1Departments of Geriatrics, and Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; Departments of 2Exercise, and Sport Science, and 3Physiology, East Carolina University, Greenville 27858; and 4Department of Medicine-Cardiology, Duke University, Durham, North Carolina 27710
Submitted 9 April 2003 ; accepted in final form 17 December 2003
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ABSTRACT |
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protein kinase C; obesity; bilateral hindlimb perfusion; gastrocnemius; phorbol esters
One possible modulator of signal transduction leading to increased muscle protein synthesis in response to insulin may be protein kinase C (PKC). Although this kinase family has been reported to interact with components of mRNA translation machinery in a number of tissues (2, 20, 23, 28), PKC has been recently implicated as a modulator of insulin signal transduction whereby increased PKC activities, particularly those of the conventional family, lead to attenuated insulin receptor tyrosine kinase and phosphatidylinositol 3-kinase activities and impaired glucose disposal (7, 15, 18). Others (1, 6) have previously demonstrated that signal transduction through these two important kinases of insulin action is impaired in obese Zucker rats. It has been suggested that Ser/Thr phosphorylation at key sites on signaling proteins may interfere with normal enzymatic activity (24, 25). To support this claim, recent reports (4, 13, 15) have demonstrated that inhibition of PKC activity partially restores signal transduction and, ultimately, glucose disposal in muscle that is otherwise insulin resistant. Furthermore, activation of PKC using phorbol esters in vitro attenuates glucose disposal even in normal muscle (4). It is unknown whether PKC modulates the insulin-mediated regulation of protein synthesis in skeletal muscle.
The purpose of this study was to determine whether or not the enhanced insulin-mediated protein-anabolic response previously observed in obese Zucker rat skeletal muscle is modulated by PKC activity. First, we examined the effect of insulin on skeletal muscle PKC activity in lean and obese Zucker rats. We then examined the effect of acute alterations in PKC activity on insulin-stimulated rates of muscle protein synthesis by use of pharmacological activation and blockade methodologies.
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METHODS |
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Bilateral hindlimb preparation.
The bilateral hindlimb preparation was as previously described (810). Briefly, following anesthesia with pentobarbital sodium (65 mg/kg ip), animals were laparatomized, and polythene catheters were placed into the left and right iliac arteries, whereby each limb received identical perfusion medium either with or without insulin and/or, when necessary, containing either an inhibitor (GF) or an activator (TPA) of PKC. Using this technique, we were able to determine PKC activity (with and without insulin) and, in subsequent studies, rates of fiber-specific muscle protein synthesis (with or without insulin) with acute inhibition or activation of PKC within each animal. Hindlimbs were perfused with a Krebs-Henseleit buffer (KHB) medium (pH 7.4) containing time-expired, washed bovine red blood cells (Hct 30%), 4% bovine serum albumin, 5 mM glucose, 2 mM phenylalanine, and all other amino acids at physiological concentrations, as described by Jefferson et al. (17). Flow rate for each hindlimb was maintained at 5 ml/min (10 ml/min for both hindlimbs combined) using flow-matched peristaltic pumps. Before the introduction of red blood cells, the KHB was filtered through a nitrocellulose filter (Millipore, 0.8 µm pore size), and, when needed for protein synthesis experiments, L-[2,3,4,5,6-3H]phenylalanine (Amersham Life Science, Arlington Heights, IL) was added to the medium to yield a final concentration of 0.5 µCi/ml. Porcine insulin (Lilly, Indianapolis, IN) was provided at 20,000 µU/ml to one or both limbs when necessary. Hindlimbs receiving treatments were counterbalanced within groups, and medium was not circulated (single-pass perfusion preparation). Lean and obese animals perfused on a given day were selected at random, with equal numbers within each group. Medium used for animals on a given day was made with sufficient volume that all animals were perfused with identical medium.
The medium and bilateral hindlimb preparation were maintained at 37°C during the perfusion protocol and gassed with humidified 95% O2-5%CO2. PO2 was maintained at >90% saturation. A 15-min washout period with KHB medium preceded the 35-min bilateral hindlimb perfusion. Insulin, along with GF (1 µg/ml for PKC inhibition studies) or TPA (5 µg/ml for PKC activation studies) was present, when indicated, for the entire 35-min perfusion period. Concentrations of GF and TPA were determined previously to be effective for the attenuation and activation, respectively, of PKC during its involvement with glucose transport in muscle (4). After the 35-min period, entire gastrocnemius muscles were excised and subsequently partitioned into red and white portions. After excision and partitioning, muscles were quick-frozen using liquid nitrogen-cooled metal tongs and stored at 80°C until assessed for PKC activity or the incorporation of tritiated phenylalanine into trichloroacetic acid-precipitable extracts (27) corrected for the specific radioactivity of the perfusion medium. Protein determinations were conducted using the bicinchoninic acid (BCA) assay (Sigma, St. Louis, MO).
Phenylalanine concentrations in the medium and liquid scintillation counting were used to establish perfusate specific radioactivity. The precise time that each muscle or portion of muscle was placed into liquid nitrogen was recorded to establish rates of protein synthesis on a per hour basis. Rates of protein synthesis are reported as nanomoles of phenylalanine incorporated per gram wet weight of muscle per hour and were calculated using the methods of Garlick et al. (11).
PKC enzyme activity.
Total PKC activity was determined by using a commercially available PKC enzyme assay system (Amersham Life Science, Piscataway, NJ) using 32P phosphorylation of a PKC-specific substrate as a marker of enzyme activity. Briefly, 50 mg of mixed gastrocnemius muscle were homogenized in 20 mM Tris (pH 7.4), 10 mM EDTA, 2 mM EGTA, 100 nM
-glycerophosphate, 1 mg/ml leupeptin, 0.1 mg/ml aprotinin, 0.1 mg/ml ovalbumin, and 50 mg phenylmethylsulfonyl fluoride. Samples were then centrifuged at 13,000 g (4°C) for 20 min, and the supernatant fraction was labeled as the cytosolic fraction. The pellet was resuspended with the aforementioned homogenization buffer with the addition of 0.5% Triton, incubated for 120 min on ice, and certrifuged as before. The supernatant fraction of this preparation was removed and labeled as the particulate (or membrane) fraction. As above, protein content was determined using a modified BCA assay. Subsequent steps for the determination in cytosolic and particulate fractions were similar to the methods as described by Cortright et al. (4).
To determine the activity of PKC in the presence of GF or TPA, we immunoblotted proteins from the particulate fraction that were run on an 8% SDS-PAGE gel that was subsequently transferred to PVDF by semidry blotting procedures. Although the regulation of PKC activity is considerably more complex, in general it is thought that, upon autophosphorylation of PKC, the enzyme is transolcated to the membrane (particulate fraction) for full activation (22). Thus we chose to focus specifically on PKC activity of specific isoforms when it was thought that the enzyme was fully active. A phosphospecific antibody simultaneously targeting various conventional and novel forms of activated PKC (PKC pan) was purchased from Cell Signaling Technology (Beverly, MA) and quantified by chemiluminescence using secondary antibodies conjugated with horseradish peroxidase. Blots were imaged using ChemiImager 5500 software (Alpha Innotech, San Leandro, CA).
Statistics. A two-way analysis of variance (treatment vs. phenotype) was used to compare means for either cytosolic or membrane PKC activity, with and without insulin, between lean and obese Zucker rats. Analysis of variance was also used to compare means of muscle protein synthesis rates with no treatment (none), with insulin, GF, or TPA, and with GF plus insulin or TPA plus insulin, respectively, in gastrocnemius muscle of lean and obese rats. Differences among means were considered significant when P < 0.05. When f ratios were significant, a Student-Newman-Keuls test was used to compare relevant means. All data are expressed as means ± SE.
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RESULTS |
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In white gastrocnemius (Fig. 3B), rates of protein synthesis were similar with the addition of GF or GF plus insulin compared with none and insulin treatments for lean animals (open bars). The addition of GF or GF plus insulin to white gastrocnemius (Fig. 3B) of obese animals (filled bars) demonstrated rates of synthesis that were lower (P < 0.05) than those with insulin alone and similar (P > 0.05) to those with none. Rates of protein synthesis in white gastrocnemius were similar (P > 0.05) for GF, GF plus insulin, and none between lean and obese rats. Together, the results above demonstrate that GF prevents the significant elevation of muscle protein synthesis by insulin in obese Zucker rats, suggesting that PKC activation is necessary for the insulin-mediated elevations of muscle protein synthesis in these animals.
Effects of TPA on muscle protein synthesis. TPA, a phorbol ester, is a potent activator of PKC and was used to determine the effect of PKC activation on muscle protein synthesis in lean and obese Zucker rats. In red gastrocnemius (Fig. 3A) of lean rats (open bars), rates of protein synthesis were lower (P < 0.05) with the addition of TPA alone compared with none, insulin, or TPA plus insulin. Rates of synthesis were similar among none, insulin, and TPA plus insulin in this phenotype. The addition of TPA to red gastrocnemius of obese animals (filled bars) demonstrated rates of synthesis that were similar to those of obese muscle with no treatment (none) and of lean muscle with TPA. The inclusion of insulin with TPA (TPA + insulin) in obese red gastrocnemius demonstrated rates of synthesis that were greater than those with none (P < 0.05) but less than those with insulin only (P < 0.05) Rates of synthesis in red gastrocnemius from obese animals with TPA plus insulin were similar to none, insulin, and TPA plus insulin of lean Zucker rats.
In white gastrocnemius from lean animals (Fig. 3B, open bars), rates of synthesis were greater (P < 0.05) for TPA plus insulin than rates of synthesis from none, insulin, or TPA only, but similar (P > 0.05) to rates of synthesis for insulin and TPA plus insulin of white gastrocnemius from obese animals (filled bars). Rates of synthesis for none, insulin, or TPA were similar in lean white gastrocnemius. In white gastrocnemius from obese animals, rates of protein synthesis were similar (P > 0.05) between TPA only and none, but lower (P < 0.05) than for insulin or TPA plus insulin. Rates of synthesis in white gastrocnemius muscles from obese animals were similar (P > 0.05) for insulin and TPA plus insulin. Together, these results suggest that insulin action on muscle protein synthesis may be a PKC-modulated event.
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DISCUSSION |
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The activation of specific isoforms of PKC by insulin has been implicated in the etiology of diabetes and/or obesity in humans (4, 7, 13, 14, 18). In muscle from individuals with diabetes, it appears that, following insulin complexation with its receptor, PKC is activated, which then impairs subsequent signal transduction, leading to diminished glucose transport. This insulin effect on PKC activity is not observed in muscle from lean humans who have a normal glucose response to insulin (15). The present study was not designed to identify the mechanism by which insulin stimulates PKC activity in muscle. However, the present study did demonstrate an insulin effect on PKC activity in both lean and obese animals. Although the overall activity was higher in the obese animals, the magnitude of change in PKC activity in response to insulin was similar between phenotypes, suggesting that total PKC activity may be a contributor to the observed phenotypic responses of insulin-mediated muscle protein synthesis. However, this conclusion may be premature, given that we cannot ascertain from the present study the specific isoforms that were activated and whether those isoforms were involved with the modulation of the translational process within a given phenotype.
The proposed mechanism for PKC's diabetogenic effect is through the phosphorylation of serine and threonine residues on important kinases in the signal transduction pathway, which then alters or diminishes the protein's function. One important kinase that has been shown to be diminished by elevated PKC activity is the tyrosine kinase of the insulin receptor (4, 13, 14, 21, 25). To support the hypothesis that PKC may alter insulin signaling, Cortright et al. (4) demonstrated improved glucose transport in response to insulin in diabetic muscle following incubation with GF, the same PKC inhibitor that was used in the present study. Furthermore, they demonstrated that glucose transport was dramatically diminished in normal muscle when it was incubated with the phorbol ester 12-deoxyphorbol 13-phenylacetate 20-acetate (a PKC agonist). The present study expands on that work, because we determined that inhibition of PKC activity via GF attenuates the enhanced insulin action on protein synthesis in obese Zucker rats. In contrast, the activation of PKC by the phorbol ester TPA facilitates rates of muscle protein synthesis in response to insulin even in normal muscle of lean rats. Together, these results suggest that glucoregulatory and muscle protein synthesis responses to insulin in muscle may be modulated by PKC.
An unexpected finding from our work is that rates of muscle protein synthesis were diminished in the presence of TPA (alone) in lean rats and that these rates were similar to what was observed, with or without TPA alone, in obese rats. At this time, we are unable to explain why activation of PKC by TPA would lead to a reduction of muscle protein synthesis without insulin in lean animals, but this reduction is consistent with rates of synthesis that are observed in obese rats that have a preexisting elevation of PKC activity. Further work is necessary to clarify this finding. Regardless, the present study is the first to demonstrate that PKC activation is necessary, although apparently not sufficient, for the insulin-mediated effects on muscle protein synthesis. An understanding of the cellular mechanisms by which PKC activity, perhaps involving specific isoforms, is linked to elevated rates of protein synthesis in the obese Zucker rats will undoubtedly provide important insights into the mechanisms by which selective metabolic responses are induced in skeletal muscle by the action of insulin.
The nonspecific effect of GF on the inhibition of PKC isoforms did not allow the present study to examine the impact of specific isoforms on altered rates of protein synthesis in response to insulin. Furthermore, we cannot be certain that the effect of GF on PKC was entirely responsible for the attenuation of muscle protein synthesis in the presence of insulin in obese rats. The mechanism by which GF attenuates PKC activity is through competitive inhibition with ATP-binding sites. Thus enzymes that have a similar ATP-binding motif may be similarly inhibited by GF. For example, a prominent enzyme that could be affected by the presence of GF in the present study was glycogen synthase kinase-3 (GSK-3), which acts as an inhibitor of both glucose disposal and mRNA translation (16). Inhibition of GSK-3 by GF should lead to an elevation of both glucose disposal and muscle protein synthesis. We did not measure GSK-3 kinase activity or glucose disposal in the present study. However, in our hands, the administration of GF led to a normalization of muscle protein synthesis. Thus we do not think that this important glucoregulatory enzyme was involved in the insulin-mediated change in muscle protein synthesis.
In contrast to GF, the use of TPA does offer some insight as to the specific isoforms that may be responsible for insulin action on muscle protein synthesis. Of the three general classes of PKC isoforms, only the conventional and novel families are sensitive to phorbol ester activation (4), suggesting that PKC isoforms in the atypical category are not involved in the protein synthetic process. The notion that conventional or novel subfamilies of PKC are necessary but not sufficient for activation of protein synthesis by insulin is consistent with our data, which used phosphospecific antibodies that target active isoforms of PKC in the conventional and novel subfamilies (Fig. 2). Activation of atypical isoforms in obese Zucker rats was not observed (data not presented). Subsequent studies from this laboratory are examining which of the PKC isoforms are activated in the presence of insulin in obese Zucker rats; additionally, efforts are being aimed at systematically determining the effects of specific PKC isoform activity on translation initiation.
In summary, the present investigation has demonstrated that insulin stimulates PKC activity in obese Zucker rats and that activation of PKC appears to be involved in the insulin-mediated elevation of muscle rates of protein synthesis in these animals. Systematic study using PKC inhibitors and activators demonstrated that the effect of insulin on muscle protein synthesis is either not observed or allowed to proceed, respectively, in obese Zucker rats. In lean rats in which there are no apparent alterations of muscle protein synthesis in response to acute insulin administration, facilitation of an insulin-mediated elevation of protein synthesis occurs with a simultaneous activation of PKC, suggesting that PKC activation (perhaps via conventional and/or novel isoforms) is necessary, although apparently not sufficient, to elicit this important anabolic effect by insulin. These results may provide insight into the hormonal control of muscle-anabolic responses, as well as having potentially important implications for our understanding of how these metabolic processes differ from normal during specific disease states such as diabetes.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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