Inhibition of glycogen synthesis by fatty acid in C2C12 muscle cells is independent of PKC-alpha , -epsilon , and -theta

R. Cazzolli, D. L. Craig, T. J. Biden, and C. Schmitz-Peiffer

Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia


    ABSTRACT
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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 beta -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-alpha , -epsilon , or -theta mutants was unable to prevent the inhibitory effects of oleate on glycogen synthesis. We conclude that neither mitochondrial lipid metabolism nor activation of PKC-alpha , -epsilon , or -theta 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


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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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 beta -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 beta -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 beta -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 beta -oxidation does not play a role in the effects of unsaturated FFA.


    MATERIALS AND METHODS
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Materials. Antibodies to PKC-alpha , PKC-beta , PKC-delta , and PKC-theta were obtained from Transduction Laboratories (Lexington, KY). Antibodies to PKC-epsilon 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-epsilon pseudo-substrate peptide (myrPSPepsilon ) 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 beta -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-alpha (kinase-dead mutation K368R), PKC-epsilon (kinase-dead mutation K436R), and PKC-theta (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 alpha -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-alpha (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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ABSTRACT
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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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Fig. 1.   Investigation of roles of glucose uptake and beta -oxidation in inhibition of glycogen synthesis in C2C12 myotubes by oleate. In each experiment, cells were pretreated in the absence and presence of oleate for 16 h in 1% FCS-EMEM, followed by serum starvation for 2 h in SF-EMEM also in the absence or presence of FFA. A: cells were incubated in Krebs-Ringer-HEPES buffer for 20 min in the absence or presence of insulin, and the uptake of 2-deoxy-[3H]glucose was then determined over 10 min. *** P < 0.005, insulin-stimulated vs. basal unpretreated cells; ** P < 0.01 (basal) and * P < 0.05 (insulin-stimulated) oleate-pretreated vs. unpretreated cells. B: cells were stimulated with insulin for 1 h in the presence of [14C]glucose. Etomoxir was absent or present during and after the serum starvation period, as indicated. Extracts were prepared in 1 M KOH, and glycogen was precipitated with ethanol and quantified by scintillation counting. All procedures were carried out as described in MATERIALS AND METHODS. ** P < 0.01, insulin-stimulated vs. basal unpretreated cells; *** P < 0.005 oleate-pretreated vs. unpretreated insulin-stimulated cells; * P < 0.05 (basal), dagger  P < 0.01 (insulin-stimulated), Dagger  P < 0.02 (insulin-stimulated + oleate) between untreated and etomoxir-treated groups.

Second, we determined whether the effects of the unsaturated fatty acid oleate were dependent on beta -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 beta -oxidation. Indeed, measurement of CO2 released from oleate under these conditions confirmed that beta -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 beta -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-alpha , 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-alpha 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-beta 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-delta 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-epsilon . 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-theta 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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Fig. 2.   Effects of free fatty acids (FFAs) on subcellular localization of protein kinase C (PKC) isoforms in C2C12 myotubes. Cells were incubated in the absence (Con) or presence of oleate (Ol), palmitate (Palm), or linoleate (Lino) for 16 h in 1% EMEM, followed by serum starvation for 2 h in SF-EMEM also in the absence or presence of the FFAs. Cytosolic (C) and membrane (M) fractions were prepared from lysates, as described in MATERIALS AND METHODS. A: proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with PKC isoform-specific antibodies (Immunoblots). Alternatively, proteins in gels were stained by Coomassie brilliant blue G250 (Coomassie stain). B: immunoblots were analyzed by densitometry, and the means of PKC levels in M and C fractions, as well as membrane-to-cytosol ratios from 3 independent experiments, are shown. * P < 0.05, *** P < 0.005 lipid-pretreated vs. unpretreated cells.

The time course of the effects of oleate on PKC-epsilon 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-epsilon 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-epsilon protein continued to decrease in the cytosolic fraction.


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Fig. 3.   Time course of PKC-epsilon translocation in response to oleate treatment. Cells were incubated in the presence of oleate for 2 h (i.e., during serum starvation only), 4 h, or 18 h, as indicated, and PKC-epsilon levels were determined in C and M fractions, as given for Fig. 2. A: an immunoblot from 1 experiment, typical of 3 carried out, is shown. B: immunoblots were analyzed by densitometry, and means of PKC-epsilon levels in M and C fractions are shown. * P < 0.05, * P < 0.02, *** P < 0.01 oleate-pretreated vs. respective unpretreated (control) cell fraction.

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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Fig. 4.   Effects of FFAs on total diacylglycerol (DAG) levels in C2C12 myotubes. A: cells were incubated in the absence (Control) or presence of FFAs, as for Fig. 1, and lipid extracts were prepared. DAGs were phosphorylated by DAG kinase in the presence of [gamma -32P]ATP, separated by thin-layer chromatography, and subjected to phosphorimaging. B: results from densitometric analysis of 2 experiments, each carried out in duplicate, are shown. All procedures were carried out as described in MATERIALS AND METHODS. *** P < 0.001 lipid-pretreated vs. unpretreated cells.

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-alpha , -beta , and -gamma , with an IC50 of 0.02 µM, and also the novel PKCs delta  and epsilon , 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-epsilon translocation, we also investigated the effect of a cell-permeant myristoylated PKC-epsilon pseudo-substrate peptide, myrPSPepsilon , 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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Fig. 5.   Effect of PKC inhibitors on inhibition of glycogen synthesis in C2C12 myotubes by oleate. Glycogen synthesis was determined in cells pretreated without or with oleate, in the absence or presence of 2 µM GF-109203X (A) or 2 µM myrPSPepsilon (B) during all incubations. * P < 0.05, ** P < 0.01, *** P < 0.001, insulin-stimulated vs. basal unpretreated cells; dagger  P < 0.05, Dagger  P < 0.002, oleate-pretreated vs. unpretreated insulin-stimulated cells; NS, nonsignificant.

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-alpha and PKC-epsilon were studied because of the greater lipid response of these enzymes. In addition, because PKC-theta expression has in fact been detected in C2C12 myoblasts in a previous study (28), although at very low levels compared with PKC-alpha , 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-theta 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 alpha  and epsilon  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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Fig. 6.   Adenovirus-mediated overexpression of PKC isoforms in C2C12 myotubes. A: C2C12 cells were incubated with increasing amounts of high-titer virus stocks, as indicated, during the 2nd day of differentiation. Recombinant viruses caused overexpression of green fluorescent protein (GFP) alone, or together with wild-type (wt) PKC-alpha , PKC-epsilon , or PKC-theta , or kinase-dead PKC-alpha (K368R), PKC-epsilon (K436R), or PKC-theta (K409R). Lysates of myotubes were prepared and subjected to immunoblotting (IB) with PKC isoform-specific antibodies. B: overexpressed histidine-tagged protein was purified with Ni2+-chelating resin and subjected to PKC assay. All procedures were carried out as described in MATERIALS AND METHODS. Similar results were obtained in 2 separate experiments.

Overexpression of wild-type PKC-alpha , PKC-epsilon , or PKC-theta 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-alpha or PKC-epsilon 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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Fig. 7.   Effect of wild-type and kinase-dead PKC isoform overexpression on inhibition of glycogen synthesis in C2C12 myotubes by oleate. Cells were infected with adenovirus for expression of PKC-alpha (A), PKC-epsilon (B), or PKC-theta (C) during differentiation, as indicated. Myotubes were pretreated in the absence or presence of oleate, and glycogen synthesis was determined, as given for Fig. 1. All procedures were carried out as described in MATERIALS AND METHODS. * P < 0.02, *** P < 0.005 oleate-pretreated vs. unpretreated insulin-stimulated cells overexpressing the same constructs; dagger  P < 0.005, PKC mutant vs. GFP in unpretreated insulin-stimulated cells; Dagger  P < 0.001, PKC mutant vs. GFP in oleate-pretreated insulin-stimulated cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, beta -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 beta -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 beta -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 beta -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-epsilon 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-theta 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.


    ACKNOWLEDGEMENTS

We thank Prof. Gottfried Baier for the provision of wild-type and mutant PKC constructs, and François Karstens for technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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