Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia
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ABSTRACT |
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We have previously
shown that glycogen synthesis is reduced in lipid-treated
C2C12 skeletal muscle myotubes and that this is
independent of changes in glucose uptake. Here, we tested whether mitochondrial metabolism of these lipids is necessary for this inhibition and whether the activation of specific protein kinase C
(PKC) isoforms is involved. C2C12 myotubes were
pretreated with fatty acids and subsequently stimulated with insulin
for the determination of glycogen synthesis. The carnitine
palmitoyltransferase-1 inhibitor etomoxir, an inhibitor of
-oxidation of acyl-CoA, did not protect against the inhibition of
glycogen synthesis caused by the unsaturated fatty acid oleate. In
addition, although oleate caused translocation, indicating activation,
of individual PKC isoforms, inhibition of PKC by pharmacological agents
or adenovirus-mediated overexpression of dominant negative PKC-
,
-
, or -
mutants was unable to prevent the inhibitory effects of
oleate on glycogen synthesis. We conclude that neither mitochondrial
lipid metabolism nor activation of PKC-
, -
, or -
plays a role
in the direct inhibition of glycogen synthesis by unsaturated fatty acids.
skeletal muscle; C2C12 cells; insulin resistance; protein kinase C; adenovirus; etomoxir
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INTRODUCTION |
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INSULIN RESISTANCE OF PERIPHERAL TISSUES is a major characteristic of type 2 diabetes and is an early defect in the development of the disease. Because skeletal muscle is the principal site of glucose disposal in response to the hormone, insulin resistance of this tissue is of primary importance. Although several factors may contribute to a reduction of insulin action in muscle, a strong correlation with increased lipid availability has been observed in several studies involving humans (7, 38), animals (20, 33, 50), and cultured cells (42, 44). Acute elevation of plasma free fatty acids (FFAs) can reduce glucose uptake in skeletal muscle, leading to an indirect inhibition of glycogen synthesis (45), but more extended elevation of FFAs also has a direct effect on glycogen synthesis (6).
The mechanisms by which lipids may inhibit glucose disposal are
not fully understood. Early work suggested that glucose oxidation is
inhibited by a process of substrate competition upon mitochondrial -oxidation of lipids as described by the glucose-fatty acid cycle (37). Glycogen synthesis may not be impaired by the same
mechanism in type 2 diabetes, and it is unclear whether
-oxidation
plays a role (36). More recent studies have suggested that
lipid-derived second messengers can activate pathways that interfere
with normal insulin signal transduction (see Ref. 39 for
review). For example, in addition to undergoing
-oxidation or
incorporation into triglyceride and phospholipids, FFAs are also
incorporated into diacylglycerol (DAG) in muscle cells, which may
inhibit insulin action through the activation of protein kinase C (PKC) isoforms.
The PKC family consists of 10 isoforms, grouped into the
classical, novel, and atypical PKCs, which exhibit differences in their
calcium and lipid dependence and in protein interactions (27). During activation, the kinases can translocate
within the cell, and the extent to which a PKC isoform is found in
membrane fractions has been shown to correlate well with its activation (56, 58). As a consequence of prolonged activation, the
kinases can also be downregulated by proteolysis.
A number of studies have linked aberrant activation of PKC to diminished insulin sensitivity, especially to that occurring together with increased lipid availability to insulin target tissues. The isoforms implicated are the classical and particularly the novel PKCs (13, 15, 17, 35, 41, 43), whereas the atypical PKCs appear to play a positive role in the regulation of glucose transport (4, 5). Alterations in novel PKCs in association with insulin resistance have been observed in muscle from the high-fat-fed rat (41, 43), lipid-infused rats (13), obese rats and humans (17, 35), and the diabetes-prone line of Psammomys obesus (15). Although PKC activity has been shown to inhibit insulin signal transduction at several sites in intact cells, ranging from the insulin receptor to more distal components (1, 11, 15, 17, 19, 34), the studies of its role in insulin resistance have so far been of a correlative nature, and the mechanisms involved have yet to be determined.
We have previously characterized a model of lipid-induced insulin
resistance by use of C2C12 myotubes pretreated
with FFAs, which reduce insulin-stimulated glycogen synthesis
(42). Whereas in other models it is usually difficult to
distinguish the direct inhibition of glycogen synthesis by
lipids from effects that are secondary to the inhibition of glucose
uptake and availability, this system has the advantage that
insulin-stimulated glycogen synthesis is independent of glucose
transport. C2C12 cells exhibit a rate of
glucose uptake that is 10-fold higher than the rate of glycogen
synthesis, whereas insulin and FFAs have little effect (42), enabling us to study the regulation of glycogen
synthesis in the absence of possible limiting effects of reduced
glucose influx. This system is therefore complementary to studies of
type 2 diabetic subjects (52) and lipid-infused subjects
(7), showing that defects in glycogen synthesis can be
independent of glucose uptake. Here, we report that unsaturated FFAs
specifically give rise to alterations in the cellular distribution of
certain PKC isoforms but that this activation has no effect on the
control of glycogen synthesis in this model. In addition, we show that -oxidation does not play a role in the effects of unsaturated FFA.
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MATERIALS AND METHODS |
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Materials.
Antibodies to PKC-, PKC-
, PKC-
, and PKC-
were obtained from
Transduction Laboratories (Lexington, KY). Antibodies to PKC-
were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Oleic acid,
linoleic acid, palmitic acid, and 2-deoxy-D-glucose were from Sigma Chemical (St. Louis, MO). Insulin was from Novo Nordisk
(Copenhagen, Denmark).
D-[U-14C]glucose,
2-deoxy-D-[2,6-3H]glucose, and
[1-14C]oleate were from Amersham (Buckinghamshire, UK).
Etomoxir was from Research Biochemicals International (Natick, MA).
Modified conventional PKC pseudo-substrate peptide (19-31,
Ser25) and myristoylated PKC-
pseudo-substrate peptide
(myrPSP
) 149-164 (Myr-Glu-Arg-Met-Arg-Pro-Arg-Lys-Arg-Gln-Gly-Ala-Val-Arg-Arg-Arg-Val) were from Auspep (Parkville, Victoria, Australia). Ni-NTA
Superflow was from Qiagen (Clifton Hill, Victoria, Australia).
Cell culture and fatty acid pretreatment. C2C12 myoblasts were maintained in MEM with Earle's salts supplemented with 2 mM glutamine, 15 mM HEPES, pH 7.5, 500 IU/ml penicillin, and 100 µg/ml streptomycin (EMEM) containing 10% fetal calf serum (FCS). For myotube formation, myoblasts were seeded at a density of 700 cells/cm2 in gelatin-coated 6- and 12-well plates or 10-cm dishes and grown to confluence over 3 days in 10% FCS-EMEM. Differentiation of confluent cells into myotubes was induced by lowering the concentration of FCS to 1%. Myotubes were used for experiments after 4 days. Lipid-containing media were prepared by conjugation of FFAs with BSA (42). Unless otherwise stated, myotubes were pretreated for 16 h in 1 ml/well or 10 ml/dish of 1% FCS-EMEM containing 5% BSA with or without FFAs, and then for 2 h in 0.5 ml/well or 5 ml/dish serum free- (SF-) EMEM, again containing 5% BSA with or without FFA. The concentrations of FFAs used (2 mM oleate, 1 mM linoleate, or 0.75 mM palmitate) have each been shown previously to reduce insulin-stimulated glycogen synthesis by 50% (42).
Preparation of cytosolic and solubilized membrane fractions. C2C12 cells were fractionated by a method we have previously used for skeletal muscle (41). Lipid-pretreated myotubes in 10-cm dishes were washed twice with ice-cold phosphate-buffered saline and scraped into 0.5-ml homogenization buffer [20 mM MOPS, pH 7.5, 250 mM mannitol, 1.2 mM EGTA, 200 µg/ml leupeptin, 2 mM benzamidine, and 2 mM phenylmethylsulfonyl fluoride (PMSF)]. Cells were sonicated (15 pulses, using a Branson 250 Sonifier and microtip at 20% duty cycle, power setting 2) and lysates centrifuged at 100,000 rpm in a Beckman TLA100 rotor for 10 min at 4°C. Supernatants were retained as the cytosolic fractions, while pellets were rinsed with 200 µl of homogenization buffer and resuspended in 500 µl of solubilization buffer [20 mM MOPS, pH 7.5, 1% (vol/vol) Triton X-100, 2 mM EDTA, 2 mM EGTA, 200 µg/ml leupeptin, 2 mM benzamidine, and 2 mM PMSF]. After incubation for 1 h at 4°C, suspensions were again centrifuged at 100,000 rpm for 10 min at 4°C. The resulting supernatants were retained as the solubilized membrane fractions. Cell lysates and fractions were immunoblotted using isoform-specific PKC antibodies, and immunoblots were quantified as previously described (41).
DAG assay. Lipid extracts were prepared from FFA-pretreated myotubes in 10-cm dishes, as previously described (42). DAG content was determined using a radiometric DAG assay kit (Amersham) according to the manufacturer's instructions. To improve resolution of phosphatidic acid by thin-layer chromatography, plates were developed in modified solvent systems (42). 32P-labeled phosphatidic acid was identified after phosphorimaging by co-migration with authentic standards.
Measurement of glucose transport. Glucose transport was determined by a method adapted from Moyers et al. (30). Lipid-pretreated myotubes in 6-well plates were incubated for 20 min in Krebs-Ringer-HEPES buffer (1 ml/well) containing 2% (wt/vol) BSA, in the absence or presence of 1 µM insulin. Glucose uptake was then measured over 10 min in the presence of 40 µM 2-deoxy-[3H]glucose (0.5 µCi/well). Cells were rapidly washed three times with ice-cold PBS and extracted in 1 ml of PBS containing 0.05% (wt/vol) SDS. After incubation at 37°C for 30 min, extracts were subjected to liquid scintillation counting. Protein determination was by bicinchoninic acid assay (Pierce). Nonspecific glucose uptake in the absence and presence of FFA was determined using 10 µM cytochalasin B and was subtracted from the total rates observed.
Measurement of glycogen synthesis. Lipid-pretreated myotubes in 12-well plates were incubated for 1 h in 0.5 ml/well of SF-EMEM containing D-[U-14C]glucose (4 µCi/ml) in the absence or presence of 100 nM insulin and FFA, and glycogen production was determined as previously described (42).
Measurement of -oxidation.
In a modification of the method described by Muoio et al.
(31), myotubes in 6-well plates were incubated for 2 h in SF-EMEM (1 ml/well) containing [14C]oleate (0.5 µCi/ml) in the presence of 10 µM etomoxir or vehicle [0.1%
(vol/vol) dimethyl sulfoxide]. The cells were then scraped into the
medium, which was transferred to 25-ml flasks containing a separate
reservoir of 2 M NaOH (1 ml). Flasks were sealed with rubber septa,
injected with 100 µl of 70% (vol/vol) perchloric acid to drive off
14CO2, and gently shaken at 37°C for 2 h. The contents of the reservoirs were then subjected to liquid
scintillation counting to determine the content of captured
14CO2.
Generation and use of recombinant adenovirus.
Histidine-tagged wild-type and kinase-dead forms of PKC-
(kinase-dead mutation K368R), PKC-
(kinase-dead mutation K436R), and
PKC-
(kinase-dead mutation K409R) were gifts from G. Baier, University of Innsbruck, Austria, and have been extensively
characterized (3, 18, 54). The pAdEasy system was used to
generate a recombinant adenovirus for the expression of these PKC
constructs, as described by He et al. (14), allowing
co-expression of green fluorescent protein (GFP) and PKC. A
pAdEasy-derived virus expressing GFP alone was used in control
infections for PKC constructs. The amount of virus required to give
80-100% infection efficiency for each virus constructed using the
pAdEasy system was individually determined by visualizing GFP
expression. All viruses were used to infect
C2C12 myotubes in 12-well plates at day
2 of differentiation, i.e., 3 days before experiments. To confirm
the activities of the overexpressed wild-type and mutant PKC proteins,
infected myotubes were lysed 3 days after infection, and
histidine-tagged PKCs were purified using Ni2+-chelating
resin (2). PKC activity was determined in the presence of
-phosphatidyl-L-serine and 1,2-dioctanoyl-sn-glycerol by
use of 5 µM of modified conventional PKC pseudosubstrate peptide
(19-31, Ser25) as substrate, with 1 mM
CaCl2 also present for assay of PKC-
(40).
Statistical analysis. All results are expressed as means ± SE and were analyzed using Student's t-test. Statistical calculations were performed using Statview SE + Graphics for Macintosh (Abacus Concepts, Berkeley, CA).
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RESULTS |
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We have previously reported (42) that pretreatment of
C2C12 myotubes with FFAs led to the inhibition
of glycogen synthesis. The saturated fatty acid palmitate inhibited PKB
signaling through the elevation of intracellular ceramide, which was
sufficient to reduce the stimulation of glycogen synthesis by insulin.
In contrast, the unsaturated fatty acids oleate and linoleate had no
effect on this pathway, and in the present study we investigated alternative mechanisms by which the unsaturated lipids might inhibit glycogen synthesis. First, although we have previously shown that glucose uptake is little affected by insulin and FFAs in this system
when measured over 1 h using 2-deoxyglucose (42), we reexamined their effects during a shorter assay period to reduce complications, such as ATP depletion, arising from the accumulation of
phosphorylated 2-deoxyglucose. In this way we were able to confirm that
insulin was able to stimulate glucose uptake by only 25% and that the
unsaturated FFA oleate had no inhibitory effect (Fig.
1A). Therefore, inhibition of
insulin-stimulated glycogen synthesis by the FFA is unlikely to arise
simply from a reduction in intracellular glucose availability.
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Second, we determined whether the effects of the unsaturated fatty acid
oleate were dependent on -oxidation of the lipid. During serum
starvation, pretreated myotubes were incubated in the absence or
presence of 10 µM etomoxir, an inhibitor of carnitine palmitoyltransferase I (22), which is essential for
transport of the lipid into mitochondria, and hence for
-oxidation.
Indeed, measurement of CO2 released from oleate under these
conditions confirmed that
-oxidation was inhibited by 41 ± 6.1% (P < 0.01) in the presence of etomoxir (not
shown). Subsequent basal and insulin-stimulated glycogen synthesis was
found to be increased, indicating that the inhibition of lipid
oxidation could indeed increase glycogen synthesis (Fig.
1B). The inhibitory effect of oleate on insulin-stimulated
glycogen synthesis was, however, still apparent in the presence of
etomoxir, indicating that
-oxidation of oleate was not necessary for
its inhibitory effect. The presence of the inhibitor during the entire
period of oleate pretreatment was also unable to reverse this
inhibition and in fact did not generate the increase in glycogen
synthesis seen after the shorter incubation (not shown).
Third, we determined the effect of FFAs on PKC activation by analysis
of the membrane translocation of PKC isoforms. Treatment of myotubes
with FFAs caused alterations in the level and cellular distribution of
PKC, as determined by immunoblotting with isoform-specific antibodies.
Representative immunoblots are shown in Fig.
2A, together with a
Coomassie-stained gel confirming uniform protein loading. Densitometry
of stained proteins (omitting the 69-kDa albumin band derived from
culture medium) indicated that lipid treatments did not reduce total
protein recovery in the cytosol: oleate, 1.24 ± 0.15 densitometric units relative to control; palmitate, 1.22 ± 0.13;
linoleate, 1.02 ± 0.15; n = 6. PKC isoform levels and distribution, expressed as the ratio of membrane-associated to
cytosolic PKC protein, are shown in Fig. 2B. Both oleate and linoleate caused a decrease in the cytosolic level of PKC-, whereas the amount of this isoform in the membrane fraction was not reduced but
in fact tended to increase. The ratio of membrane-bound to cytosolic
PKC-
therefore increased, although the total levels were reduced,
which could be interpreted as the result of chronic activation and
downregulation of the enzyme. In contrast, palmitate treatment did not
significantly affect the levels or distribution of this isoform.
PKC-
was poorly detected in the myotube fractions, making
quantitation difficult, but results from immunoblotting indicated that
pretreatment with lipid tended to reduce the levels of this enzyme.
Cytosolic PKC-
was reduced by all FFAs, but to a greater degree with
oleate or linoleate, such that the latter also significantly altered
the distribution of this kinase. The greatest effect of the unsaturated
FFAs on PKC distribution was seen on PKC-
. Although total levels of
the kinase were reduced, the ratio of membrane to cytosolic protein was
increased 2.8-fold by oleate and 6.2-fold by linoleate, indicating PKC
activation. In contrast to our studies of rat muscle, we were unable to
detect PKC-
in myotube fractions by immunoblotting (not shown),
suggesting that this isoform either is expressed at very low levels or
is in fact absent.
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The time course of the effects of oleate on PKC- levels and
distribution was also determined to distinguish membrane translocation from cytosolic downregulation. Myotubes were preincubated for 2, 4, or
18 h with the FFA, and PKC-
levels were again determined by
immunoblotting of cytosolic and membrane fractions (Fig.
3). We observed a 2.5- to 3-fold increase
in the amount of the kinase recovered in the membrane fraction, which
was maintained for
18 h, indicating that the active fraction of the
kinase was not downregulated by longer preincubation with oleate but
was still preserved at maximal levels. In contrast, PKC-
protein
continued to decrease in the cytosolic fraction.
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These observations suggested that treatment of the myotubes with oleate
and linoleate led to the intracellular accumulation of lipid activators
of PKC; they are consistent with the hypothesis that unsaturated FFAs
may inhibit glycogen synthesis through the action of one or more of
these kinases. Because DAG can be generated from FFAs and is known to
stimulate PKC translocation, we determined the total level of DAG in
cells treated with the different lipids. Surprisingly, oleate and
linoleate gave only limited increases in DAG levels, which did not
attain statistical significance, whereas palmitate treatment caused a
>4-fold increase in total intracellular DAG (Fig.
4).
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To examine the role of PKC in the effect of oleate on glycogen
synthesis, we first employed a pharmacological inhibitor of this family
of kinases. GF-109203X (bisindolylmaleimide I) inhibits several
isoforms, including conventional PKC-, -
, and -
, with an
IC50 of 0.02 µM, and also the novel PKCs
and
,
each with an IC50 of 0.2 µM (25, 53). The
presence of this inhibitor during oleate pretreatment failed to reverse
the effects of the lipid on insulin-stimulated glycogen synthesis (Fig.
5A). Because the effects of
unsaturated FFAs were most obvious on PKC-
translocation, we also
investigated the effect of a cell-permeant myristoylated PKC-
pseudo-substrate peptide, myrPSP
, on the inhibition of glycogen
synthesis by oleate. MyrPSPs have previously been used to determine PKC
isoform-dependent effects (46, 49, 51). This inhibitor was
also unable to prevent the decrease in insulin-stimulated glycogen
synthesis (Fig. 5B).
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To confirm the negative results obtained by the use of the
pharmacological PKC inhibitors, we also determined the effects of
adenovirus-mediated expression of wild-type and kinase-dead forms of
certain isoforms. PKC- and PKC-
were studied because of the
greater lipid response of these enzymes. In addition, because PKC-
expression has in fact been detected in C2C12
myoblasts in a previous study (28), although at very low
levels compared with PKC-
, and because it has been strongly
associated with insulin resistance in several reports, we also
investigated the effects of this isoform. Preliminary experiments
indicated that ectopically expressed PKC-
also translocates in
response to unsaturated FFA pretreatment (not shown). The kinase-dead
PKC mutants have previously been shown to act in an
isoform-specific dominant negative fashion when overexpressed
(3, 18, 54).
Initial characterization of adenovirus-infected myotubes indicated that
the expression of PKC isoforms and
could be elevated up to
100-fold above the endogenous levels depending on the amount of virus
used (Fig. 6A). In subsequent
experiments, we used an amount of virus giving 80-90% infection
efficiency, as determined by the co-expression of GFP, which resulted
in 10- to 30-fold PKC overexpression. Kinase assays of the
affinity-purified overexpressed proteins confirmed the lack of activity
of the kinase-dead PKC mutants, in contrast to the results obtained
with the wild-type forms (Fig. 6B). A virus causing
expression of GFP alone was used for control purposes in further
experiments.
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Overexpression of wild-type PKC-, PKC-
, or PKC-
did not have
any significant effects on glycogen synthesis in untreated C2C12 myotubes and also did not potentiate the
effect of oleate (Fig. 7). In agreement
with the results obtained using pharmacological inhibitors of PKC,
overexpression of the kinase-dead PKC isoforms did not reverse the
effects of the lipid. Thus overexpression of any PKC mutant did not
prevent a significant decrease in insulin-stimulated glycogen synthesis
caused by oleate pretreatment. Overexpression of either PKC-
or
PKC-
kinase-dead mutants in fact gave rise to a minor but
significant decrease in insulin-stimulated glycogen synthesis. We also
examined the effect of wild-type and mutant PKC overexpression in
myotubes pretreated with linoleate and again were unable to potentiate
or reverse the effects of this unsaturated FFA (not shown).
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DISCUSSION |
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We have previously observed differences in the inhibitory effects
of saturated and unsaturated FFAs on glucose disposal in a model of
lipid-induced insulin resistance employing
C2C12 myotubes, and we have found evidence for
the role of ceramide and protein phosphatase 2A in mediating the effect
of the saturated FFA palmitate (8, 42). In this study, we
have examined possible mechanisms, involving inhibition of glucose
uptake, -oxidation, or PKC activation, for the inhibition of
insulin-stimulated glycogen synthesis by unsaturated FFAs. Glucose
uptake was little affected by insulin or FFAs in this system and was
not studied further. The use of the CPT I inhibitor etomoxir indicated
that
-oxidation was not required for the inhibitory effect of
oleate, suggesting that the lipid did not decrease glycogen synthesis
by means of the glucose-fatty acid cycle or any other pathway involving
mitochondrial lipid metabolism.
The metabolism of FFAs by -oxidation is essential for the inhibition
of glucose oxidation and glucose uptake by the glucose-fatty acid cycle
(37), but the role of mitochondrial metabolism in the
effects of lipids on glycogen synthesis is not clear (36). By preventing the transport of long-chain acyl-CoA derivatives of FFA
into mitochondria by use of etomoxir, we were able to demonstrate that
this process is not necessary for the effect of unsaturated FFA we have
observed on glycogen synthesis, indicating that the inhibitory effects
of these lipids are mediated outside the mitochondria. Short-term
treatment with the inhibitor did, however, elevate both basal and
insulin-stimulated glycogen synthesis, suggesting that
-oxidation
can depress glycogen synthesis in C2C12
myotubes, even if it does not mediate the effects of oleate
pretreatment. Long-term etomoxir treatment did not elevate glycogen
synthesis, suggesting that a buildup of FFA derivatives in the cytosol
of myotubes leads to inhibitory effects. This is in agreement with a
recent study showing that both high-fat feeding and etomoxir treatment
lead to intramyocellular lipid accumulation and insulin resistance in
rats (12).
Our findings that unsaturated FFAs increased the proportion of specific
PKC isoforms in solubilized membrane fractions from C2C12 myotubes indicate that the lipids caused
chronic activation of these enzymes, whereas the reductions in total
kinase levels suggest an additional effect of PKC downregulation by
proteolysis. Because only cytosolic levels of PKCs were reduced, this
may indicate that the isoforms continuously translocate to the
membrane, where they are activated but eventually degraded, such that
the cytosolic pool is depleted while the membrane pool appears
constant. This possibility is supported by the time course of PKC-
translocation in response to oleate, showing that only cytosolic levels
of the kinase are reduced with time. Such activation corresponds well with the alterations in PKC that we observed in fat-fed rats
(41) and is consistent with our initial hypothesis that
PKC plays a role in mediating lipid-induced insulin resistance in this model.
Because unsaturated FFAs had the greatest effects on PKC isoforms, we were surprised to find that total DAG levels in lipid-treated myotubes were elevated only when the saturated FFA palmitate was present, whereas the unsaturated FFAs oleate and linoleate had little effect. This specific effect of palmitate on DAG has, however, been previously observed in smooth muscle cells (16, 24, 57) and cultured human muscle (29). The lack of effect of palmitate on PKC distribution, despite the elevated DAG level induced, is consistent with the concept that polyunsaturated DAG species are the true activators of PKC (55), since DAG derived from palmitate will most likely comprise saturated acyl chains. Our data suggest that pretreatment with unsaturated FFAs leads to the elevation of minor but physiologically active pools of polyunsaturated DAG, although it is possible that intramyocellular elevation of other lipid species is responsible for the observed PKC translocation. Unsaturated FFAs themselves have been shown to stimulate PKC activity in vitro (21, 26, 32). The minor effects of palmitate on PKC, despite the large elevation of DAG, are in agreement with some but not all studies of PKC activation by lipid pretreatment of cultured cells (16, 24, 29, 57).
Using either pharmacological or molecular biological approaches, we were unable to find evidence for the involvement of PKC in the inhibitory effects of unsaturated FFAs on glycogen synthesis. Despite the apparent lack of effect of the kinase-dead PKC mutants, it is likely that they were able to exert dominant negative effects on endogenous PKC isoforms, given the high levels of overexpression achieved with adenovirus and their previously reported effects in other cells (3, 18, 54). The data derived by each of our approaches support the conclusion that, although unsaturated FFAs cause PKC translocation, this does not account for the concomitant inhibition of glycogen synthesis in C2C12 myotubes.
In agreement with the findings reported here, several PKC inhibitors
had no effect on glycogen synthase activity in rat adipocytes and L6
myotubes (47). Experiments carried out with incubated muscle, however, indicated an inhibitory role of PKC that was independent of glucose uptake (23). Insulin signaling
through the PKB pathway was also inhibited at the level of insulin
receptor tyrosine phosphorylation, insulin receptor
substrate-1-associated phosphatidylinositol 3-kinase, PKB, and glycogen
synthase kinase-3 (GSK-3) (23). Although we did not
observe inhibition of PKB and GSK-3 phosphorylation in
C2C12 cells treated with unsaturated FFAs
(42), this difference is unlikely to be related to the lack of expression of PKC- in C2C12
myotubes, because overexpression of this isoform did not give rise to
inhibition of glycogen synthesis (Fig. 7) or of PKB phosphorylation
(unpublished results). Other studies have reported increased muscle
glycogen synthesis in the presence of PKC inhibitors, and such
discrepancies may stem from the nonspecific actions of these agents.
For example, the PKC inhibitor RO 31-8220 has been shown to
activate c-Jun NH2-terminal kinase, or JNK, and hence
glycogen synthesis, in adipocytes and L6 myotubes independently of
effects on PKC (47). These problems are avoided in our
experiments in which kinase-dead PKC mutants are used.
It must be emphasized that we have investigated FFA effects on glycogen synthesis in a system in which such glucose disposal is independent of glucose transport into the cell: under the conditions employed here, C2C12 cells exhibit insulin-insensitive glucose uptake, which is little affected by FFAs (42). Thus, although decreased glucose availability can lead to reduced glycogen synthesis (9), we have shown that FFAs can also inhibit glycogen synthesis in myotubes through a distinct mechanism, in agreement with other human studies (7, 52). Although we are also able to conclude that PKC translocation does not play a role in the inhibition of glycogen synthesis, we cannot exclude the possibility that one or more PKC isoforms would inhibit insulin-stimulated glucose transport in intact muscle. Indeed, other studies have reported PKC-mediated inhibition of glucose transport in insulin-sensitive models (10, 29, 48). Although we have not observed unsaturated FFA-mediated inhibition of the PKB pathway (42), PKC activation resulting from lipid oversupply may inhibit other insulin signals that regulate glucose transport but not glycogen synthesis.
Furthermore, whereas we show that the incorporation of glucose into glycogen is reduced by preincubation with FFAs, our data do not distinguish between the inhibition of glycogen synthase and the stimulation of glycogen phosphorylase. FFAs could exert their effects through the regulation of either (or both) of these metabolic enzymes, and these possibilities remain to be determined.
In summary, we have refuted two possible mechanisms for the inhibition of glycogen synthesis in a defined model of unsaturated FFA-induced insulin resistance. First, we have shown that mitochondrial lipid oxidation is unnecessary to account for the reduction observed in glycogen synthesis in response to oleate pretreatment. Second, although we demonstrated that unsaturated FFAs gave rise to specific alterations in PKC translocation, these were also unrelated to the reduced glycogen synthesis. The underlying mechanism for the inhibition of glycogen synthesis by these lipids, independent of decreased glucose uptake, therefore remains to be determined.
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ACKNOWLEDGEMENTS |
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We thank Prof. Gottfried Baier for the provision of wild-type and mutant PKC constructs, and François Karstens for technical assistance.
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FOOTNOTES |
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This work was supported by the National Health and Medical Research Council of Australia and in part by a research grant from Aza Research Pty. Ltd.
Address for reprint requests and other correspondence: C. Schmitz-Peiffer, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010, Australia (E-mail: c.schmitz-peiffer{at}garvan.org.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 5, 2002;10.1152/ajpendo.00487.2001
Received 31 October 2001; accepted in final form 4 February 2002.
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