Endocrinology Vol. 141, No. 8 2773-2778
Copyright © 2000 by The Endocrine Society
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 Activity1
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
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:
dcooper{at}com1.med.usf.edu
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Abstract
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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 although 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.
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Introduction
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PROTEIN 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 (
, ßI, ßII, 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 µ], which require only phospholipids (see reviews in
Refs. 1, 2, 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).
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
insulin-mediated glucose transport in skeletal muscle and glycogen
metabolism in liver (7, 8, 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
PKC-dependent 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 differentiation 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.
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Materials and Methods
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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 µg/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 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 ß-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 (ß-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 ß-actin on
its 5'- and 3'-ends. The mimic will specifically compete for primer
binding sites with the target PKC or ß-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 µg) 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 µg/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
µCi/µmol) 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.
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 ß-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.
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Results
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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 examined 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). Although 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 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
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 phosphorylation 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
insulin-stimulated 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.
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Discussion
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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 up-regulated 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 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 insulin-resistant 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, PKCßII 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
consistent 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.
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Acknowledgments
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We thank Dr. Harald Mischak for providing pMV-7 and pMV-7PKC
plasmids.
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Footnotes
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1 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.). 
Received October 30, 1999.
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J. Chen, G. Lu, and Q. J. Wang
Protein Kinase C-Independent Effects of Protein Kinase D3 in Glucose Transport in L6 Myotubes
Mol. Pharmacol.,
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A. G. Kayali, D. A. Austin, and N. J. G. Webster
Rottlerin Inhibits Insulin-Stimulated Glucose Transport in 3T3-L1 Adipocytes by Uncoupling Mitochondrial Oxidative Phosphorylation
Endocrinology,
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[Abstract]
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M. Caruso, M. A. Maitan, G. Bifulco, C. Miele, G. Vigliotta, F. Oriente, P. Formisano, and F. Beguinot
Activation and Mitochondrial Translocation of Protein Kinase Cdelta Are Necessary for Insulin Stimulation of Pyruvate Dehydrogenase Complex Activity in Muscle and Liver Cells
J. Biol. Chem.,
November 21, 2001;
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Top
Abstract
Introduction
