the fact that this
inhibitor readily entered these cells and effectively inhibited
DAG-sensitive PKCs in these experiments. Similarly, L379196, in
concentrations up to 10 µM, failed to inhibit 2-DOG
uptake in 3T3/L1 adipocytes, BC3H-1 myocytes and rat soleus muscles
(Fig. 12). Goe 6796, a selective
inhibitor of PKC-, ß and (24), also failed to inhibit 2-DOG
uptake in these cells (data not shown). These findings provided further
evidence suggesting that PKC-ß1, PKC-ß2 and
probably most other DAG-sensitive PKCs, are not likely to contribute
importantly to insulin effects on glucose transport.
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, this inhibitor elicted similar
dose-dependent inhibition curves for insulin-stimulated 2-DOG uptake in
non-down-regulated and TPA-down-regulated L6 myotubes. Inasmuch as
PKC-ß2 and other DAG-sensitive PKCs were markedly
depleted in TPA-treated cells, it may be surmised that the inhibition
of insulin-stimulated glucose transport by CG53353 cannot be attributed
to the inhibition of PKC-ß2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß, may operate downstream of PI 3-kinase (see
26), the full retention of insulin effects on glucose transport and
translocation of both GLUT4 and GLUT1 to the plasma membrane in L6
myotubes (ref 3, and present findings) and certain other cells,
e.g. 3T3/L1 cells (27), after prolonged phorbol ester
treatment and marked depletion of DAG-sensitive PKCs, argues against
their involvement in glucose transport. Indeed, as shown presently in
L6 myotubes, TPA provoked marked losses of all detectable DAG-sensitive
PKCs (, ß1, ß2, , ) without
altering insulin effects on glucose transport. Moreover, contrary to
findings in BC3H-1 myocytes (6, 18), TPA did not induce increases in
PKC-ß2 mRNA or protein in L6 myotubes; it therefore seems
clear that retention of the PKC-ß2 isoform is unlikely to
explain the retention of insulin effects on glucose transport in
TPA-down-regulated myotubes. As stated above, from the present and previous (3) findings in down-regulated cells, it seems most unlikely that DAG-sensitive PKCs serve as required kinases that operate downstream of PI 3-kinase in insulin-stimulated glucose transport. As may be surmised, this conclusion differs from that of another study in which transient expression of a nine amino acid, but not a sixteen amino acid, C-terminal, truncated form of PKC-ß2, led to apparently complete inhibition of insulin-stimulated glucose transport in L6 myotubes (5). However, it is presently not clear why the sixteen amino acid-truncated form of PKC ß2 failed to inhibit insulin effects on glucose transport, as this form was enzymatically even less active than the nine amino acid-truncated form. This discrepancy was explained by postulating that the smaller truncated form of PKC-ß2 could function as a dominant-negative inhibitor of intact endogenous PKC-ß2 by virtue of an amino acid sequence that was present in the nine amino-acid form and was required for recognition of substrate or an intracellular receptor (5). This explanation is plausible but speculative. In contrast to findings of Chalfant et al. (5), our findings with PKC inhibitors and PKC depletion seem to be less subject to speculative interpretation, and, in our view, strongly suggest that PKC-ß2 is not required for insulin stimulation of glucose transport. Similarly, our finding that acute effects of TPA on glucose transport were blocked by wortmannin and LY294002 leaves little doubt that DAG-sensitive PKCs, including PKC-ß2, cannot serve to directly activate the glucose transport process during TPA treatment in L6 myotubes. Nevertheless, we are currently examining the role of PKC-ß in studies of muscles of mice in which PKC-ß has been knocked out by homologous recombination methodology.
Further support for our contention that a DAG-insensitive, rather than
a DAG-sensitive, PKC is more likely to be involved in insulin
stimulation of glucose transport derives from two other lines of
evidence, i.e. 1) the PKC- pseudosubstrate and CG53353, a
general PKC inhibitor (5), inhibited insulin-stimulated 2-DOG uptake in
cells largely depleted of DAG-sensitive PKCs (i.e. after
24-h TPA treatment); and 2) inhibitory effects of a highly selective
PKC-ß inhibitor, LY379196, on insulin-stimulated 2-DOG uptake were
observed only at concentrations that were far in excess of those
required to inhibit PKC-ß and probably most other DAG-sensitive PKCs.
Obviously, the higher concentrations of LY379196 that inhibited 2-DOG
uptake may have inhibited PKC-, other atypical PKCs, e.g.
, and/or other related kinases. Along the latter lines, it may be
noted that we have previously reported that, at concentrations of 5
µM or greater, R0 31–8220, a relatively nonspecific PKC
inhibitor, blocks insulin-stimulated glucose transport in L6 myotubes
(1). Of further interest, we have found that comparably high
concentrations of RO 31–8220 inhibit immunoprecipitated PKC- (our
unpublished data), whereas considerably lesser concentrations of RO
31–8220 have been reported to inhibit PKC- and PKC-ß (see Ref.28).
Presently, we were able to obtain evidence that insulin activates
PKC- by a PI 3-kinase dependent mechanism in L6 myotubes. This
apparent dependency on PI 3-kinase may at least in part reflect the
fact that PKC- has been found to be directly activated in
vitro by polyphosphoinositides (see Refs. 29 and 30) that are
generated through PI 3-kinase action. In further support of this
possibility, we have recently found in studies of rat adipocytes (our
unpublished data) that PI-3,4-(P04)2 and
PI-3,4,5-(P04)3 activate control, but not
insulin-stimulated, PKC- immunoprecipitates, thereby diminishing or
abolishing the difference in activity between these immunoprecipitates.
With respect to activating mechanisms, we have also recently found that
insulin and the above-stated polyphosphoinositides stimulate the
phosphorylation, as well as the enzymatic activation, of PKC- in rat
adipocytes; however, it is presently not clear if this phosphorylation
contributes to the enzymatic activation of PKC-. Further studies
will be required to see if the activation of PI 3-kinase is sufficient
to account for insulin-induced activation of PKC-.
Similar to findings in rat adipocytes (7) and 3T3/L1 adipocytes (8), we
found that TPA activated a membrane form of PI 3-kinase in L6 myotubes.
However, it should be noted that, unlike insulin, TPA does not activate
PI 3-kinase through IRS-1, at least in rat adipocytes (see Ref.7).
Needless to say, it is presently unclear as to how TPA activates PI
3-kinase, and what specific PI 3-kinase isoform is activated by TPA.
Nevertheless, our findings with both wortmannin and LY294002 strongly
suggested that PI 3-kinase activation is required for glucose transport
effects of TPA in L6 myotubes. This somewhat surprising finding
provides clear evidence that the simple demonstration of TPA effects on
glucose transport does not necessarily imply that DAG-sensitive PKCs
participate directly in activating this and other processes. As alluded
to above, a more likely scenario, at least in L6 myotubes, is that TPA
activates PI 3-kinase through DAG-dependent PKCs, and PI 3-kinase, in
turn, apparently through factors that are clearly distinct from
DAG-sensitive PKCs, is largely responsible for activating glucose
transport. Whether PKC- operates downstream of PI 3-kinase during
TPA action is presently uncertain.
Finally, we recognize the fact that our findings with stable and
transient expression of kinase-inactive PKC- can only be taken as
suggestive evidence that PKC- acts as a downstream effector for PI
3-kinase in the regulation of glucose transport. Nevertheless, in view
of the inhibitory effects of the PKC- pseudosubstrate, at least one
PKC, or a closely related protein kinase, appears to be required for
insulin-stimulated glucose transport. Moreover, because DAG-sensitive
PKCs are unlikely candidates, it is reasonable to suggest that one or
more DAG-insensitive PKCs, e.g. PKC- and/or other
atypical PKCs or related protein kinases, may be involved in the
activation of glucose transport in L6 myotubes by insulin. Clearly,
more work will be needed to test the involvement of PKC- and others
DAG-insensitive protein kinases in insulin-stimulated glucose
transport.
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Footnotes |
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Received February 20, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S.-F. Yan, J. Lu, Y. S. Zou, W. Kisiel, N. Mackman, M. Leitges, S. Steinberg, D. Pinsky, and D. Stern Protein Kinase C-beta and Oxygen Deprivation. A NOVEL Egr-1-DEPENDENT PATHWAY FOR FIBRIN DEPOSITION IN HYPOXEMIC VASCULATURE J. Biol. Chem., April 14, 2000; 275(16): 11921 - 11928. [Abstract] [Full Text] [PDF]
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L. Braiman, A. Alt, T. Kuroki, M. Ohba, A. Bak, T. Tennenbaum, and S. R. Sampson Protein Kinase C{delta} Mediates Insulin-Induced Glucose Transport in Primary Cultures of Rat Skeletal Muscle Mol. Endocrinol., December 1, 1999; 13(12): 2002 - 2012. [Abstract] [Full Text]
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L. M. NERI, A. M. MARTELLI, P. BORGATTI, M. L. COLAMUSSI, M. MARCHISIO, and S. CAPITANI Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-{zeta} translocation to the nucleus of NGF-treated PC12 cells FASEB J, December 1, 1999; 13(15): 2299 - 2310. [Abstract] [Full Text]
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M. P. Sajan, M. L. Standaert, G. Bandyopadhyay, M. J. Quon, T. R. Burke Jr., and R. V. Farese Protein Kinase C-zeta and Phosphoinositide-dependent Protein Kinase-1 Are Required for Insulin-induced Activation of ERK in Rat Adipocytes J. Biol. Chem., October 22, 1999; 274(43): 30495 - 30500. [Abstract] [Full Text] [PDF]
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G. Bandyopadhyay, M. L. Standaert, M. P. Sajan, L. M. Karnitz, L. Cong, M. J. Quon, and R. V. Farese Dependence of Insulin-Stimulated Glucose Transporter 4 Translocation on 3-Phosphoinositide-Dependent Protein Kinase-1 and Its Target Threonine-410 in the Activation Loop of Protein Kinase C-{zeta} Mol. Endocrinol., October 1, 1999; 13(10): 1766 - 1772. [Abstract] [Full Text]
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M. L. Standaert, G. Bandyopadhyay, L. Galloway, J. Soto, Y. Ono, U. Kikkawa, R. V. Farese, and M. Leitges Effects of Knockout of the Protein Kinase C {beta} Gene on Glucose Transport and Glucose Homeostasis Endocrinology, October 1, 1999; 140(10): 4470 - 4477. [Abstract] [Full Text]
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L. Lavoie, C. J. Band, M. Kong, J. J. M. Bergeron, and B. I. Posner Regulation of Glycogen Synthase in Rat Hepatocytes. EVIDENCE FOR MULTIPLE SIGNALING PATHWAYS J. Biol. Chem., October 1, 1999; 274(40): 28279 - 28285. [Abstract] [Full Text] [PDF]
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M. L. Standaert, G. Bandyopadhyay, L. Perez, D. Price, L. Galloway, A. Poklepovic, M. P. Sajan, V. Cenni, A. Sirri, J. Moscat, et al. Insulin Activates Protein Kinases C-zeta and C-lambda by an Autophosphorylation-dependent Mechanism and Stimulates Their Translocation to GLUT4 Vesicles and Other Membrane Fractions in Rat Adipocytes J. Biol. Chem., September 3, 1999; 274(36): 25308 - 25316. [Abstract] [Full Text] [PDF]
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G. J. Etgen, K. M. Valasek, C. L. Broderick, and A. R. Miller In Vivo Adenoviral Delivery of Recombinant Human Protein Kinase C-zeta Stimulates Glucose Transport Activity in Rat Skeletal Muscle J. Biol. Chem., August 6, 1999; 274(32): 22139 - 22142. [Abstract] [Full Text] [PDF]
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B. Yu, L. A. Poirier, and L. E. Nagy Mobilization of GLUT-4 from intracellular vesicles by insulin and K+ depolarization in cultured H9c2 myotubes Am J Physiol Endocrinol Metab, August 1, 1999; 277(2): E259 - E267. [Abstract] [Full Text] [PDF]
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Q. Wang, R. Somwar, P. J. Bilan, Z. Liu, J. Jin, J. R. Woodgett, and A. Klip Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts Mol. Cell. Biol., June 1, 1999; 19(6): 4008 - 4018. [Abstract] [Full Text] [PDF]
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M. L. Standaert, G. Bandyopadhyay, M. P. Sajan, L. Cong, M. J. Quon, and R. V. Farese Okadaic Acid Activates Atypical Protein Kinase C (zeta /lambda ) in Rat and 3T3/L1 Adipocytes. AN APPARENT REQUIREMENT FOR ACTIVATION OF GLUT4 TRANSLOCATION AND GLUCOSE TRANSPORT J. Biol. Chem., May 14, 1999; 274(20): 14074 - 14078. [Abstract] [Full Text] [PDF]
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M. L. Standaert, G. Bandyopadhyay, E. K. Antwi, and R. V. Farese RO 31-8220 Activates c-Jun N-Terminal Kinase and Glycogen Synthase in Rat Adipocytes and L6 Myotubes. Comparison to Actions of Insulin Endocrinology, May 1, 1999; 140(5): 2145 - 2151. [Abstract] [Full Text]
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D. R. Tyson, J. T. Swarthout, and N. C. Partridge Increased Osteoblastic c-fos Expression by Parathyroid Hormone Requires Protein Kinase A Phosphorylation of the Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein at Serine 133 Endocrinology, March 1, 1999; 140(3): 1255 - 1261. [Abstract] [Full Text]
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J. J. Evans Modulation of Gonadotropin Levels by Peptides Acting at the Anterior Pituitary Gland Endocr. Rev., February 1, 1999; 20(1): 46 - 67. [Abstract] [Full Text]
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Z. A. Khayat, T. Tsakiridis, A. Ueyama, R. Somwar, Y. Ebina, and A. Klip Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC Am J Physiol Cell Physiol, December 1, 1998; 275(6): C1487 - C1497. [Abstract] [Full Text] [PDF]
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 Y. Izumi, T. Hirose, Y. Tamai, S.-i. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K. J. Kemphues, and S. Ohno An Atypical PKC Directly Associates and Colocalizes at the Epithelial Tight Junction with ASIP, a Mammalian Homologue of Caenorhabditis elegans Polarity Protein PAR-3 J. Cell Biol., October 5, 1998; 143(1): 95 - 106. [Abstract] [Full Text] [PDF]
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Y. Kanoh, G. Bandyopadhyay, M. P. Sajan, M. L. Standaert, and R. V. Farese Thiazolidinedione Treatment Enhances Insulin Effects on Protein Kinase C-zeta /lambda Activation and Glucose Transport in Adipocytes of Nondiabetic and Goto-Kakizaki Type II Diabetic Rats J. Biol. Chem., May 26, 2000; 275(22): 16690 - 16696. [Abstract] [Full Text] [PDF]
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G. Bandyopadhyay, M. P. Sajan, Y. Kanoh, M. L. Standaert, T. R. Burke Jr., M. J. Quon, B. C. Reed, I. Dikic, L. E. Noel, C. B. Newgard, et al. Glucose Activates Mitogen-activated Protein Kinase (Extracellular Signal-regulated Kinase) through Proline-rich Tyrosine Kinase-2 and the Glut1 Glucose Transporter J. Biol. Chem., December 22, 2000; 275(52): 40817 - 40826. [Abstract] [Full Text] [PDF]
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N. A. Patel, C. E. Chalfant, J. E. Watson, J. R. Wyatt, N. M. Dean, D. C. Eichler, and D. R. Cooper Insulin Regulates Alternative Splicing of Protein Kinase C beta II through a Phosphatidylinositol 3-Kinase-dependent Pathway Involving the Nuclear Serine/Arginine-rich Splicing Factor, SRp40, in Skeletal Muscle Cells J. Biol. Chem., June 15, 2001; 276(25): 22648 - 22654. [Abstract] [Full Text] [PDF]
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F. Oriente, P. Formisano, C. Miele, F. Fiory, M. A. Maitan, G. Vigliotta, A. Trencia, S. Santopietro, M. Caruso, E. Van Obberghen, et al. Insulin Receptor Substrate-2 Phosphorylation Is Necessary for Protein Kinase Czeta Activation by Insulin in L6hIR Cells J. Biol. Chem., September 28, 2001; 276(40): 37109 - 37119. [Abstract] [Full Text] [PDF]
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