Inhibition of insulin signaling and glycogen synthesis by
phorbol dibutyrate in rat skeletal muscle
Yenshou
Lin1,3,
Samar I.
Itani1,
Theodore G.
Kurowski1,
David J.
Dean1,
Zhijun
Luo1,
Gordon C.
Yaney1,2, and
Neil B.
Ruderman1,3
1 Diabetes and Metabolism Unit and 2 Obesity Research
Center, Department of Medicine, and 3 Department of Physiology,
Boston University Medical Center, Boston, Massachusetts 02118
 |
ABSTRACT |
Numerous studies have shown a
correlation between changes in protein kinase C (PKC) distribution
and/or activity and insulin resistance in skeletal muscle. To
investigate which PKC isoforms might be involved and how they affect
insulin action and signaling, studies were carried out in rat soleus
muscle incubated with phorbol esters. Muscles preincubated for 1 h
with 1 µM phorbol 12,13-dibutyrate (PDBu) showed an impaired ability
of insulin to stimulate glucose incorporation into glycogen and a
translocation of PKC-
, -
I, -
, and -
, and probably -
II,
from the cytosol to membranes. Preincubation with 1 µM PDBu decreased
activation of the insulin receptor tyrosine kinase by insulin and to an
even greater extent the phosphorylation of Akt/protein kinase B and
glycogen synthase kinase-3. However, it failed to diminish the
activation of phosphatidylinositol 3'-kinase by insulin. Despite these
changes in signaling, the stimulation by insulin of glucose transport
(2-deoxyglucose uptake) and glucose incorporation into lipid and
oxidation to CO2 was unaffected. The results indicate that
preincubation of skeletal muscle with phorbol ester leads to a
translocation of multiple conventional and novel PKC isoforms and to an
impairment of several, but not all, events in the insulin-signaling
cascade. They also demonstrate that these changes are associated with
an inhibition of insulin-stimulated glycogen synthesis but that, at the
concentration of PDBu used here, glucose transport, its incorporation
into lipid, and its oxidation to CO2 are unaffected.
protein kinase C; phorbol dibutyrate; Akt/protein kinase B; glycogen synthase kinase-3; soleus muscle
 |
INTRODUCTION |
IT HAS BEEN SUGGESTED
THAT protein kinase C (PKC) activation plays a role in the
downregulation of insulin signaling and the development of insulin
resistance in skeletal muscle (see review in Refs. 8 and
28). Previous studies have demonstrated alterations in the activity
and/or distribution of PKC isoforms in skeletal muscle in a variety of
insulin-resistant states, including those induced by fat feeding
(29), fructose ingestion (10), and glucose
infusion (25). In addition, alterations in PKC
distribution have been observed in insulin-resistant muscle of
fa/fa and Goto-Kakizaki (GK) rats (2).
Studies employing phorbol esters (i.e., PKC activators) to investigate
the relationship between PKC and insulin action in skeletal muscle have
primarily focused on glucose transport. Thus it has been reported that,
in the absence of insulin, phorbol 12-myristate 13-acetate (PMA), also
referred to as 12-O-tetradecanoylphorbol 13-acetate (TPA),
stimulates glucose transport in rat epitrochlearis (16,
17) and extensor digitorum longus muscles (14) but has only a minimal effect on glucose transport in rat soleus muscle (33). Variable effects of phorbol esters on
insulin-stimulated glucose transport have been reported (14, 16,
33). Glycogen synthesis has been less studied, although PMA has
been shown to inhibit glycogen synthase activity (17) and
glycogen synthesis (33) in muscles incubated in the
absence of added insulin. The use of phorbol esters to evaluate the
role of PKC as a central factor in the pathogenesis of insulin
resistance in muscle (6, 7, 31) has been limited.
In the present study, we investigated the effects of preincubation with
the phorbol ester phorbol 12,13-dibutyrate (PDBu) on insulin-stimulated
glucose uptake and metabolism and on PKC distribution in rat
soleus muscle. In addition, the effects of PDBu on key steps in the
insulin-signaling cascade, including insulin receptor (IR),
phosphatidylinositol 3'-kinase (PI3K), Akt/protein kinase B (PKB), and
glycogen synthase kinase-3 (GSK3), were examined.
 |
MATERIALS AND METHODS |
Experimental animals and muscle incubation.
Male Sprague-Dawley rats weighing 50-65 g, purchased from Charles
River Breeding laboratories (Wilmington, MA), were maintained on a
12:12-h light-dark cycle in a temperature-controlled (19-21°C) animal room. All rats were fasted but had free access to water during
the 18-20 h before they were killed. On the experimental day, rats
were anesthetized with pentobarbital sodium (4-6 mg/100 g body wt
ip), and soleus muscles from both limbs were isolated. Muscles were
initially preincubated in Krebs-Henseleit solution (KHS) supplemented
with 6 mM glucose and were continuously gassed with 95%
O2-5% CO2 for 20 min. After this, they were
incubated for 1 h with vehicle (0.1% DMSO) or various
concentrations of calphostin C and then for 0.5-2 h with various
concentrations of PDBu (or another phorbol ester) as described in
RESULTS. Because the inhibitory activity of calphostin C is
light dependent, these incubations were performed in clear glass tubes
under the laboratory (cool-white fluorescent) light source.
Subsequently, muscles were washed in KHS containing 6 mM glucose for 2 min and then incubated in KHS containing 6 mM glucose with or without
10 mU/ml insulin for 30 min. At the end of these incubations, muscles
were blotted with a piece of gauze and then quick-frozen in liquid
nitrogen. They were stored at
80°C before subsequent measurements
were performed.
Measurement of glucose uptake and glucose disposition.
Deoxy-[3H]glucose uptake and [U-14C]glucose
incorporation into glycogen and lipid and oxidation to CO2
and lactate release were measured as described previously
(18).
Tissue extraction for PKC studies.
The method used for tissue extraction and PKC solubilization is
modified from Schmitz-Peiffer et al. (29). Muscles
(25-35 mg) were homogenized and extracted in 500 µl of an
ice-cold homogenizing buffer containing 250 mM sucrose, 50 mM Tris (pH
7.5), 2 mM EDTA, 0.4 mM EGTA, 10 mM dithiothreitol (DTT), 5 µg/ml
leupeptin, 4 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 2 mM diisopropyl fluorophosphate, 2.5 µg/ml pepstatin A, 10 mM sodium
fluoride (NaF), and 1 mM
-glycerophosphate. A portion of the
homogenate (200 µl) was centrifuged at 140,000 g for
1 h at 4°C, and the supernatant (cytosolic fraction) was removed
and stored in liquid nitrogen. The pellet was resuspended in 200 µl
of homogenizing buffer containing 0.2% (wt/wt) of the detergent
decanoyl-N-methyl-glucamide, known as MEGA-10. After sitting
for 1 h at 4°C, the extract was centrifuged again at 140,000 g for 1 h, and the supernatant that contained
solubilized protein (membrane fraction) was removed. All fractions were
stored at
80°C before assay.
PKC immunoblotting and activity assay.
Protein was determined with the detergent-tolerant protein assay kit
Bio-Rad DC (Hercules, CA). Equal amounts (20-30 µg protein) of
the cytosol and membrane fractions from each muscle were subjected to
10% SDS-PAGE by the method of Laemmli (24). Protein was
transferred to nitrocellulose membranes (Amersham, Piscataway, NJ) with
a semi-dry transfer apparatus following a method outlined by the manufacturer (Owl Scientific, Cambridge, MA). After transfer, the
nitrocellulose membranes were blocked with 5% nonfat dried milk in a
buffer of 1.5 M NaCl and 50 mM Tris, pH 7.4, containing 0.05% Tween-20
(TBS-T) overnight at 4°C. The membranes were probed with
isozyme-specific antibodies against PKC (polyclonal Ab:
,
I,
II,
,
,
,
,
,
, and
; Santa Cruz Biotechnology,
Santa Cruz, CA) for 2 h at room temperature. After 4× 5-min
washes in TBS-T buffer, membranes were incubated with horseradish
peroxidase-conjugated goat anti-rabbit antibody (Boehringer Mannheim,
Mannheim, Germany) for 45 min at room temperature. Visualization of
bands was obtained by chemiluminescence as outlined by the manufacturer
(Pierce Chemical, Rockford, IL). The optimal band intensity was
determined to be linear with increasing time of exposure and amount of
sample loaded (data not shown), as determined by the NIH image analysis
software (free distribution by the National Institutes of Health,
Bethesda, MD).
In some studies, muscles incubated to determine the effects of PDBu and
calphostin C on glycogen synthesis were homogenized as described above
and fractionated into cytosol and total particulate fractions for
measurement of PKC activity. Activity was measured using the Biotrak
System from Amersham. Given that we wished to measure only intrinsic
PKC activity, peptide substrate, but no PKC activators
(phosphatidylserine and phorbol ester), was added to the assay
samples. After phosphorylation, substrate peptides were spotted on
filter disks, washed, and counted as previously outlined
(19).
Akt/PKB immunoprecipitation and activity assay.
The processing of muscles used to assay Akt/PKB activity was as
described previously (23). In brief, immunoprecipitates of
Akt/PKB were obtained by incubating the muscle extract with an
Akt/PKB-specific antibody (provided by Dr. P. Tsichlis, Jefferson Medical College) that had been preincubated with Sepharose-A beads for
2 h. The beads were then washed, and activity was assessed by the
phosphorylation of the substrate peptide "crosstide" (Upstate Biotechnology, Lake Placid, NY).
IR, Akt, and GSK3
Western blot.
Muscles were weighed (average 25-35 mg) and homogenized in 1 ml of
ice-cold buffer containing 250 mM sucrose, 20 mM Tris (pH 7.4), 10 mM
NaF, 5 mM EDTA, 2 mM Na3VO4, 2 mM
NH4MbO4, 1 mM
-glycerophosphate, 1 mM DTT, 1 µM microcystin-LR, and 10 µl/ml Protease Inhibitor Cocktail P8340.
Igepal CA630 was added to each sample to achieve a final concentration
of 1% (vol/vol), and the sample was solubilized for 1.5 h at
4°C. The resultant lysate was centrifuged at 15,000 g for
5 min, and the supernatant was used in Western blot assay of IR, Akt,
GSK3, and various anti-phosphorylated residue antibodies.
PI3K assay.
Muscle lysates were incubated with an antibody against IRS-1 and the
immunocomplex precipitated by adding trisacryl protein A beads
(Pierce). PI3K was assayed according to Kelly et. al. (20).
Statistical analysis.
Data are presented as means ± SE. Treatment effects were
evaluated using a two-tailed Student's t-test. A
P value <0.05 was considered to be statistically significant.
 |
RESULTS |
Preincubation with phorbol ester impairs insulin-stimulated
glycogen synthesis.
To assess the effect of PKC activation on insulin action, rat soleus
muscles were incubated with phorbol esters. As shown in Fig.
1, preincubation with the PKC activators
PMA and PDBu, but not the inactive phorbol ester 4-
phorbol,
significantly inhibited the ability of insulin to stimulate glycogen
synthesis. Figure 2 shows that maximal
inhibition of the response to insulin by 1 µM PDBu was evident after
1 h of preincubation, whereas a lower concentration of 0.1 µM
required 2 h to demonstrate any effect. At these same time points,
the phorbol ester did not have an effect on this parameter in the
absence of insulin.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of preincubation with phorbol esters on
insulin-stimulated glycogen synthesis. Soleus muscles were preincubated
for 60 min in a control medium (0.1% DMSO) or a medium containing a
phorbol ester. Incorporation of [14C]glucose into
glycogen was then measured over 20 min in the presence of 10 mU/ml of
insulin and 6 mM glucose. PMA, phorbol 12-myristate 13-acetate; PDBu,
phorbol 12,13-dibutyrate. Values are means ± SE;
n = 4. *P < 0.05.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Time course and dose response of PDBu incubation of
insulin (Ins)-stimulated glycogen synthesis. Soleus muscles were
preincubated with different concentrations of PDBu for the indicated
time periods. The incorporation of [14C]glucose into
glycogen in the presence and absence of insulin was determined as
described in Fig. 1. Values are means ± SE; n = 4. *P < 0.05.
|
|
Incubation with PDBu does not inhibit glucose
uptake.
In contrast to glycogen synthesis, PDBu had no effect on
insulin-stimulated 2-deoxyglucose uptake, glucose incorporation into total lipid, or oxidation to CO2. PDBu treatment also did
not alter these parameters in the absence of insulin (Fig.
3).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of preincubation with PDBu on glucose disposition.
Soleus muscles were preincubated, and [14C]glucose
incorporation into glycogen and total lipid and its oxidation into
CO2 were determined subsequently, as described in legend to
Fig. 1. Uptake of 2-deoxy-D-[1,2-3H]glucose
was determined under similar conditions in a separate experiment (see
MATERIALS AND METHODS). Values are means ± SE; n = 6. *P < 0.01.
|
|
Impairment of insulin-stimulated glycogen synthesis by
PDBu is partially reversed by preincubation with
calphostin C.
Preincubation with calphostin C, a PKC inhibitor that competes with
phorbol ester at the diacylglycerol binding site (IC50 of 50 nM) (5, 21), had no effect on glycogen synthesis in either the presence or the absence of insulin (Fig.
4). On the other hand, it partially
(30~40%) reversed the inhibitory effect of 1 µM PDBu on
insulin-stimulated glycogen synthesis.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of preincubation with calphostin C on the
impairment of insulin-stimulated glycogen synthesis induced by PDBu.
Soleus muscles were preincubated for 20 min in 6 mM glucose and then
for 60 min in 6 mM glucose supplemented with either DMSO (final
concentration 0.1%) or calphostin C. They were then incubated for
1 h in a medium containing PDBu and 6 mM glucose, washed for 2 min
in a glucose-free medium, and then incubated for 30 min with a medium
containing 6 mM glucose and [14C]glucose. Values are
means ± SE; n = 4-5. *P < 0.05.
|
|
Distribution and activity of PKC between cytosolic
and membrane fractions.
As shown in Fig. 5, decreases in PKC-
,
-
I, -
II, -
, and -
, but not -
, were observed in the
cytosol after preincubation with PDBu in both the presence and the
absence of insulin. In the case of PKC-
, -
I, -
, and -
(but
not -
II or -
), this was associated with an increase in
membrane-associated isoforms. PKC-
also showed an additional band in
the membrane fraction after incubation with PDBu. Calphostin C competes
with phorbol ester at the C1 binding site of PKC. As is also shown in
Fig. 5, the distribution of PKC isoforms was not affected by calphostin C alone, and surprisingly, calphostin C did not reverse the
translocation of PKCs caused by PDBu.

View larger version (76K):
[in this window]
[in a new window]
|
Fig. 5.
Protein kinase C (PKC) distribution in soleus muscles
preincubated with PDBu and/or calphostin C. Muscles were incubated as
described in MATERIALS AND METHODS. After the incubation,
muscles were quickly frozen in liquid nitrogen and stored at 80°C.
At a later time, they were homogenized and fractionated. Equal amounts
of protein from each fraction were loaded and separated by 8%
SDS-PAGE. After transfer to polyvinylidene difluoride (PVDF), membranes
were immunoblotted with antibodies to specific PKC isoforms. Actin was
used as a control. Each blot shown is representative of 1 experiment
that was repeated 4 times.
|
|
PKC activity in the membrane and cytosol fractions from these muscles
was assayed without the addition of activators (see METHODS). As shown in Fig. 6,
kinase activity in the cytosol was not altered by incubation with
either PDBu or PDBu plus calphostin in muscles incubated with insulin
(Fig. 6A). In contrast, activity in the membrane fraction
was increased by incubation with PDBu, and this increase in activity
was totally inhibited by calphostin C (Fig. 6B).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.
PKC activity in cytosol (A) and membrane
(B) fractions of soleus muscle preincubated with PDBu and/or
calphostin C. The intrinsic activity of PKC in the 2 fractions was
assayed in the absence of activators (phosphatidylserine and phorbol
ester), as described in MATERIALS AND METHODS and legend to
Fig. 4. Values are means ± SE; n = 4. *P < 0.05.
|
|
Alterations in the insulin-signaling cascade induced by
PDBu.
To identify a defect in insulin signaling caused by PDBu, several
signaling molecules thought to be involved in insulin action were
examined. As shown in Fig. 7A,
the abundance of Akt/PKB was not altered by PDBu; however, the gel
shift of Akt/PKB caused by insulin, as well as its phosphorylation on
threonine 308 and serine 473 and the insulin-stimulated increase in its
activity, was substantially depressed by PDBu (Fig. 7,
A-D).

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of preincubation with PDBU on the abundance,
phosphorylation, and activity of Akt/PKB in soleus muscle. After
incubation, as described in legend to Fig. 4 (except no calphostin C),
preincubated muscles were quickly frozen and stored at 80°C. At a
later time, they were homogenized in Akt/PKB buffer, and the
15,000-g supernatant was collected (see MATERIALS AND
METHODS). A: equal amounts of protein were separated
on 8% SDS-polyacrylamide gels, and the gels were transferred to a PVDF
membrane. The membrane was blotted with Akt/PKB antibody. B
and C: after stripping, the membrane was probed with
anti-phospho-Thr308 Akt/PKB and
anti-phospho-Ser473 Akt/PKB antibody. D:
supernatant was immunoprecipitated (IP) with Akt/PKB antibody and
conjugated to beads, and Akt/PKB activity was determined using
crosstide as substrate (see MATERIALS AND METHODS). Each
point represents means ± SE of 5 muscles. *P < 0.05.
|
|
As shown in Fig. 8, preincubation with
PDBu did not alter the abundance of the insulin receptor, but it
significantly decreased its tyrosine phosphorylation (P < 0.05). The phosphorylation of GSK3-
and -
, downstream
molecules of Akt/PKB, was also depressed by PDBu. As with the insulin
receptor and Akt/PKB, no change in GSK3 abundance was observed (Fig.
9).
PI3K activity tended to be diminished; however, this was not
statistically significant (Fig. 10).

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of preincubation with PDBu on tyrosine
phosphorylation of the insulin receptor (IR). Muscles incubated as
described in Fig. 7 were quickly frozen and stored in 80°C. Muscles
were homogenized, and the 15,000-g supernatant was
collected. A: equal amounts of protein were separated on 6%
SDS-polyacrylamide gels, and the gel was transferred to a PVDF
membrane, which was blotted with anti-IR antibody. At left:
molecular mass markers. B: after membrane was stripped, it
was probed with anti-phosphotyrosine antibody (representative blot in
insert). Bars represent means ± SE of 5 muscles.
*P < 0.05.
|
|

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of preincubation with PDBu on glycogen synthase
kinase-3 (GSK3) phosphorylation. Muscles were incubated and processed
as described in legend to Fig. 7. They were homogenized in GSK3 buffer,
and the 15,000-g supernatant was collected. A and
B: immunoblots of GSK3- and GSK3- , respectively, after
separation on 10% SDS-polyacrylamide gels. At left,
molecular mass markers. C: immunoblots of
GSK3 phosphorylated on Ser9 and Ser21
(top). Each bar (middle and bottom)
represents mean ± SE of 4 muscles. *P < 0.05.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of preincubation with PDBu on
phosphatidylinositol 3-kinase (PI3K) activity. Muscles were incubated
and processed as described in legend to Fig. 7. PI3K activity was
assayed in an insulin receptor substrate-1 (IRS-1) immunoprecipitate
from the muscle lysate. Results are means ± SE; n = 5.
|
|
Effect of PDBu on glucose uptake is dose dependent.
Because there was no change in insulin-stimulated glucose uptake and
PI3K activity in muscles incubated with 1 µM PDBu, we next examined
the effect of incubating muscle with a higher concentration of PDBu. As
shown in Fig. 11, a small but
significant decrease in insulin-stimulated 2-deoxyglucose uptake (20%,
P < 0.01) was observed in soleus incubated with 3 µM, but not 1 µM, PDBu. In contrast, insulin-stimulated glycogen
synthesis was similarly depressed at the two PDBu concentrations.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 11.
Effect of different concentrations of PDBu on glucose
uptake and glucose disposition. Muscles were incubated and processed as
described in legend to Fig. 3. A: glucose uptake;
B: incorporation of [14C]glucose into
glycogen; C: conversion of [14C]glucose into
lactate. See MATERIALS AND METHODS for details. Values are
means ± SE; n = 5. *P < 0.01.
|
|
 |
DISCUSSION |
The key findings in this study are as follows. First, insulin
action was selectively inhibited in muscles preincubated with PDBu.
Insulin-stimulated glycogen synthesis was diminished, but 2-deoxyglucose uptake and glucose incorporation into total lipid and
its oxidation to CO2 were unaffected. Second, the
impairment of insulin-stimulated glycogen synthesis induced by PDBu was
associated with changes in the distribution of PKC-
, -
I, -
II,
-
, and -
and with decreases in the ability of insulin to
phosphorylate the insulin receptor, Akt/PKB, and GSK3 and to increase
Akt/PKB activity.
Previous studies of the effects of preincubation with phorbol esters on
insulin-stimulated glucose transport in rat skeletal muscle have
yielded variable results (Table 1). Thus
it was shown that PMA stimulates 3-methylglucose transport in rat
epitrochlearis muscle both in the absence of insulin (17)
and in the presence of insulin at a submaximal concentration
(16), whereas at a supraphysiological insulin
concentration it had no effect (16). Similar findings were
observed in the extensor digitorum longus muscle (14). In
contrast, TPA (33) (the same compound as PMA) and PDBu
(this study) had minimal if any effect on 2-deoxyglucose uptake in rat
soleus muscle in either the presence or the absence of insulin.
Incubation with another phorbol ester, 12-deoxyphorbol 13-phenylacetate
20-acetate, caused a three- to fourfold increase in 3-methylglucose
uptake both in the presence of insulin at submaximal and maximal
concentrations and in its absence (16). Collectively, these results suggest that the disparate findings in the literature may
be related to differences in the type of muscle and phorbol ester used
and in experimental design. Also, in general they suggest that phorbol
ester effects on glucose transport are most evident in muscles
incubated either in the absence of insulin or when insulin was present
at a physiological concentration. To our knowledge, the effects of
phorbol esters on insulin-stimulated glycogen synthesis in rat muscle
were not examined in any of these studies.
Our results indicate that preincubation with PDBu leads to a selective
inhibition of insulin-stimulated glycogen synthesis. Prior studies bear
only indirectly on this finding. Sowell et al. (33) also
observed an inhibition of glycogen synthesis, but not glucose
transport, in rat soleus muscle; however, these experiments were
carried out only in the absence of insulin. Likewise, Avignon et al.
(2) reported that RO 31-8220, a PKC inhibitor, increases glycogen synthesis, but not glucose uptake, in both the
presence and the absence of insulin in incubated rat soleus muscle. In
addition, they found that RO 31-8220 reversed the impairment of
insulin-stimulated glycogen synthesis in soleus muscle of the GK rat
but had no effect on glycogen synthesis in the absence of insulin
(2). In general, these observations suggest that PKC is a
negative regulator of the glycogen synthetic pathway, even when it does
not affect glucose transport.
The molecular mechanism by which PKCs inhibit insulin-stimulated
glycogen synthesis remains to be determined. One possibility is that
activation of PKC (e.g., by a phorbol ester) directly leads to
phosphorylation and inhibition of glycogen synthase, as has been
reported to occur in other tissues (1, 3). Against this
notion, however, we found that PDBu caused no inhibition of glycogen
synthesis in soleus muscle incubated in the absence of added insulin
(Fig. 3), although, as noted in the preceding paragraph, others have
observed such an effect. Another possibility is that PDBu acts by
inhibiting one or more steps in the insulin-signaling cascade. In
keeping with such a mechanism, preincubation with this phorbol ester
decreased the ability of insulin to phosphorylate Akt/PKB (90%) and
GSK3 (35%) and to increase Akt/PKB activity (inhibited by 50%). In
contrast, it failed to diminish the activation of PI3K by insulin and
diminished insulin receptor tyrosine phosphorylation by only 30%.
Thus, at the high concentration of insulin used, activation of the
insulin receptor tyrosine kinase, although somewhat diminished in
muscles incubated with PDBu, was sufficient to allow a normal
activation of PI3K. The normal activation of PI3K by insulin in solei
incubated with PDBu was surprising, because Akt/PKB phosphorylation and
activity were diminished, and PI3K is generally thought to be one of
its upstream activators. A similar dissociation between PI3K and Akt
activation by insulin in muscle has been observed in both EDL of the GK
rat (32) and EDL of control rats preincubated in a
hyperglycemic medium (not containing insulin) for 2-4 h
(23). A noteworthy finding in both of these studies was
that the ability of insulin to stimulate glucose incorporation into
glycogen was impaired; however, as in the present study, insulin-stimulated glucose transport was maintained. What accounts for
the dissociation of PI3K and Akt/PKB in these situations remains to be
determined, as does the reason inhibition of Akt/PKB is associated with
inhibition of glucose incorporation into glycogen but not glucose
transport into the muscle cell. This could occur if Akt/PKB is involved
in the regulation by insulin of glycogen synthesis but not glucose
transport. Early studies with cultured cells (15, 22, 34)
suggested that Akt/PKB plays a key role in mediating insulin-stimulated
glucose transport; however, this has recently been questioned
(13). Alternatively, insulin-stimulated glycogen synthesis
could simply be more sensitive to inhibition of the insulin receptor
tyrosine kinase and Akt than are glucose transport and lipid synthesis,
and/or it may simply be an earlier event in the evolution of insulin
resistance in the muscle cell. The fact that both glucose transport and
glycogen synthesis are inhibited concurrently in a wide variety of in
vivo models in which insulin signaling at the level of its receptor and
Akt is impaired (30, 35) is consistent with this notion.
On the basis of immunoblots with specific antibodies, preincubation
with PDBu caused a translocation of PKC-
, -
I, -
, -
, and
possibly -
II, from the cytosol to a membrane fraction (Fig. 5).
Whether one, some, or all of these isoforms mediated the observed changes in insulin signaling and action or whether the alterations in
PKC distribution are an epiphenomenon remains to be determined. Studies
in other insulin-resistant muscles do not answer this question. Changes
in the distribution of PKC-
and -
, but not other isoforms, have
been observed in skeletal muscle of intact rats in a number of
insulin-resistant states, including those induced by fat feeding
(29), fructose ingestion (10), and glucose
infusion (25). In contrast, alterations in PKC-
, -
, -
, and -
have been described in muscle of fa/fa and GK
rats (2). In rats made insulin resistant by raising plasma
free fatty acid levels during a euglycemic hyperglycemic clamp, altered PKC-
distribution has been reported (12). In denervated
muscle, in which, as in PDBu-treated muscle, the ability of insulin to stimulate glycogen synthesis but not glucose transport is impaired, PKC-
and, to a lesser extent, PKC-
, were altered
(26). For the most part, these reports suggest that
changes in the distribution of novel PKC isoforms correlate most
closely with the development of insulin resistance in skeletal muscle.
Incubation with calphostin C partially prevented the impairment of
insulin-stimulated glycogen synthesis caused by PDBu (Fig. 4).
Interestingly, calphostin C did so without reversing the effect of the
phorbol ester on PKC isoform intracellular translocation (Fig. 5), but
it totally inhibited the activation of membrane-associated PKC activity
caused by PDBu (Fig. 6). Furthermore, it did so without affecting PKC
activity in the cytosol. Given its lipophilic nature, these findings
suggest that calphostin C taken up by the muscle was predominantly
restricted to the membrane compartment during the period of incubation.
The apparent absence of calphostin C in the cytosol could account for
its failure to affect PKC translocation. It could also explain why it
only partially reversed the effect of PDBu on insulin-stimulated
glycogen synthesis.
That translocation/activation of one or more PKC isoforms can lead to
insulin resistance has been suggested by studies in tissues other than
skeletal muscle, including murine adipocytes (11),
fibroblasts (4), vascular cells, and heart
(27). In addition, multiple insulin-resistant states
associated with altered PKC distribution or activity have been observed
in muscles of both experimental animals (see review in Ref.
28) and humans (9). How these findings relate
to the observations of PKC in the present study remains to be determined.
In conclusion, our results show that insulin-stimulated glycogen
synthesis, but not glucose uptake, is impaired in soleus muscle
preincubated with 1 µM PDBu. This impairment of insulin action was
associated with alterations in the distribution of PKC-
, -
I,
-
II, -
, and -
, but not -
, and with decreased tyrosine phosphorylation of the insulin receptor. In addition, insulin-induced phosphorylation of Akt/PKB and GSK3 was depressed. The data strongly suggest that translocation and/or activation of one or more PKC isoforms causes defects in insulin signaling that lead to a selective impairment of insulin-stimulated glycogen synthesis.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grants DK-49147 and DK- 19514.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: N. B. Ruderman, Chief, Diabetes and Metabolism Research Unit, Boston Medical Center, 650 Albany St., EBRC Rm. 820, Boston, MA 02118 (E-mail:
nruderman{at}medicine.bu.edu).
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.
Received 17 August 2000; accepted in final form 15 February 2001.
 |
REFERENCES |
1.
Ahmad, Z,
Lee FT,
DePaoli-Roach A,
and
Roach PJ.
Phosphorylation of glycogen synthase by the Ca2+- and phospholipid-activated protein kinase (protein kinase C).
J Biol Chem
259:
8743-8747,
1984[Abstract/Free Full Text].
2.
Avignon, A,
Yamada K,
Zhou X,
Spencer B,
Cardona O,
Saba-Siddique S,
Galloway L,
Standaert ML,
and
Farese RV.
Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis.
Diabetes
45:
1396-404,
1996[Abstract].
3.
Blackmore, PF,
Strickland WG,
Bocckino SB,
and
Exton JH.
Mechanism of hepatic glycogen synthase inactivation induced by Ca2+-mobilizing hormones. Studies using phospholipase C and phorbol myristate acetate.
Biochem J
237:
235-242,
1986[ISI][Medline].
4.
Bossenmaier, B,
Mosthaf L,
Mischak H,
Ullrich A,
and
Haring HU.
Protein kinase C isoforms beta 1 and beta 2 inhibit the tyrosine kinase activity of the insulin receptor.
Diabetologia
40:
863-866,
1997[ISI][Medline].
5.
Bruns, RF,
Miller FD,
Merriman RL,
Howbert JJ,
Heath WF,
Kobayashi E,
Takahashi I,
Tamaoki T,
and
Nakano H.
Inhibition of protein kinase C by calphostin C is light-dependent.
Biochem Biophys Res Commun
176:
288-293,
1991[ISI][Medline].
6.
Caro, JF,
Jenquin M,
and
Long S.
Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity.
Mol Cell Biochem
109:
115-118,
1992[ISI][Medline].
7.
Chen, KS,
Heydrick S,
Kurowski T,
and
Ruderman NB.
Diacylglycerol-protein kinase C signalling in skeletal muscle: a possible link to insulin resistance.
Trans Assoc Am Physicians
104:
206-212,
1991[Medline].
8.
Considine, RV,
and
Caro JF.
Protein kinase C: mediator or inhibitor of insulin action?
J Cell Biochem
52:
8-13,
1993[ISI][Medline].
9.
Cortright, RN,
Azevedo JL, Jr,
Zhou Q,
Sinha M,
Pories WJ,
Itani SI,
and
Dohm GL.
Protein kinase C modulates insulin action in human skeletal muscle.
Am J Physiol Endocrinol Metab
278:
E553-E562,
2000[Abstract/Free Full Text].
10.
Donnelly, R,
Reed MJ,
Azhar S,
and
Reaven GM.
Expression of the major isoenzyme of protein kinase-C in skeletal muscle, nPKC theta, varies with muscle type and in response to fructose-induced insulin resistance.
Endocrinology
135:
2369-2374,
1994[Abstract].
11.
Frevert, EU,
and
Kahn BB.
Protein kinase C isoforms epsilon, eta, delta and zeta in murine adipocytes: expression, subcellular localization and tissue-specific regulation in insulin-resistant states.
Biochem J
316:
865-871,
1996[ISI][Medline].
12.
Griffin, ME,
Marcucci MJ,
Cline GW,
Bell K,
Barucci N,
Lee D,
Goodyear LJ,
Kraegen EW,
White MF,
and
Shulman GI.
Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade.
Diabetes
48:
1270-1274,
1999[Abstract].
13.
Guilherme, A,
and
Czech MP.
Stimulation of IRS-1-associated phosphatidylinositol 3-kinase and Akt/protein kinase B but not glucose transport by beta1-integrin signaling in rat adipocytes.
J Biol Chem
273:
33119-33122,
1998[Abstract/Free Full Text].
14.
Guma, A,
Camps M,
Palacin M,
Testar X,
and
Zorzano A.
Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level.
Biochem J
268:
633-639,
1990[ISI][Medline].
15.
Hajduch, E,
Alessi DR,
Hemmings BA,
and
Hundal HS.
Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells.
Diabetes
47:
1006-1013,
1998[Abstract].
16.
Hansen, PA,
Corbett JA,
and
Holloszy JO.
Phorbol esters stimulate muscle glucose transport by a mechanism distinct from the insulin and hypoxia pathways.
Am J Physiol Endocrinol Metab
273:
E28-E36,
1997[Abstract/Free Full Text].
17.
Henriksen, EJ,
Rodnick KJ,
and
Holloszy JO.
Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium.
J Biol Chem
264:
21536-21543,
1989[Abstract/Free Full Text].
18.
Heydrick, SJ,
Ruderman NB,
Kurowski TG,
Adams HB,
and
Chen KS.
Enhanced stimulation of diacylglycerol and lipid synthesis by insulin in denervated muscle. Altered protein kinase C activity and possible link to insulin resistance.
Diabetes
40:
1707-1711,
1991[Abstract].
19.
Itani, SI,
Zhou Q,
Pories WJ,
MacDonald KG,
and
Dohm GL.
Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity.
Diabetes
49:
1353-1358,
2000[Abstract].
20.
Kelly, KL,
Ruderman NB,
and
Chen KS.
Phosphatidylinositol-3-kinase in isolated rat adipocytes. Activation by insulin and subcellular distribution.
J Biol Chem
267:
3423-3428,
1992[Abstract/Free Full Text].
21.
Kobayashi, E,
Nakano H,
Morimoto M,
and
Tamaoki T.
Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C.
Biochem Biophys Res Commun
159:
548-553,
1989[ISI][Medline].
22.
Kohn, AD,
Summers SA,
Birnbaum MJ,
and
Roth RA.
Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation.
J Biol Chem
271:
31372-31378,
1996[Abstract/Free Full Text].
23.
Kurowski, TG,
Lin Y,
Luo Z,
Tsichlis PN,
Buse MG,
Heydrick SJ,
and
Ruderman NB.
Hyperglycemia inhibits insulin activation of Akt/protein kinase B but not phosphatidylinositol 3-kinase in rat skeletal muscle.
Diabetes
48:
658-663,
1999[Abstract].
24.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
25.
Laybutt, DR,
Schmitz-Peiffer C,
Saha AK,
Ruderman NB,
Biden TJ,
and
Kraegen EW.
Muscle lipid accumulation and protein kinase C activation in the insulin-resistant chronically glucose-infused rat.
Am J Physiol Endocrinol Metab
277:
E1070-E1076,
1999[Abstract/Free Full Text].
26.
Lin, Y,
Yaney GC,
and
Ruderman NB.
Altered protein kinase C distribution, but normal Akt activation in insulin resistant, denervated muscle (Abstract).
Diabetes
48:
A64,
1999[ISI].
27.
Naruse, K,
and
King GL.
Protein kinase C and myocardial biology and function.
Circ Res
86:
1104-1106,
2000[Free Full Text].
28.
Ruderman, NB,
Saha AK,
Vavvas D,
and
Witters LA.
Malonyl-CoA, fuel sensing, and insulin resistance.
Am J Physiol Endocrinol Metab
276:
E1-E18,
1999[Abstract/Free Full Text].
29.
Schmitz-Peiffer, C,
Browne CL,
Oakes ND,
Watkinson A,
Chisholm DJ,
Kraegen EW,
and
Biden TJ.
Alterations in the expression and cellular localization of protein kinase C isozymes epsilon and theta are associated with insulin resistance in skeletal muscle of the high-fat-fed rat.
Diabetes
46:
169-178,
1997[Abstract].
30.
Shepherd, PR,
and
Kahn BB.
Glucose transporters and insulin action
implications for insulin resistance and diabetes mellitus.
N Engl J Med
341:
248-257,
1999[Free Full Text].
31.
Shmueli, E,
Alberti KG,
and
Record CO.
Diacylglycerol/protein kinase C signalling: a mechanism for insulin resistance?
J Intern Med
234:
397-400,
1993[ISI][Medline].
32.
Song, XM,
Kawano Y,
Krook A,
Ryder JW,
Efendic S,
Roth RA,
Wallberg-Henriksson H,
and
Zierath JR.
Muscle fiber type-specific defects in insulin signal transduction to glucose transport in diabetic GK rats.
Diabetes
48:
664-670,
1999[Abstract].
33.
Sowell, MO,
Treutelaar MK,
Burant CF,
and
Buse MG.
Minimal effects of phorbol esters on glucose transport and insulin sensitivity of rat skeletal muscle.
Diabetes
37:
499-506,
1988[Abstract].
34.
Tanti, JF,
Grillo S,
Gremeaux T,
Coffer PJ,
Van Obberghen E,
and
Le Marchand-Brustel Y.
Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes.
Endocrinology
138:
2005-2010,
1997[Abstract/Free Full Text].
35.
Zierath, JR,
Krook A,
and
Wallberg-Henriksson H.
Insulin action in skeletal muscle from patients with NIDDM.
Mol Cell Biochem
182:
153-160,
1998[ISI][Medline].
Am J Physiol Endocrinol Metab 281(1):E8-E15
0193-1849/01 $5.00
Copyright © 2001 the American Physiological Society