0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society
Vol. 141, No. 8 Printed in U.S.A.
Protein Kinase C Expression Is Increased upon Differentiation of Human Skeletal Muscle Cells: Dysregulation in Type 2 Diabetic Patients and a Possible Role for Protein Kinase C in Insulin-Stimulated Glycogen Synthase Activity* CHARLES E. CHALFANT, THEODORE P. CIARALDI, JAMES E. WATSON, SVETLANA NIKOULINA, ROBERT R. HENRY, AND DENISE R. COOPER Departments of Biochemistry and Molecular Biology (C.E.C., D.R.C.) and Internal Medicine (D.R.C.), University of South Florida College of Medicine, and The James A. Haley Veterans Hospital (J.E.W., D.R.C.), Tampa, Florida 33612; and San Diego Veterans Hospital (S.N., R.R.H.), and Department of Medicine, University of California (T.P.C., S.N., R.R.H.), San Diego, La Jolla, California 92093 ABSTRACT Protein kinase C (PKC) is a key enzyme in regulating a variety of cellular functions, including growth and differentiation. PKC is the most abundant PKC isoform expressed in skeletal muscle; however, its role in differentiation and metabolism is not clear. We examined the effect of muscle cell differentiation on PKC expression in human skeletal muscle cells from normal and type 2 diabetic subjects. Low levels of PKC messenger RNA (mRNA) and protein were detected in human myoblasts from both types of subjects. Upon differentiation into myotubes, PKC mRNA and protein were increased 12-fold in myotubes from normal subjects. In human skeletal muscle cells obtained from type 2 diabetic subjects, increases in PKC mRNA and protein were not observed upon differentiation into myotubes al-
though expression of other markers of differentiation and fusion increased. Cells from type 2 diabetic subjects also exhibited decreased insulin-stimulated glycogen synthase activity. To determine whether the up-regulation of PKC was important for the metabolic actions of insulin, PKC was overexpressed in L6 rat skeletal muscle cells. Increased expression of PKC occurred with differentiation of skeletal muscle myoblasts to myotubes. Glycogen synthase activity was further increased in L6 myotubes stably transfected with the complementary DNA for PKC. The decreased expression of PKC found in cells from type 2 diabetic subjects may be linked to insulin resistance and decreased glycogen synthase activity. (Endocrinology 141: 2773– 2778, 2000)
P
PKC, a nPKC, has a unique tissue distribution and is the predominant isotype expressed in skeletal muscle, hematopoietic tissues, platelets, and testis (5). Little is known about the physiological consequences of PKC expression and its regulation in differentiating cells. Recently, PKC has been implicated in T cell activation and regulation of gene expression, suggesting a role for PKC in ligand-mediated signal transduction (6). Previous studies have shown that activation of some PKC isoforms can modulate both insulinmediated glucose transport in skeletal muscle and glycogen metabolism in liver (7–9). The role of PKC in regulating glycogen synthase (GS) activation is complex, as it can phosphorylate at least two distinct sites within GS, but can also phosphorylate GS kinase-3 to inhibit its activity (8). The roles of specific PKC isozymes to regulate muscle glycogen metabolism have not been reported. Specifically, PKC is rapidly activated and translocated in response to insulin in rat skeletal muscle (7). It has also been suggested that PKCdependent mechanisms might contribute to the development of insulin resistance (7). Insulin-stimulated GS activity in human skeletal muscle cells obtained from human type 2 diabetic subjects is reduced compared with that in healthy controls. These cells fuse in normal culture conditions and retain defects in basal and insulin-stimulated GS activity (10). Here we examine the effect of cell fusion and differenti-
ROTEIN KINASE C (PKC) mediates cellular responses elicited by hormones, neurotransmitters, and growth factors. Molecular cloning has revealed that PKC represents a multigene family, to date consisting of 11 different isozymes encoded by 10 distinct genes. Depending on the cofactor requirements, PKCs can be subdivided into 3 groups: classical or cPKCs (␣, I, II, and ␥), which require diacylglycerol (DAG), phospholipids, and Ca2⫹ for full activity; novel or nPKCs (␦, ⑀, , and ), which are phospholipid and DAG dependent, but Ca2⫹ independent; and atypical or aPKCs [, (), and ], which require only phospholipids (see reviews in Refs. 1–3). Activation of cPKCs and nPKCs involves their translocation from the cytoplasm to various cell membranes and cytoskeletal structures after binding of DAG, which is generated by agonist-induced hydrolysis of phosphatidylinositol-bis-phosphate, phosphatidylcholine, or a phosphatidylinositol-containing glycan or from the de novo synthesis of phosphatidic acid (4). Received October 30, 1999. Address all correspondence and requests for reprints to: Denise R. Cooper, Ph.D., J. A. Haley Veteran’s Hospital (VAR 151), 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail:
[email protected]. usf.edu. * The work was supported by the Medical Research Service of the Department of Veterans Affairs (to D.R.C., R.R.H., and T.P.C.) and the NSF (Grant 9318124, to D.R.C.).
2773
PKC EXPRESSION IN HUMAN SKELETAL MUSCLE CELLS
2774
ation on PKC expression in human skeletal muscle cells and the role of PKC on GS activity using rat L6 skeletal muscle cells that overexpress PKC. Materials and Methods Human subjects Nondiabetic and type 2 diabetic subjects were recruited for muscle biopsy. Muscle biopsies were performed on the lateral portion of the quadriceps femoris (vastus lateralis) under lidocaine anesthesia using a 5-mm diameter side-cutting needle (11). All subjects were in the fasting state at the time of biopsy. Glucose tolerance was determined from a 75-g oral glucose tolerance test, as previously described, within 24 h of the biopsy (10). Subject characteristics are summarized in Table 1. The diabetic group was significantly older than the nondiabetics. Although the diabetic subjects were also more obese, this difference was not statistically significant. Fasting hyperglycemia and an exaggerated glucose response to the oral glucose tolerance test confirmed glucose intolerance and insulin resistance in the diabetic subjects (Table 1). The experimental protocol was approved by the committee on human investigation of the University of California (San Diego, CA). Informed written consent was obtained from all subjects after explanation of the protocol.
Human muscle cell cultures The method for muscle cell isolation and clonal growth has been described in detail previously (12). At confluence, cells were fused for 4 days in ␣MEM containing 2% FBS, 1% fungibact, 100 U/ml penicillin, and 100 g/ml streptomycin. Fusion media were changed every 2 days. All studies were carried out on first passage cultures. The extent of differentiation was established by fluorescent microscopic observation of multinucleation and striation patterns of skeletal myosin fast (heavy chain), and induction of sarcomeric-specific ␣-actin protein was shown by Western blot as described below (11). For both groups, 85–90% of the cells were multinucleated.
Rat L6 skeletal muscle cell culture Rat L6 skeletal myoblasts (obtained from Dr. Amira Klip, The Hospital for Sick Children, Toronto, Canada) were grown in ␣MEM supplemented with 10% FBS to confluence. Cells were fused by changing medium to ␣MEM supplemented with 2% FBS for 4 days postconfluence, with medium changed daily. The extent of differentiation was established by observation of multinucleation of 85–90% of cells. Myotubes were incubated in ␣MEM with 0.1% BSA for 6 h and placed in PBS with 0.1% BSA just before treatment with insulin.
Competitive RT-PCR Total cellular RNA was obtained using a single step method (13). Generation of single stranded complementary DNA (cDNA) templates for RT-PCR was carried out on human skeletal muscle cell total RNA TABLE 1. Clinical characteristics of human subjects Nondiabetic (n ⫽ 4)
Age (yr) BMI Fasting glucose (mM) Fasting insulin (U/ml) 2 h OGTT Glucose (mM) Insulin (U/ml) Glycogen synthase FV basal FV ⫹ insulin
Type 2 diabetic (n ⫽ 4)
41.0 ⫾ 1.2 27.0 ⫾ 1.6 5.0 ⫾ 0.2 7⫾1
50.7 ⫾ 3.5a 31.7 ⫾ 2.0 9.9 ⫾ 1.9a 52 ⫾ 34
5.8 ⫾ 0.3 82 ⫾ 50
16.7 ⫾ 2.2a 79 ⫾ 38
0.124 ⫾ 0.027 0.163 ⫾ 0.030
0.083 ⫾ 0.020 0.120 ⫾ 0.040
BMI, Body mass index; OGTT, 75-g oral glucose tolerance test; FV, fractional velocity of glycogen synthase activity in cultured muscle cells. a P ⬍ 0.01 vs. nondiabetic subjects.
Endo • 2000 Vol 141 • No 8
using the SuperScript II preamplification kit. For quantitative RT-PCR, 5% of the cDNA was amplified in the presence of 10-2–10-5 attomoles of mimic DNA using primers specific for human PKC or -actin and Taq DNA polymerase (Fig. 1). The PKC-specific primers are a sense primer (5⬘-CCTTCTTCCCACAGCCCACAT-3⬘) and an antisense primer corresponding to the V5 region of PKC (5⬘-GCACTCAACAT-CATCGTCCAT-3⬘). After amplification in a Biometra Trioblock thermocycler (actin and PKC: 94 C, 1 min; 58 C, 1 min; and 72 C, 3 min, for 35 cycles), 20% of the PCR reactions were resolved on 1% agarose gels. Ethidium bromide-stained PCR products were quantified by scanning densitometry. The mimic (competitor) for PKC was constructed using neutral piece of DNA containing primer sequences for either PKC or -actin on its 5⬘- and 3⬘-ends. The mimic will specifically compete for primer binding sites with the target PKC or -actin cDNA. Extension rates of competitor mimics are within 5% of the target cDNA extension rate. A constant amount of mimic was amplified with each reaction after a series of dilutions to determine cellular concentration ranges. The competitive RT-PCR assay was linear over 5 log dilutions of the competitor.
Western analysis L6 and human skeletal muscle cell lysates (40 g) were subjected to 9% PAGE-SDS (10). Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with Tris-buffered saline and 0.1% Tween-20 containing 5% nonfat dried milk, washed, and incubated with a polyclonal antibody against PKC or, in the case of human skeletal muscle cell lysates, sarcomeric-specific ␣-actin. After incubation with antigoat or antimouse IgG-horseradish peroxidase, detection was performed using enhanced chemiluminescence.
Overexpression of PKC in L6 rat skeletal muscle cells L6 myoblasts were stably transfected with pMV7 (empty vector control) or pMV7 (with the cDNA insert for PKC) using calcium phosphate/DNA precipitate for 16 h. Cells were then washed twice with PBS and placed in medium for 48 h (14, 15). Stable transfectants were selected in the presence of 750 g/ml G418 and maintained in bulk cultures. Overexpression was evaluated by Western blot analysis, as described above, and in L6 cells by a 3- to 4- fold increase in total PKC activity toward histone III-S, assayed as previously described (16).
GS activity GS activity was measured in extracts of cells (grown on 100-mm plates) incubated in serum-free medium containing 5.5 mm d-glucose for 1 h followed by 30-min treatment with insulin. After rinsing three times with cold PBS, GS buffer (10) was added to cultures, and cells were scraped into Eppendorf tubes and sonicated (Vibra Cell, Sonics & Materials, Danbury, CT) at 50% output for 30 sec at 4 C. GS activity was assayed at a physiological concentration of substrate (0.3 mm UDP[14C]glucose) and 0.1 and 10 mm concentrations of the allosteric activator, glucose-6-phosphate (17). Enzyme activity is expressed as fractional velocity (FV) derived from a ratio of nanomoles per mg protein/ min (specific activity) at 0.1 mm glucose-6-phosphate divided by specific activity at 10 mm glucose-6-phosphate.
[1,2-3H]2-Deoxy-D-glucose (2-deoxyglucose) uptake L6 myoblasts were grown and differentiated as described above in 24-well plates. Before [3H]2-deoxyglucose uptake, cells were switched to ␣MEM with 0.1% BSA for 6 h. [3H]2-Deoxyglucose uptake was assayed as previously described (20). Cells were preincubated for 10 min with Dulbecco’s PBS (DPBS) with 1% BSA, insulin (1–100 nm), or the vehicle; DPBS plus BSA were added, and cells were incubated for an additional 20 min at 37 C. Uptake was measured by the addition of 10 nmol [3H]2-deoxyglucose (50 –150 Ci/mol) followed by incubation for 6 min at 37 C. The uptake was terminated by aspiration of medium, and cell monolayers were washed three times with cold DPBS. Cells were lysed with 1 ml 1% SDS, and radioactivity was determined by liquid scintillation counting. 2-Deoxyglucose uptake refers to transport of the analog across the plasma membrane operating in tandem with its phosphorylation by hexokinase.
PKC EXPRESSION IN HUMAN SKELETAL MUSCLE CELLS
2775
FIG. 1. Structure of PKC and regions amplified by RT-PCR. A schematic representation of PKC, deduced from cDNA sequence analysis, is shown. C1–C4 represent the conserved regions common to nPKCs. V1–V5 are variable regions specific for each PKC isoform. Arrows indicate where oligonucleotide primer sequences correspond in the PKC cDNA. The PKC RT-PCR product is 779 bp.
Materials ␣MEM, antibiotics, G418, oligonucleotide primers, and SuperScript II Pre-amplification kit were obtained from Life Technologies, Inc. (Gaithersburg, MD). Porcine insulin, BSA, FBS, and sarcomeric-specific ␣-actin antibody were purchased from Sigma (St. Louis, MO). Taq polymerase was obtained from Perkin-Elmer Corp. (Foster City, CA). The Mimic Construction Kit and -actin primers were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). PKC antibody and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody binds to an epitope corresponding to an amino acid sequence in the carboxyl-terminus of PKC (apparent Mr of 82 kDa) and does not cross-react with other PKC isoforms. The enhanced chemiluminescence (ECL) reagent was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). UDP-[14C]glucose was obtained from NEN Life Science Products (Boston, MA). All other biochemicals were purchased from the usual vendors.
Results PKC messenger RNA (mRNA) levels increased upon differentiation of human skeletal muscle cells
The expression of PKC mRNA was examined in response to differentiation of human skeletal muscle cells from nondiabetic subjects using competitive RT-PCR to quantify relative amounts of mRNA. The competitive RT-PCR assay amplified the PKC regions shown in Fig. 1. Upon differentiation of human skeletal myoblasts into multinucleated myotubes, PKC mRNA levels increased 12.1 ⫾ 2.1-fold (Fig. 2). PKC protein levels increased upon differentiation of human skeletal muscle cell
To examine whether the effects of differentiation on PKC mRNA levels were also reflected by increased protein levels in nondiabetic subjects, a polyclonal antibody specific for PKC was used. PKC immunoreactive protein levels were increased to a similar extent as PKC mRNA levels upon differentiation of human skeletal muscle cells (9.6 ⫾ 1.8-fold; Fig. 3). Thus, the increase in PKC mRNA expression was directly reflected at the protein level and was coordinately regulated with cell fusion and differentiation, as demonstrated by the increased expression of sarcomeric ␣-actin in the same samples (Fig. 3). PKC mRNA and protein levels were not significantly increased upon differentiation of human skeletal muscle cells from type 2 diabetic patients
Defects in PKC expression have been implicated in insulin action and insulin resistance, although data in human skeletal muscle cells were not provided (7, 18, 22). We ex-
FIG. 2. Competitive RT-PCR analysis of PKC mRNA levels in human skeletal muscle cells. Total RNA from human skeletal muscle cells was extracted, reverse transcribed, and subjected to competitive PCR using synthetic competitors for PKC primer binding sites. The PKC target length was 779 bp, and the mimic target length was 564 bp. Aliquots of the reactions were resolved by fractionation on agarose gels and visualized via ethidium bromide staining. Lanes 1– 4 are total RNA from normal human skeletal myoblasts (undifferentiated), and lanes 5– 8 are total RNA from normal human skeletal myotubes (fused and differentiated). Lanes 1 and 5 contain 10⫺2 attomoles of PKC mimic, lanes 2 and 6 contain 10⫺3 attomoles of PKC mimic, lanes 3 and 7 contain 10⫺4 attomoles of PKC mimic, and lanes 4 and 8 contain 10⫺5 attomoles of PKC mimic. PKC mRNA levels were normalized using a competitive RT-PCR assay for -actin. Shown is a representative analysis that was repeated on four other occasions with different human skeletal muscle cell mRNA.
amined PKC mRNA in skeletal muscle cells obtained from normal and type 2 diabetic patients. Unlike skeletal muscle cells from normal subjects, where 10- to 14-fold increases in PKC mRNA levels were noted, PKC mRNA did not significantly increase upon differentiation of myoblasts to multinucleated myotubes when derived from type 2 diabetic subjects (Fig. 4). PKC protein levels were also determined in myoblasts and myotubes from normal and diabetic subjects (Fig. 3). PKC protein levels were not significantly increased upon differentiation to myotubes in cells from type 2 diabetic subjects even though increased sarcomeric ␣-actin expression demonstrated that the cells had fully differentiated. Protein levels increased upon differentiation to the same extent in both groups. Thus, the lower PKC expression in myotubes from type 2 diabetics is a specific event and is not due to impaired differentiation. GS activity in human muscle cells
Cells from the subjects analyzed for PKC expression were also assayed for GS activity. In type 2 diabetic subjects, both basal and insulin-stimulated GS activities were lower, as reflected by fractional velocity (Table 1). Al-
2776
PKC EXPRESSION IN HUMAN SKELETAL MUSCLE CELLS
Endo • 2000 Vol 141 • No 8
that age had no significant impact on GS activity (not shown). Overexpression of PKC in L6 rat skeletal muscle cells increases insulin-stimulated GS activity
FIG. 3. Effects of differentiation on PKC and sarcomeric ␣-actin protein expression in human skeletal muscle cells. Whole cell lysates (20 g) from human skeletal muscle cells from nondiabetic subjects were prepared as described in Materials and Methods and subjected to SDS-PAGE fractionation. Western blots were developed as described in Materials and Methods. mb, Undifferentiated myoblasts; mt, fused and differentiated myotubes. Shown are representative results of eight separate cell preparations from eight different experiments. PKC protein levels increased 20-fold in this case, as determined by densitometry. The range was 10- to 20-fold for the subjects analyzed.
FIG. 4. Effect of differentiation on PKC mRNA levels in human skeletal muscle cells from normal individuals (n ⫽ 4) and type 2 diabetic patients (n ⫽ 4). Total RNA from human skeletal muscle cells was extracted, reverse transcribed, and subjected to competitive PCR using synthetic competitors for PKC primer binding sites. mb, Undifferentiated myoblasts; mt, fused and differentiated myotubes. Data are the mean ⫾ SE (attomoles of PKC mRNA).
though the values were not significant for this group due to subject variability, a larger sampling of type 2 diabetic patients that included these specific subjects did demonstrate a significant impairment in GS activity (10). Although the diabetic subjects studied here were older than the normal subjects, data from other subjects suggested
As skeletal muscle cells from type 2 diabetic patients demonstrated decreased levels of PKC and lower levels of insulin-stimulated GS activity (Table 1) (10, 12), it was likely that PKC activation could be related to GS activation. To test this hypothesis, we examined the effect of stable PKC overexpression in L6 myotubes, a cell line expressing lower levels of PKC than human skeletal muscle cells. PKC levels increased upon differentiation of L6 myoblasts to myotubes by 3.5-fold, and 10- to 12-fold higher levels of PKC were achieved in L6 myotubes stably transfected to overexpress PKC, compared with levels in control myotubes (Fig. 5). The 10- to 12-fold increase in PKC expression was similar to the overall increase noted during differentiation of myotubes from nondiabetic human subjects (Fig. 4). L6 myoblasts overexpressing PKC (L6) readily fused to form multinucleated myotubes. L6 myotubes exhibited elevated basal levels of GS activity compared with control L6 myotubes (Table 2). The fractional velocity (or ratio of GS activity at 0.1 vs. 10 mm glucose-6-phosphate) increased approximately 75% in response to insulin in L6 myotubes. L6 myotubes transfected with the empty vector as a control demonstrated only a 25% increase in GS fractional velocity in response to insulin (Table 2). The increase in activity was probably due to phosphor-
FIG. 5. Characterization of PKC protein expression in stably transfected L6 skeletal muscle cells by Western blot analysis. Cell lysates (10 g) from L6 skeletal muscle cells were prepared as described in Materials and Methods, and Western blot analysis was performed using antiserum for PKC, where a single immunoreactive band corresponding to PKC was observed. mb, Undifferentiated L6 myoblasts; mt, vector control myotubes; mt, L6 myotubes stably transfected with PKC. The fold increase in PKC expression with differentiation as determined by densitometric analysis is shown below. Similar results were obtained in three separate preparations of L6 myoblasts, myotubes, and cells overexpressing PKC.
PKC EXPRESSION IN HUMAN SKELETAL MUSCLE CELLS
ylation changes in GS, as an increase in specific activity at 0.1 mmol/liter glucose-6-phosphate and no significant differences in maximal activity at 10 mm glucose-6-phosphate were noted. Thus, PKC overexpression and its increased expression occurring with differentiation of human myotubes correlated with increases in GS activity in a both rat L6 and human skeletal muscle myotubes from nondiabetic subjects. Effects of PKC overexpression in L6 myotubes on insulinstimulated 2-deoxyglucose uptake
The overexpression of PKC could also act to increase the uptake of glucose into L6 cells and could be reflected in increases in GS activity. To determine whether this was the case, the effects of insulin on 2-deoxyglucose uptake were determined (Table 3). There was no significant difference in the uptake of [3H]2-deoxyglucose between L6 and L6 myotubes. The overexpression of PKC appeared to be associated with effects on basal and insulin-stimulated GS activation rather than increased glucose flux into cells. Discussion
PKC is the most abundant PKC isoform in skeletal muscle and has been implicated to play a role in insulin action and insulin resistance (5, 7). We found PKC expression upregulated during differentiation in human skeletal muscle cells, with increased PKC mRNA and immunoreactive protein noted after fusion of myoblasts to multinucleated myotubes. We also found that skeletal muscle cells obtained from type 2 diabetic subjects expressed lower levels of PKC even upon differentiation. No differences in myotube fusion and differentiation were observed in cells from type 2 diabetic subjects compared with those in normal human skeletal muscle cells, as reflected by number of multinucleated cells and the expression of sarcomeric-specific ␣-actin. Our study differs from a report in embryonic myotubes in which PKC expression decreased upon differentiation of fetal myoblasts into myotubes (19). The difference in results may be explained by the use of fetal cells, whereas myoblasts from adult subjects were studied here. Cells from adult subjects may express a different profile of PKC isozymes. These TABLE 2. Basal and insulin-stimulated glycogen synthase activity in L6 and L6 myotubes Rat skeletal muscle cell type L6 (vector control)
Basal state (nmol/mg protein䡠min) 0.1 mmol/liter G-6-P 2.03 ⫾ 0.23 10 mmol/liter G-6-P 42.70 ⫾ 4.85 FV basal 0.048 ⫾ 0.022 Insulin-stimulated state (nmol/mg protein䡠min) 0.1 mmol/liter G-6-P 3.04 ⫾ 0.44 10.0 mmol/liter G-6-P 47.11 ⫾ 4.90 FV ⫹ insulin 0.064 ⫾ 0.003
L6 (overexpression)
4.78 ⫾ 0.08a 61.38 ⫾ 7.63 0.080 ⫾ 0.008a 5.94 ⫾ 0.21a 45.51 ⫾ 7.70 0.140 ⫾ 0.028a
Values are the mean ⫾ SEM. Basal state, No insulin added to serum-free media; stimulated state, 20 nM insulin added to serumfree medium for 30 min. Glycogen synthase activity at 0.1 and 10 mM G-6-P is expressed as fractional velocity (FV). FV was calculated as the ratio of activity measured at 0.1 and 10 mM G-6-P. a P ⬍ 0.05 vs. corresponding activity in vector control cells (L6) by Student’s t test (n ⫽ 3).
2777
TABLE 3. Insulin effects on 2-deoxyglucose transport in L6 and L6 myotubes 2-Deoxyglucose uptake (pmol/mg protein䡠min)
Control Insulin (20 nM) Insulin (200 nM)
L6 (vector control)
L6 (overexpressers)
323 ⫾ 30 662 ⫾ 7 784 ⫾ 31
279 ⫾ 50 558 ⫾ 55 832 ⫾ 94
L6 vector control and L6 myoblasts were grown in 24-well plates until confluent and then differentiated into myotubes as described. Data are expressed as the mean ⫾ SE (picomoles per mg protein) for triplicate determinations. The experiment was repeated on three occasions with similar results.
studies used different methods to visualize PKC expression. Moreover, data for skeletal muscle tissue taken from embryonic and fetal limbs do not correspond with the cell culture data showing high expression of PKC in these differentiated tissues, but decreased expression in differentiated myotubes (19). In our studies PKC did not appear to function in the differentiation process of human skeletal muscle cells, as skeletal muscle cells from diabetic subjects fused normally, and the overexpression of PKC in rat myoblasts did not interfere with cell proliferation before fusion to differentiated myotubes. Thus, an increase in PKC may act as a final marker for muscle cell differentiation but not necessarily be required for the process. Reaven and co-workers reported that increases in PKC immunoreactive protein in the cell membranes of white muscle (but not red muscle) occurred in fructose-induced insulinresistant rats (18). They theorized that PKC activation in fructose-fed rats was a possible cause of insulin resistance in skeletal muscle (18). They suggested that PKC acted as an inhibitor of insulin action, possibly by phosphorylation of the insulin receptor. However, activities of metabolic enzymes such as GS were not reported. Our data suggested that increased PKC expression was a normal event in skeletal myoblast differentiation and that myotubes from type 2 diabetic patients demonstrated impaired PKC expression. Skeletal myotubes from type 2 diabetic patients also demonstrated impaired glucose transport and GS activation in response to insulin, which would argue against increased PKC activation being involved in insulin resistance (18). Other PKC isoforms, PKCII and , have been implicated in insulin-stimulated glucose transport (20, 21). As decreased expression of PKC might also be associated with impaired insulin-stimulated GS activity and glucose uptake, we examined the effects of PKC overexpression on metabolic effects of insulin. The overexpression of PKC in L6 rat skeletal muscle cells resulted in increased basal GS activity and increased effects of insulin on GS activation, but it had no effect on glucose uptake. Thus, one could hypothesize a potential role for PKC in the regulation of GS activity in both the presence and absence of insulin. The results of using PKC inhibitors to evaluate GS activity are varied, with reports of stimulation as well as inhibition of GS activity (22, 24). As no specific PKC inhibitors are available, we relied on overexpression of the isoform to augment its function. Whether the effect of PKC activity is directly on GS, on GS kinase-3, or on other regulatory enzymes is unknown. The data are con-
2778
PKC EXPRESSION IN HUMAN SKELETAL MUSCLE CELLS
sistent with an effect on GS kinase-3 that would result in the dephosphorylation and subsequent activation of GS. Further physiological evidence for the hypothesis that PKC regulates GS activity comes from the observation that PKC expression is low in L6 myotubes compared with that in human skeletal muscle cells. L6 myotubes demonstrated lower levels of GS activity in the presence and absence of insulin compared with that in human myotubes. When L6 cells were stably transfected with the cDNA, PKC levels approximating those noted in human myotubes were achieved, and increases in basal and insulin-stimulated GS fractional velocity were noted. The increase in GS activation was probably not due to increased glucose uptake, because PKC had no effect on insulin-stimulated 2-deoxyglucose uptake. Our studies support other reports that insulin activated and translocated PKC in skeletal muscle (7, 23). Another report suggested that PKC may subsequently inhibit insulin receptor tyrosine kinase activity in the presence of insulin receptor substrate-1, and PKC may have phosphorylated insulin receptor substrate-1 (25). In these studies PKC may have altered insulin signaling. Other studies suggested a possible role for the isozyme in insulin metabolic effects, and they also demonstrated lower PKC levels in skeletal muscle of insulin-resistant type 2 diabetic (GK) rats (23). In type 2 diabetes, impaired insulin action is associated with defects in glycogen synthesis and GS activity. The human protein kinase C gene was recently sequenced and shown to span 62 kb of chromosome 10p15. It is composed of 15 coding exons and 14 introns. In the future, directed searches for potential genetic polymorphisms and/or abnormalities may reveal more about the defect in its expression in type 2 diabetic subjects (25). Our results suggest that GS activity may be regulated via a PKC pathway in human and rat skeletal muscle cells. This study further associates lower GS activity with decreased expression of PKC in skeletal muscle cells from type 2 diabetic subjects and associates increased GS activity with PKC expression in skeletal muscle. Acknowledgments We thank Dr. Harald Mischak for providing pMV-7 and pMV-7PKC plasmids.
6. 7.
8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18.
19.
20.
21. 22. 23.
References 1. Stabel S, Parker PJ 1991 Protein kinase C. Pharmacol Ther 51:71–95 2. Nishizuka Y 1992 Intracellular signaling of hydrolysis of phospholipids and activation of protein kinase C. Science 258:607– 614 3. Johannes F-J, Prestle J, Eis S, Oberhagemann P, Pfizenmaier K 1994 PKC is a novel, atypical member of the protein kinase C family. J Biol Chem 269:6140 – 6148 4. Toker A, Meyer M, Reddy KK, Falck JR, Aneja R, Aneja S, Parra A, Burns DJ, Ballas LM, Cantley LC 1994 Activation of protein kinase C family members by novel polyphosphoinositides PtdIns-3, 4-P2 and PtdIns-3,4,5-P3. J Biol Chem 269:32358 –32367 5. Osada S-I, Mizuno K, Saido TC, Suzuki K, Kuroki T, Ohno S 1992 A new
24.
25.
26.
Endo • 2000 Vol 141 • No 8
member of the protein kinase C family, nPKC, predominantly expressed in skeletal muscle. Mol Cell Biol 12:3930 –3938 Monks CR, Kupfer H, Tamir I, Barlow A, and Kupfer A 1997 Selective modulation of protein kinase C during T-cell activation. Nature 385:83– 86 Yamada K, Avignon A, Standaert ML, Cooper DR, Spencer B, Farese RV 1995 Effects of insulin on the translocation of protein kinase C and other protein kinase C isoforms in rat skeletal muscles: comparisons with effects in adipocytes. Biochem J 308:177–180 Pugazhenthi S, Khandelwal RL 1995 Regulation of glycogen synthase activation in isolated hepatocytes. Mol Cell Biochem 149/150:95–101 Nakabayashi H, Chan K-FJ, Huang K-P 1987 Role of protein kinase C in the regulation of rat liver glycogen synthase. Arch Biochem Biophys 252:81–90 Henry RR, Ciaraldi TP, Abrams-Carter L, Mudaliar S, Park KS, Nikoulina SE 1996 Glycogen synthase activity is reduced in cultured skeletal muscle cells of non-insulin-dependent diabetes mellitus subjects. J Clin Invest 98:1231–1236 Henry RR, Abrams L, Nikoulina S, Ciaraldi TP 1995 Insulin action and glucose metabolism in non-diabetic control and NIDDM subjects: comparison using human skeletal muscle cell cultures. Diabetes 44:936 –946 Ciaraldi TP, Abrams L, Nikoulina S, Mudaliar S, Henry RR 1996 Glucose transport in cultured human skeletal muscle cells: regulation by insulin and glucose in nondiabetic and non-insulin-dependent diabetes mellitus subjects. J Clin Invest 96:2820 –2827 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-choloform extraction. Anal Biochem 162:156 –159 Sambrook J, Fitsch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Goodnight J, Mischak H, Kolch W, Mushinski JF 1995 Immunocytochemical localiztion of eight protein kinase C isozymes overexpressed in NIH 3T3 fibroblasts. J Biol Chem 270:9991–10001 Cooper DR, Khalakdina A, Watson JE 1993 Chronic effects of glucose on insulin signaling in vascular smooth muscle cells. Arc Biochem Biophys 302:490 – 498 Thornburn AW, Gumbiner B, Bulacan F, Brechtel G, Henry RR 1991 Multiple defects of muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus. J Clin Invest 87:489 – 495 Donnelly R, Reed MJ, Azhar S, Reaven GM 1994 Expression of the major isozyme of protein kinase-C in skeletal muscle, nPKC, varies with muscle type and in response to fructose-induced insulin resistance. Endocrinology 135:2369 –2374 Zappelli F, Willems D, Osada S, Ohno S, Wetsel WC, Molinaro M, Cossu G, Bouche M 1996 The inhibition of differentiation caused by TGFbeta in fetal myoblasts is dependent upon selective expression of PKC: a possible molecular basis for myoblast diversification during limb histogenesis. Dev Biol 180:156 –164 Chalfant CE, Ohno S, Konno Y, Fisher AA, Watson JE, Cooper DR 1996 A carboxy-terminal deletion mutant of PKCII inhibits insulin-stimulated 2deoxyglucose uptake in L6 rat skeletal muscle cells. Mol Endocrinol 10:1273– 1281 Bandyopadhyay G, Standaert ML, Zao L, Yu B, Avignon A, Galloway L, Karnam P, Moscat J, Farese RV 1997 Activation of Protein Kinase C (␣,, and ) by insulin in 3T3/L1 cells. J Biol Chem 272:2551–2558 Standaert ML, Bandyopadhyay G, Antwi EK, Farese RV 1999 RP 31– 8220 activates c-jun N-terminal kinase and glycogen synthase in rat adipocytes and L6 myotubes. Comparison to actions of insulin. Endocrinology 140:2145–2151 Avignon A, Yamada K, Zhou X, Spencer B, Cardona O, Saba-Siddique S, Galloway L, Standaert ML, Farese RV 1996 Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki, obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis. Diabetes 45:1396 –1404 Gali RR, Pugazhenthi S, Khandelwal RL 1993 Reciprocal effects of the protin kinase C inhibitors staurosporine and H-7 on the regulation of glycogen synthase and phosphorylase in the primary culture of hepatocytes. Metabolism 42:1475–1480 Kellerer M, Mushack J, Seffer E, Mischak H, Ullrich A, Haring HU 1998 Protein kinase C isoforms ␣, ␦ and require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells). Diabetologia 41:833– 848 Kofler K, Kochl S, Parson W, Utermann EM, Baier G 1998 Molecular characterization of the human protein kinase C theta gene locus (PRKCQ). Mol Gen Genet 259:398 – 403
