1 Diabetes and Metabolism Unit and 2 Department of Physiology, Boston University Medical Center, Boston, Massachusettes 02118; and 3 Department of Medicine, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Denervation has been shown to impair the
ability of insulin to stimulate glycogen synthesis and, to a lesser
extent, glucose transport in rat skeletal muscle. Insulin binding to
its receptor, activation of the receptor tyrosine kinase and
phosphatidylinositol 3'-kinase do not appear to be involved. On the
other hand, it has been shown that denervation causes an increase in
the total diacylglycerol (DAG) content and membrane-associated protein
kinase C (PKC) activity. In this study, we further characterize these changes in PKC and assess other possible signaling abnormalities that
might be related to the decrease of glycogen synthesis. The results
reveal that PKC- and -
, but not -
or -
, are increased in
the membrane fraction 24 h after denervation and that the timing of these changes parallels the impaired ability of insulin to stimulate
glycogen synthesis. At 24 h, these changes were associated with a
65% decrease in glycogen synthase (GS) activity ratio and decreased
electrophoretic mobility, indicative of phosphorylation in GS in
muscles incubated in the absence of insulin. Incubation of the
denervated soleus with insulin for 30 min minimally increased glucose
incorporation into glycogen; however, it increased GS activity
threefold, to a value still less than that of control muscle, and it
eliminated the gel shift. In addition, insulin increased the apparent
abundance of GS kinase (GSK)-3 and protein phosphatase (PP)1
in the
supernatant fraction of muscle homogenate to control values, and it
caused the same increases in GSK-3 and Akt/protein kinase B (PKB)
phosphorylation and Akt/PKB activity that it did in nondenervated
muscle. No alterations in hexokinase I or II activity were observed
after denervation; however, in agreement with a previous report,
glucose 6-phosphate levels were diminished in 24-h-denervated soleus,
and they did not increase after insulin stimulation. These results
indicate that alterations in the distribution of PKC-
and -
accompany the impairment of glycogen synthesis in the 24-h-denervated
soleus. They also indicate that the basal rate of glycogen synthesis
and its stimulation by insulin in these muscles are diminished despite
a normal activation of Akt/PKB and phosphorylation of GSK-3. The
significance of the observed alterations to GSK-3 and PP1
distribution remain to be determined.
denervation; soleus muscle; novel protein kinase C; Akt/protein kinase B; glycogen synthase kinase 3; protein phosphatase-1; glycogen synthase
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INTRODUCTION |
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DENERVATION OF SKELETAL MUSCLE for as few as 6-24 h has been shown to impair the ability of insulin to stimulate glycogen synthesis and, to a lesser extent, glucose transport (8, 10, 20, 35). Although defects in glycogen synthase (GS) activation have been reported (8), the mechanism by which insulin action is impaired is not clear. Thus binding of insulin to its receptor and activation of the receptor tyrosine-kinase (9) and phosphatidylinositol 3'-kinase (PI3K) (11) do not appear to be affected, suggesting that events distal to PI3K activation are involved.
It has been shown that denervation causes an increase in the total
diacylglycerol (DAG) content and membrane-associated protein kinase C
(PKC) activity (20). Alterations in the activity and/or distribution of novel (PKC- and -
, nPKC) and occasionally
conventional (c)PKC isoforms have been demonstrated in skeletal muscle
in a variety of insulin-resistant states, including those induced by fat feeding (33), fructose ingestion (15),
and glucose infusion (24). In addition, alterations in PKC
distribution have been observed in insulin-resistant muscle of
fa/fa and Goto Kakizaki rats (1). Which PKC
isoform(s) is altered in the denervated muscle is not known.
It has also been reported that GS activity is impaired in denervated rat muscle in both basal and insulin-stimulated states (8). GS, the rate-limiting enzyme in glycogen synthesis, is regulated by covalent modification and allosterical regulation (23, 34). Covalent modification occurs by phosphorylation, as GS can be phosphorylated on at least six residues by a variety of kinases that cumulatively inhibit its activity. One of these kinases, GS kinase (GSK)-3, is phosphorylated and inactivated by insulin. It has been proposed that this results in the disinhibition of GS by diminishing its phosphorylation (14, 17, 41). It has also been suggested that GS dephosphorylation and activation can result from the insulin-stimulated activation of glycogen-targeted protein phosphatase-1 (PP1) (29).
In the present study, we have attempted to address the mechanism of GS
inhibition after sciatic nerve section by asking the following
questions. 1) What are the PKC isoforms responsible for the
observed alteration in membrane-associated PKC activity? 2)
Is the timing of changes in PKC distribution consistent with the
development of insulin resistance and documented elevation of
malonyl-CoA? 3) Do alterations in distal events in the
insulin-signaling cascade or in the distribution of GS, GSK-3, and
PP1 occur after denervation? Finally, when initial data confirmed an
earlier report that the concentration of glucose 6-phosphate was
diminished in the denervated soleus, we asked whether this might be
related to a decrease in hexokinase activity.
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MATERIALS AND METHODS |
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Experimental animals and denervation. 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. Sciatic nerve section was performed under anesthesia with pentobarbital sodium (4-6 mg/100 g body wt ip) at various times before an experiment, as detailed in RESULTS. A sham operation was performed on the contralateral limb. All rats were fasted but had free access to water during the 18-20 h before they were killed. In all instances, rats were killed at 12:00 noon.
Incubation protocol.
On the experimental day, rats were reanesthetized, and soleus muscles
from both limbs were isolated. Muscles were initially incubated for 30 min at 37°C in Krebs-Henseleit solution (KHS) continuously gassed
with 95% O2-5% CO2 and supplemented with 6 mM
glucose and then for 2 min in glucose-free KHS. After that, they were
transferred to KHS containing 6 mM glucose (with
[U-14C]glucose when the measurements of glucose
disposition were performed) and 10 mU/ml insulin as indicated. At the
end of a 30-min incubation in this medium, muscles were quickly blotted
with a piece of gauze and quick-frozen in liquid nitrogen. The muscles
were stored at 80°C until the measurement of glucose disposition
and other assays were performed.
Measurement of glucose disposition. [U-14C]glucose incorporation in glycogen and lipid and its oxidation to CO2 were measured as described previously (20).
PKC immunoblotting.
The processing of muscles used for tissue extraction and PKC
solubilization was as described previously (26). Briefly,
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 (PMSF), 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) removed and stored in
liquid nitrogen. The pellet was resuspended in 200 µl of homogenizing
buffer containing 0.2% (wt/vol) of the detergent decanoyl-N-methylglucamide (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.
Akt/PKB immunoprecipitation and activity assay. The processing of muscles used to assay Akt/PKB activity was as described previously (21). 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 1 h. The beads were then washed and activity assessed by the phosphorylation of the substrate peptide "crosstide" (Upstate Biotechnology, Lake Placid, NY). In the Western blot, an equal amount of protein was loaded, separated by SDS-PAGE, and blotted with anti-phospho-Ser473 antibody (New England Biolabs, Beverly, MA).
Assay of GS activity and Western blot. Muscles were homogenized in 50 mM HEPES (pH 7.8), 10 mM EDTA, and 100 mM NaF plus protease inhibitors and then subjected to centrifugation for 10 min at 2,500 g. GS activity in the supernatant was measured as described previously (6, 25). Briefly, 25-50 µl of supernatant were assayed in a final volume of 100 µl of GS buffer containing 5 mM UDP-glucose and 1 µCi/ml UDP-[U-14C]glucose, in both the presence and absence of 10 mM glucose 6-phosphate (G-6-P). Samples were incubated at 37°C and then placed on ice for 15 min. Ninety microliters of the reaction mixture were spotted on GF/A filters, dried for 3 s, and then placed in 70% ethanol on ice. Filters were washed for 20 min at 4°C and then washed two more times in 70% ethanol at room temperature. Filters were air-dried, and UDP-[U-14C]glucose incorporation into glycogen was measured by liquid scintillation counting. In the Western blot, an equal amount of protein was loaded, separated by SDS-PAGE, and blotted with anti-GS antibody (a generous gift from Dr. J. Lawrence Jr., University of Virginia). GS blots were visualized using HRP-conjugated secondary antibody followed by chemiluminescence as outlined by the manufacturer (Amersham).
GSK-3 Western blot.
Muscles were weighed (average 25-35 mg) and homogenized in 1 ml of
ice-cold buffer containing (in mM) 50 HEPES (pH 7.4), 1 EGTA, 1 EDTA,
10 -glycerolphosphate, 5 sodium pyrophosphate, 100 KCl + 1 DTT,
and 0.5 Na3VO4 and 10 µl/ml protease
inhibitor cocktail P8340 added just before use. Triton X-100 was added
to each sample to achieve a final concentration of 0.5% (vol/vol) and
the sample solubilized for 1 h at 4°C. The resultant lysate was
centrifuged at 15,000 g for 10 min, and the supernatant was used in either Western blot assay for GSK-3 or the activity measurement.
PP1 Western blot.
For PP1
assay, the processing was as described previously
(7). Briefly, muscle was homogenized in PP1
homogenization buffer [50 mM HEPES (pH 7.2), 2 mM EDTA, 0.2%
2-mercaptoethanol, and 2 mg/ml glycogen] plus 10 µg/ml aprotinin, 1 mM benzamidine, and 0.1 mM PMSF added just before use. The homogenate
was centrifuged at 10,000 g to yield supernatant for Western
blot assay. In Western blot, an equal amount of protein was loaded,
separated by SDS-PAGE, and blotted with anti-PP1
antibody (a
generous gift from Dr. J. Lawrence, Jr.). PP1
blots were visualized
using HRP-conjugated secondary antibody followed by chemiluminescence
as outlined by the manufacturer (Amersham Pharmacia).
G-6-P measurement. Muscles (25-35 mg) were homogenized in 1 ml of ice-cold 6% perchloric acid for 1 min. The precipitated protein was pelleted by centrifugation at 5,000 g for 15 min. The supernatant was neutralized with KHCO3, and G-6-P was assayed spectrophotometrically as described by Lowry and Passoneau (27).
Hexokinase activity measurement. Muscles (25-30 mg) were homogenized in 10-fold volume of homogenizing buffer (in mM: 20 Tris · HCl, 900 KCl, 10 MgCl2, 2 EDTA, and 10 glucose and 0.5% Tween 20). The homogenate was centrifuged at 10,000 g for 30 min, and the supernatant was split in half to measure total hexokinase activity and heat-stable hexokinase (hexokinase I). Hexokinase activity was assayed as described by Postic et al. (31). Hexokinase II activity was subsequently calculated by subtracting hexokinase I from total hexokinase activity.
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.
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RESULTS |
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Impairment of insulin-stimulated GS at 24 h, but not 6 h,
after denervation in the soleus muscle.
In keeping with previous data (8, 20), glucose
incorporation into glycogen in both the presence and absence of insulin was significantly decreased in the 24-h-denervated muscle, whereas glucose incorporation into lipid was enhanced. Six hours after denervation, neither of these alterations was evident (Fig.
1).
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Alteration of nPKC distribution in muscle 24 h, but not
2-12 h, after denervation.
As noted previously (20), an increase in
membrane-associated PKC activity has been observed in muscle 24 h
after denervation; however, the PKC isoforms responsible for this are
not known. To answer this question, the cellular distribution of
various PKC isoforms was compared in sham-operated and denervated
muscle. Twenty-four hours after denervation, the abundance of PKC-
and -
(P < 0.05), but not PKC-
or -
, was
increased in the total homogenate, suggesting an increase in net
synthesis. As shown in Fig.
2B, this was due to an
increase in the abundance of these isoforms in the membrane fraction
(P < 0.05) without a corresponding decrease in the
cytosolic fraction.
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Activation of Akt/PKB by insulin is unaffected by denervation.
The ability of insulin to activate its receptor tyrosine kinase
(9) and PI3K (11) is not diminished in
24-h-denervated muscle. This plus the differential effect of insulin on
glucose incorporation into glycogen and lipid synthesis in these
muscles suggest that more distal events in the insulin-signaling
pathway are involved. As shown in Fig. 4,
this event did not appear to involve Akt/PKB. Thus both basal Akt/PKB
activity and insulin-stimulated activation of this kinase were the same
in denervated and control soleus muscles. Likewise, the phosphorylation
of Ser473 on Akt/PKB, an indicator of its activation by
insulin, was unaffected by denervation.
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GS activity is impaired by denervation.
The abundance and activity of GS were measured in control and
24-h-denervated muscles incubated in the presence and absence of
insulin. Figure 5A shows that
the activity of the active form of the enzyme (expressed as the
activity ratio G-6-P/+G-6-P) was decreased by
60% in denervated muscle incubated with a medium devoid of insulin.
Surprisingly, insulin caused a greater percentage increase in the
activity of GS in the denervated soleus than it did in control muscles,
although activity was still significantly higher in the control muscle.
Total GS activity (Fig. 5B) was also slightly lower in
denervated than in control muscle, although in the presence of insulin
this difference was not statistically significant. None of these
changes was associated with an alteration in the abundance of GS (Fig.
5C). On the other hand, an immunoblot of GS showed a gel
shift in the denervated soleus that was not observed after incubation
with insulin (Fig. 5C).
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Effects of denervation and insulin on GSK-3 and PP1 distribution
and GSK-3 phosphorylation.
The abundance of GSK-3 and, to an even greater extent, its
phosphorylation on Ser9 were markedly decreased in the
15,000-g supernatant of 24-h-denervated soleus incubated in
the absence of insulin (Fig. 6).
Incubation with insulin reversed both of these abnormalities. As shown
in Fig. 7, denervation caused a similar
alteration in PP1
distribution, which was also reversed by insulin.
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The concentration of G-6-P was diminished in denervated muscle in
the presence and absence of insulin, whereas hexokinase I and II
activities were unchanged.
The failure of insulin to stimulate glycogen synthesis in denervated
muscle, despite its ability to phosphorylate GSK-3 and activate GS,
caused us to examine the effect of denervation on the concentration of
G-6-P. In keeping with the findings of Burant et al.
(8), the concentration of G-6-P was diminished
by 50% in the 24-h-denervated soleus, and insulin failed to increase its concentration as it did in a control muscle (Fig.
8A). Thus, after insulin
stimulation, the concentration of G-6-P was 70% lower in
the denervated soleus. As shown in Fig. 8B, these
differences were not paralleled by differences in the activity of
hexokinase I or II between control and denervated muscle.
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DISCUSSION |
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The increases in the concentration of DAG and PKC activity have been associated with insulin resistance in skeletal muscle in several model systems, including the denervated muscle (20). Denervation causes a dichotomy in insulin action: the ability of insulin to stimulate glucose incorporation into glycogen is markedly inhibited, whereas its ability to direct glucose into DAG and lipids is enhanced. These findings plus the observation that membrane-associated PKC activity is increased 24 h after denervation (20) led to the hypothesis that an increased formation of complex lipids, such as DAG, could initiate the events leading to insulin resistance (impaired glycogen synthesis) in denervated muscle by causing a sustained activation of one or more conventional or novel PKC isoforms (12, 20). Thus, at various time points after sciatic nerve sectioning, we examined changes in glucose disposition, PKC isoform distribution, and the activity of metabolic enzymes and signaling molecules to better understand how insulin-stimulated glycogen synthesis is inhibited.
The distribution of individual PKC isoforms in denervated soleus muscle
was examined by Western blot. We focused on PKC-, -
, and -
,
because they are the predominant isoforms expressed in skeletal muscle
and changes in their distribution have been found in a variety of
insulin-resistant states (1, 15, 24, 33), and on the
atypical isoform PKC-
, which is thought to play an important role in
the regulation of glucose transport (38) and protein
synthesis (28) by insulin. The results revealed that only
the DAG-sensitive nPKC isoforms PKC-
and -
were affected by
denervation; their abundance in both the whole homogenate and the
membrane fraction was increased at 24 h (Fig. 2B). In
contrast, no changes in the mass of PKC-
and PKC-
were observed.
Given that PKC-
is also DAG sensitive, it is unclear why it did not respond in similar fashion. One possibility is that changes in DAG did
not colocalize with PKC-
; alternatively, changes in intracellular calcium as well as in DAG might be required to alter its membrane targeting. Irrespective of the explanation, the observation that the
changes in PKC-
and PKC-
abundance and distribution are not
observed at 2 and 12 h after denervation, when insulin-stimulated glycogen synthesis is still normal, suggests that these alterations in
PKC could play a causal role. The increase in total lipid synthesis also temporally correlates with a repartitioning of free fatty acids
into complex lipids such as phosphatidic acid and DAG, as might
be expected after a rise in malonyl-CoA (32). Although this increase in malonyl-CoA peaked between 6 and 8 h after
denervation, it persisted at twice the control levels for more than
24 h.
Activation of nPKCs might impair insulin action by a number of
mechanisms. Thus increased PKC activity has been shown in certain settings to serine-phosphorylate the insulin receptor, leading to
decreases in the ability of insulin to stimulate glucose transport and
various of its metabolic effects (2, 5, 18, 39). Against
such a mechanism being operative in 24-h-denervated muscle, inhibition
of early steps in the insulin-signaling cascade (receptor tyrosine
kinase, PI3K, Akt/PKB) was not observed in these muscles (Refs.
9 and 11 and this study). In addition,
insulin-stimulated glycogen synthesis was severely impaired after
denervation, whereas the ability of insulin to stimulate glucose
transport and overall glucose utilization was only minimally diminished
and its ability to stimulate glucose incorporation into lipid was
enhanced (Ref. 20 and this study). Collectively, these
findings suggest that the decrease in glycogen synthesis in denervated
muscle 1) is not secondary to decreased glucose transport
and, 2) if it is related to impaired signaling, either a
distal event in the cascade mediated by insulin or changes in an early
event are involved. Whether the observed alteration in PKC- and -
contributed to these changes remains to be determined, although it is
noteworthy that incubation of the soleus with 1 µM of phorbol
dibutyrate for 1 h, which activates both cPKC and nPKC isoforms,
inhibits insulin-stimulated glycogen synthesis but not glucose
transport (26, 37). Furthermore, inhibition of the insulin
receptor tyrosine kinase and PI3K were minimal in these muscles,
whereas a clear-cut decrease in Akt/PKB phosphorylation was observed
(26).
In 24-h-denervated solei incubated in a medium devoid of insulin, the
activity of the active form of GS is diminished by 60% and
electrophoretic mobility of GS is altered, consistent with phosphorylation. Unexpectedly, these changes were associated with a
decrease in the abundance of both PP1 and GSK-3 in the
15,000-g supernatant of the muscle homogenate and with a
decreased phosphorylation of GSK-3 in this fraction. Presumably, the
net effect of these changes was to increase GS phosphorylation.
Although insulin stimulated glycogen synthesis only minimally in the
24-h-denervated soleus, it activated Akt/PKB and GS to the same extent
that it did in nondenervated muscles. In addition, it eliminated the
gel shift of GS seen in the absence of insulin, and it restored the
abundance of PP1 and GSK-3 in the muscle supernatant to control
values. The net effect appeared to be activation of GS most likely
related to its dephosphorylation. Studies to assess translocation
between the supernatant and pellet fraction of these molecules are
needed to explore the basis for these changes in subcellular distribution.
In keeping with the earlier study by Burant et al. (8), insulin did not increase the concentration of G-6-P in denervated muscle as it did in control muscle (Fig. 8A). As a result, the concentration of G-6-P in the denervated muscle incubated with insulin was only 25% of that of a control muscle. This persistently low concentration of G-6-P offers one explanation for the limited effect of insulin on the rate of glucose incorporation into glycogen in situ in the incubated soleus after denervation (Fig. 1) despite its pronounced effect on GS activity (Fig. 5). Interestingly, this result points to the fact that 24-h denervation did not affect the ability of insulin to increase the intrinsic activity of GS, as had been previously thought (7). The reason for this failure to increase the G-6-P concentration despite an apparently normal stimulation of glucose uptake and glycolysis (13, 20) in the denervated soleus is not known. The data in this study suggest that it is not attributable to a decrease in hexokinase activity, with the caveat that this reflects the intrinsic activity of the enzyme. The efficient phosphorylation of glucose by hexokinase requires its coupling to mitochondria, and this is increased by muscle contraction (3, 32, 42). Therefore, one might speculate that denervation results in a less efficient coupling and a decrease in the in situ activity of hexokinase. Another hypothetical possibility is that insulin-stimulated glucose transport directed to glycogen, vs. down the glycolytic pathway, is selectively depressed. There are no data in the literature relevant to this subject; however, it has been demonstrated that insulin and exercise recruit GLUT4 from different pools (30). Whether these transporters differentially direct glucose taken up by the muscle cell to different sites has not been studied.
That denervation leads to an impairment of insulin-stimulated glycogen synthesis, and sometimes glucose transport in muscle, has long been appreciated (8, 10, 35). The results of different studies are somewhat difficult to compare because of such variables as muscle type, duration of denervation, and nutritional state (8, 35, 36). In an experiment with rat diaphragm 1 day after denervation, Smith and Lawrence (35) observed a 50% decrease in glucose incorporation into glycogen and a decreased activity ratio for GS in both the presence and absence of insulin. In addition, they failed to observe a decrease in 2-deoxyglucose uptake at that time but did so 3 days after denervation. Later studies suggest that this secondary decrease in glucose transport is related to late-occurring decreases in the abundance of GLUT4 (4, 13, 19), as well as alterations in insulin signaling (16, 40). The same investigators demonstrated that, in an incubated epitrochlearis muscle prelabeled with 32P, activation of GS by insulin is associated with the dephosphorylation of GS on at least two sites. In muscle denervated for 3 days, they found that the ability of insulin to do this was lost, as was its ability to activate GS (36). Studies in muscle denervated for 1 day were not reported.
In conclusion, our results show that the inhibition of
insulin-stimulated glycogen synthesis in rat soleus muscle 24 h
after denervation correlates with altered distribution of PKC- and PKC-
and an increase in glucose incorporation into lipids. The reason that the ability of insulin to stimulate glucose incorporation into glycogen is depressed in these muscles remains an enigma. Data
from this study suggest that it is not due to an impaired ability of
insulin to alter Akt/PKB and GSK-3 phosphorylation or to activate GS.
The results also suggest that a redistribution of GSK-3 and PP1
occurs in denervated muscle; however, its relation to the
pathophysiology of impaired glycogen synthesis is unclear, but it may
relate to the phosphorylation state of GS.
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ACKNOWLEDGEMENTS |
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We thank T. Kurowski, Dr. D. Dean, and V. Kaushik for their technical assistance and K. Tornheim for helpful discussion.
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FOOTNOTES |
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Support in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49147 and a grant from the Juvenile Diabetes Foundation.
Address for reprint requests and other correspondence: N. B. Ruderman, 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.
April 16, 2002;10.1152/ajpendo.00390.2001
Received 29 August 2001; accepted in final form 12 April 2002.
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