1 Noll Physiological Research Center, The Pennsylvania State University, University Park, Pennsylvania 16802; and 2 Departments of Experimental Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Physiological stressors such as sepsis and
tissue damage initiate an acute immune response and cause transient
systemic insulin resistance. This study was conducted to determine
whether tumor necrosis factor- (TNF-
), a cytokine produced by
immune cells during skeletal muscle damage, decreases insulin
responsiveness at the cellular level. To examine the molecular
mechanisms associated with TNF-
and insulin action, we measured
insulin receptor substrate (IRS)-1- and IRS-2-mediated
phosphatidylinositol 3-kinase (PI 3-kinase) activation, IRS-1-PI
3-kinase binding, IRS-1 tyrosine phosphorylation, and the
phosphorylation of two mitogen-activated protein kinases (MAPK, known
as p42MAPK and
p44MAPK) in cultured
C2C12
myotubes. Furthermore, we determined the effects of TNF-
on
insulin-stimulated 2-deoxyglucose (2-DG) uptake. We observed that
TNF-
impaired insulin stimulation of IRS-1- and IRS-2-mediated PI
3-kinase activation by 54 and 55% (P < 0.05), respectively. In addition, TNF-
decreased
insulin-stimulated IRS-1 tyrosine phosphorylation by 40%
(P < 0.05). Furthermore, TNF-
repressed insulin-induced p42MAPK
and p44MAPK tyrosine
phosphorylation by 81% (P < 0.01).
TNF-
impairment of insulin signaling activation was accompanied by a
decrease (P < 0.05) in 2-DG uptake
in the muscle cells (60 ± 4 vs. 44 ± 6 pmol · min
1 · mg
1). These data suggest
that increases in TNF-
may cause insulin resistance in skeletal
muscle by inhibiting IRS-1- and IRS-2-mediated PI 3-kinase activation
as well as p42MAPK and
p44MAPK tyrosine phosphorylation,
leading to impaired insulin-stimulated glucose uptake.
tumor necrosis factor-; phosphatidylinositol 3-kinase; mitogen-activated protein kinase
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INTRODUCTION |
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THE STRESS of tissue damage is characterized by an
alteration in body homeostasis, an acute immune response, and
mononuclear cell release of cytokines, including tumor necrosis
factor- (TNF-
), interleukin (IL)-1, and IL-6 (4, 34).
Furthermore, physiological stress resulting from muscle damage is
accompanied by the development of whole body transient insulin
resistance (1, 2, 7, 20). Recent studies performed in our laboratory
(19, 21) have confirmed an increase in insulin secretion after the
physiological stress induced by eccentric exercise in young subjects.
Therefore, an increase in pancreatic
-cell secretion may be a
mechanism to compensate for the transient systemic insulin resistance
frequently associated with eccentric exercise-induced muscle damage (1, 20). However, the molecular mechanisms associated with stress-induced insulin resistance are not known. Recent publications suggest that
TNF-
may impair the insulin signaling pathway in cultured adipocytes
and hepatocytes (10, 15). However, the effects of TNF-
on the
insulin signaling pathway in skeletal muscle are less clear. In
addition, TNF-
infusion in rodents has been shown to induce whole
body insulin resistance during euglycemic-hyperinsulinemic clamps (22).
Thus increased production of TNF-
by mononuclear cells could provide
a link between muscle tissue damage and transient insulin resistance,
possibly through a mechanism associated with altered regulation of the
insulin signaling pathway.
The insulin signaling pathway is comprised of a complex array of
protein kinases and protein phosphatases that regulate insulin action
(5, 33). Insulin receptor substrate (IRS)-1 and IRS-2 are docking
proteins that are pivotal in initiating the pleiotropic effects of
insulin through phosphatidylinositol 3-kinase (PI 3-kinase; see Refs. 5
and 29) and mitogen-activated protein kinases (MAPK)
p42MAPK and
p44MAPK (5). Both PI 3-kinase and
MAPK are activated by insulin via tyrosine phosphorylation and have
been shown to be involved in the regulation of glucose uptake and
glycogen synthesis, respectively (3, 5, 8). Thus the purpose of this
study was to determine the effects of TNF- on insulin stimulation of
IRS-1- and IRS-2-mediated PI 3-kinase activation,
p42MAPK/p44MAPK
tyrosine phosphorylation, and 2-deoxyglucose (2-DG) uptake in skeletal
muscle cells.
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METHODS |
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Materials.
C2C12
myoblasts were purchased from the American Type Culture Collection
(Rockville, MD). Recombinant mouse TNF- was purchased from Genzyme
Diagnosis (Cambridge, MA). Anti-active MAPK polyclonal antibody was
purchased from Promega (Madison, WI). Monoclonal antiphosphotyrosine
antibody PY20 and monoclonal antiserum to the p85 regulatory subunit of
the PI 3-kinase were purchased from Transduction Laboratories
(Lexington, KY). Polyclonal antiserum to rabbit IRS-1 and IRS-2 were
obtained from Upstate Biotechnology (Lake Placid, NY). Okadaic acid was
obtained from Biomol Research Laboratories (Plymouth Meeting, PA).
Phosphatidylserine and phosphatidylinositol were purchased from Avanti
Polar Lipids. All biochemicals, 2-DG, cell culture reagents, and FBS
were from Sigma (St. Louis, MO), and radiochemicals
(2-[3H]DG and
32P labeled) were from Du Pont-New
England Nuclear. Protein assay reagents were from Bio-Rad Laboratories,
and chemiluminescence reagents were from Amersham.
Cell culture. C2C12 myoblasts were cultured in 100-mm dishes in an atmosphere of 5% CO2 at 37°C in DMEM supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin (100 U/ml) to reach 100% confluence. Myoblast differentiation was induced with DMEM supplemented with 5% horse serum, L-glutamine, and penicillin/streptomycin for 72 h. Differentiated myotubes were then starved for 5 h in serum-free DMEM before treatment.
Insulin and TNF- treatment.
Serum-free myotubes were treated with insulin (100 nM) for 3 or 15 min,
with TNF-
(10 ng/ml) alone for 1 h, or with TNF-
for 1 h followed
by insulin for 3 or 15 min. All of the treatments were performed in
duplicate in three independent experiments. Medium was then removed,
and cells were washed two times with ice-cold PBS. Cell lysates were
obtained by scraping the cells in lysate buffer [1% Triton
X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 2 mM
NaVO4, 1 mM benzamidine, 0.2 M
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and 10 µg/ml of
antipain, pepstatin, aprotinin] or in PI 3-kinase
immunoprecipitation buffer [50 mM HEPES, 137 mM NaCl, 1 mM MgCl2, 1 mM
CaCl2, 10 mM sodium pyrophosphate,
10 mM NaF, 2 mM EDTA, 2 mM NaVO4,
1% Nonidet P-40 (NP-40), 10% glycerol, 2 µg/ml aprotinin, 5 µg/ml
leupeptin, 1.5 mg/ml benzamidine, 0.2 M AEBSF, 10 µg/ml antipain, and
0.5 µg/ml pepstatin]. Cell lysates were then spun down, and the
cell pellet/debris was discarded. Supernatant protein concentration was
determined by the Bio-Rad DC protein assay.
Determination of IRS-1- and IRS-2-associated PI
3-kinase activity. A 1-mg sample of cell lysate was
immunoprecipitated with either 4 µg of IRS-1 or IRS-2 polyclonal
antibodies, rocking overnight at 4°C. A 40-µl sample of slurry
protein A-Sepharose was added to the immunoprecipitate for 2 h, and
immunocomplexes were obtained by brief centrifugation at 9,000 rpm and
washed three times in PBS-1% NP-40, two times in 500 mM LiCl-100 mM
Tris, pH 7.6, and one time in 10 mM Tris · HCl, pH
7.4, 100 mM NaCl, and 1 mM
trans-1,2-diaminoacylclohexane-N,N,N',N'-tetraacetic acid. The pellets were spun down one more time and washed in PI 3-kinase adenosine assay buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 10 mM
MgCl2, 0.5 mM EGTA, and 120 µM
adenosine). The final pellet was resuspended in 40 µl of PI 3-kinase
adenosine assay buffer. A 50-µl sample of phosphatidylinositol and
phosphatidylserine was dried down in a nitrogen stream and sonicated in
100 µl of 20 mM HEPES-1 mM EDTA, pH 7.4. The lipid mixture was kept
on ice, and 5 µl of this mixture (2 µg/µl of
phosphatidylinositol) were added to each sample. The solution was mixed
by sonication and incubated for 10 min at 30°C on a heat block. A
mixture consisting of 170 µCi of
[-32P]ATP and 280 µM unlabeled ATP was prepared, and the reaction was started by adding
5 µl of this mixture to each sample. After 10 min at 30°C, the
reaction was stopped by the addition of 200 µl 1 N HCl to each
sample. The phosphatidylinositol 3-phosphate (PI3P) was extracted with 160 µl
chloroform-methanol (1:1). The phases were separated by centrifugation,
and the lower organic phase was removed and separated by TLC. The
radioactivity incorporated into PI3P was determined by
phosphorimaging of the TLC plates.
Determination of IRS-1 tyrosine phosphorylation and IRS-1 binding to p85 by immunoprecipitation and immunoblotting. A total of 300 µg of cell lysate was immunoprecipitated with rabbit antiserum to IRS-1, rocking overnight at 4°C. A 50-µl slurry of protein A-Sepharose was added to the immunoprecipitates, and incubation was continued for 1 h at 4°C followed by brief centrifugation at 9,000 rpm. The agarose pellets were then washed three times with immunoprecipitation buffer. A 50-µl sample of 2× Laemmli buffer was added, and the samples were boiled for 5 min at 100°C. Immunoprecipitates were run on 8% SDS-PAGE blotted to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore) following the same procedure as that used for p42MAPK and p44MAPK. The blots were then probed with anti-phosphotyrosine PY20, rabbit polyclonal anti-IRS-1, and mouse polyclonal anti-p85 antibodies followed by an incubation with their respective secondary antibodies bound to horseradish peroxidase. Immunodetection was performed by enhanced chemiluminescence (ECL; Amersham) following the manufacturer's instructions.
Determination of p42MAPK and p44MAPK tyrosine phosphorylation. A total of 100 µg of cell lysate was mixed with 2× Laemmli buffer, boiled at 100°C for 5 min, loaded on 10% SDS-PAGE under reducing conditions, and blotted to Immobilon-P PVDF membranes (Millipore) for 3 h with 300 mA current at 4°C. Membranes were blocked for 90 min in blocking buffer [10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20 plus 5% nonfat dry milk (1% BSA for phosphotyrosine blots)]. The blot was then probed with anti-active MAPK polyclonal antibody to recognize phosphorylated forms of both p42MAPK and p44MAPK. An anti-rabbit antibody bound to horseradish peroxidase was then used, and immunodetection was performed by ECL.
Determination of glucose uptake by
C2C12 skeletal
muscle cells.
Glucose uptake was assayed using 2-DG. Glucose uptake measurements were
performed in duplicate and in three independent experiments. After 5 h
of serum starvation, cells were incubated with insulin (100 nM) for 30 min, with TNF- (10 ng/ml) for 1 h, or with TNF-
for 1 h followed
by insulin for 30 min. After TNF-
and insulin treatment, cells were
washed two times with wash buffer (20 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, and 1 mM
CaCl2). Cells were then
incubated in buffer transport solution (wash buffer containing 0.5 mCi
2-[3H]DG/ml and 10 mM
2-DG) for 10 min. Uptake was terminated by aspiration of the solution.
Cells were then washed three times, and radioactivity associated with
the cells was determined by cell lysis in 0.05 M NaOH, followed by
scintillation counting. Aliquots of cell lysates were used for protein
content determination. 2-DG uptake was expressed as picomoles per
minute per milligram of protein.
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RESULTS |
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Effects of TNF- on IRS-1-mediated PI 3-kinase
activity in insulin-stimulated
C2C12 myotubes.
It has been suggested that PI 3-kinase activation is a critical step in
the regulation of the insulin signaling pathway and glucose uptake
(29). Therefore, we questioned whether TNF-
would decrease insulin
stimulation of PI 3-kinase activation. Values were expressed as
percentage of insulin-stimulated activity. Insulin treatment alone for
3 min increased (P < 0.001)
IRS-1-mediated PI 3-kinase activation (Fig.
1, lane 2 vs. lane 1, 100 vs. 11 ± 5%).
TNF-
alone did not alter IRS-1-mediated PI 3-kinase activity when
compared with nontreated control cells (Fig. 1, lane
3 vs. lane 1, 13 ± 6 vs. 11 ± 5%). However, pretreatment with TNF-
for 1 h
resulted in a significant decrease (P < 0.05) in IRS-1-mediated PI 3-kinase activity (Fig. 1,
lane 2 vs. lane
4, 100 vs. 46 ± 18%).
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Effects of TNF- on IRS-2-mediated PI 3-kinase
activity in insulin-stimulated
C2C12 myotubes.
We then examined whether IRS-2 could provide an alternative signaling
pathway for TNF-
downregulation of IRS-1-mediated PI 3-kinase
activation. Similar results as those obtained for IRS-1-mediated PI
3-kinase activation were observed for IRS-2. Insulin increased (P < 0.001) IRS-1-mediated PI
3-kinase activation (Fig. 2,
lane 2 vs. lane
1, 100 vs. 10 ± 4%). As observed in the data from
IRS-1, TNF-
alone had no effect on IRS-2-mediated PI 3-kinase
activity (Fig. 2, lane 3 vs.
lane 1, 14 ± 5 vs. 10 ± 4%).
However, TNF-
did decrease (P < 0.05) insulin stimulation of IRS-2-mediated PI 3-kinase activation
(Fig. 2, lane 2 vs.
lane 4, 100 vs. 45 ± 10%).
Although it has been suggested that IRS-2 provides alternative pathways
to compensate for impaired IRS-1 in IRS-1-deficient mice (25, 31), our
study suggests that IRS-2 does not provide alternative insulin
signaling to overcome the downregulation of IRS-1 by TNF-
.
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Effects of TNF- on IRS-1-p85 binding in
insulin-stimulated
C2C12 myotubes.
The enzyme PI 3-kinase is composed of two subunits, a regulatory p85
subunit and a catalytic p110 subunit (5). IRS-1 binding to the
regulatory p85 subunit is critical for the activation of PI 3-kinase
(29). Thus, to determine the effect of TNF-
on insulin stimulation
of IRS-1-p85 binding, cell lysates were subjected to
immunoprecipitation with anti-IRS-1 antibody, and Western blot analysis
was performed with an anti-p85 antibody. Insulin treatment for 3 min
increased (P < 0.001) IRS-1-bound PI
3-kinase (Fig. 4, lane
2 vs. lane 1, 100 vs.
6 ± 3%). In accordance with the data obtained for IRS-1 and PI
3-kinase, TNF-
impaired insulin-induced binding of IRS-1-p85 (Fig.
4, lane 2 vs. lane
4, 100 vs. 42 ± 13%). TNF-
alone did not alter
IRS-1-p85 binding when compared with control cells (Fig. 4,
lane 3 vs. lane
1, 15 ± 5 vs. 6 ± 3%).
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Effect of TNF- on p42MAPK
and p44MAPK tyrosine phosphorylation.
Both p42MAPK and
p44MAPK are downstream molecules
in the insulin signaling pathway and have been reported to be involved
in the regulation of glycogen synthesis (3, 5, 8). Insulin treatment
for 15 min increased (P < 0.001)
tyrosine phosphorylation on
p42MAPK and
p44MAPK in the cultured
C2C12
myotubes used in these experiments (Fig. 5,
lane 2 vs. lane
1, 100 vs. 16 ± 6% and 100 vs. 10 ± 4%,
respectively). TNF-
alone did not alter tyrosine phosphorylation on
p42MAPK and
p44MAPK when compared with control
cells (Fig. 5, lane 3 vs.
lane 1, 8 ± 5 vs. 16 ± 6% and
9 ± 7 vs. 10 ± 4%, respectively). However, TNF-
treatment
decreased (P < 0.01)
insulin-stimulated p42MAPK and
p44MAPK tyrosine phosphorylation
(Fig. 5, lane 2 vs.
lane 4, 100 vs. 19 ± 10% and 100 vs. 21 ± 8%, respectively).
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DISCUSSION |
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Insulin resistance is a complex metabolic abnormality that affects the
ability of peripheral tissues to respond to insulin. TNF-, a
cytokine produced by mononuclear cells during stress (4, 34), has been
closely associated with inhibition of the insulin signaling pathway and
insulin resistance (10, 15). TNF-
has previously been reported to
diminish insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation in
hepatocytes and adipocytes, resulting in impaired insulin action (16,
18, 26). It has been suggested that PI 3-kinase activation by IRS-1 and
IRS-2 is essential for insulin-mediated glucose uptake by the cell (12, 24). Therefore, we examined the effects of TNF-
on PI 3-kinase activation to better understand the cross-talk between TNF-
and insulin action in muscle cells. Skeletal muscle is the major site for
insulin-mediated glucose disposal (9). We show that insulin stimulation
of IRS-1- and IRS-2-mediated PI 3-kinase activity is decreased by
TNF-
in cultured
C2C12
myotubes. Furthermore, TNF-
decreased insulin stimulation of
cellular glucose uptake. Thus impaired insulin stimulation of PI
3-kinase activation by elevated levels of TNF-
after the stress of
tissue damage could result in decreased glucose uptake and transient
insulin resistance.
It has been suggested that IRS-1 is a pivotal molecule in the
regulation of the insulin signaling pathway (29). Insulin activation of
IRS-1 through tyrosine phosphorylation results in IRS-1 binding with
the p85 regulatory subunit of PI 3-kinase. Pretreatment with TNF-
for 1 h impaired insulin stimulation of IRS-1 and IRS-1-p85 binding,
with no change in IRS-1 total protein. These results are strongly
supported by previous studies performed in adipocytes and hepatocytes
(10, 14, 18) and in models in vivo (14), in which insulin-induced
tyrosine phosphorylation of IRS-1 is impaired by TNF-
. However, our
results extend these findings to skeletal muscle, which is responsible
for >85% of insulin-mediated glucose disposal in the body (9).
It has been suggested that PI 3-kinase activation is required for
insulin-mediated glucose uptake (6). Furthermore, impaired PI 3-kinase
activation has been associated with decreased GLUT-4 translocation (11)
and insulin resistance (13). Thus PI 3-kinase activation is key in the
regulation of insulin-mediated glucose uptake. PI 3-kinase is activated
by tyrosine-phosphorylated IRS in response to insulin binding to its
receptor in insulin-dependent tissues (17, 29). Two IRS complexes,
IRS-1 and IRS-2, have been identified and have been shown to be
structurally and functionally similar (30). Therefore, we questioned
whether TNF- could impair insulin stimulation of IRS-1- and
IRS-2-mediated PI 3-kinase activation in skeletal muscle cells. We
found that insulin-dependent PI 3-kinase activation was impaired by
TNF-
in both IRS-1- and IRS-2-mediated PI 3-kinase activation.
Although it has been suggested that IRS-2 provides an alternative
pathway for insulin signal transduction in IRS-1-deficient mice (25,
32), it has also been reported that insulin-induced PI 3-kinase
activation is significantly decreased in muscle, but not in liver, from
IRS-1-deficient mice compared with those in wild-type mice (35). In our
study, pretreatment with TNF-
repressed insulin stimulation of
IRS-1- and IRS-2-mediated PI 3-kinase activation by 54 and 55%,
respectively, in myotubes stimulated with insulin, indicating that
IRS-2 does not compensate for the downregulation by TNF-
of
IRS-1-activated PI 3-kinase. These data suggest that IRS-1- and
IRS-2-mediated PI 3-kinase activation are equally diminished by TNF-
in muscle cells. Previous studies have suggested that insulin
stimulation of IRS-2-mediated PI 3-kinase activation occurs faster and
more transiently than insulin stimulation of IRS-1-mediated PI 3-kinase
activation (23). Our study suggests that IRS-1 and IRS-2 activation is
synchronously stimulated by insulin and is impaired by TNF-
in our
model of C2C12
myotubes. Thus, in differentiated muscle, insulin stimulation of both
IRS-1- and IRS-2-mediated PI 3-kinase activation is minimized by
TNF-
. Impaired insulin activation of PI 3-kinase could lead to a
decrease in glucose uptake and therefore insulin resistance.
To further investigate the potential role played by TNF- on insulin
resistance, we evaluated the effects of TNF-
on insulin-mediated glucose uptake. TNF-
downregulation of PI 3-kinase activation was
accompanied by impairment of insulin-stimulated glucose uptake. These
results suggest that elevated TNF-
may be a key component in the
development of insulin resistance in skeletal muscle by inhibiting
IRS-1 tyrosine phosphorylation and PI 3-kinase activation, leading to
decreased insulin stimulation of glucose uptake. TNF-
also caused
significant reductions in basal glucose uptake. Previous investigations
have shown a decrease in GLUT-1 protein content in 3T3-L1 adipocytes
exposed to TNF-
(28). GLUT-1 is the glucose transporter responsible
for basal glucose uptake (5). Thus a decrease in GLUT-1 protein content
after TNF-
treatment could be responsible for the reductions
observed in basal glucose uptake in our model of
C2C12
skeletal muscle cells.
IRS-1 tyrosine phosphorylation has also been implicated in signal
transduction from the insulin receptor downstream to the MAPK,
p42MAPK and
p44MAPK. These two isoforms of
MAPK have been associated with the regulation of glycogen synthesis (5,
8, 27). In the present study, TNF- impairs insulin-induced
p42MAPK and
p44MAPK tyrosine phosphorylation
in cultured muscle cells. Furthermore, the extent to which TNF-
downregulates p42MAPK and
p44MAPK (81%) is greater than
TNF-
impairment of IRS-1-mediated PI 3-kinase activation (54%).
These data suggest that the intracellular components involved in
insulin-mediated glycogen synthesis may be more sensitive to the
effects of TNF-
than the upstream molecules of the insulin signaling
pathway involved in glucose uptake. Therefore, inhibition of
p42MAPK and
p44MAPK could be one of the major
components of the insulin signaling pathway downregulated by TNF-
.
In summary, these data indicate that TNF- impairs insulin
stimulation of IRS-1- and IRS-2-mediated PI 3-kinase activation, p42MAPK/p44MAPK
tyrosine phosphorylation, and 2-DG uptake in muscle cells. IRS-2 was
unable to compensate for the downregulation of IRS-1-mediated PI
3-kinase activation by TNF-
, suggesting that IRS-2 is not an
alternative pathway to IRS-1 in the presence of increased TNF-
in
muscle cells. Impaired insulin activation of
p42MAPK/p44MAPK
by elevated levels of TNF-
could result in decreased glycogen synthesis. Thus less glucose available for storage and impaired glycogen synthesis activity could result in a failure to synthesize glycogen stores in skeletal muscle. Further studies need to be performed to determine whether TNF-
is a key factor in the
development of the systemic insulin resistance resulting from muscle
tissue damage in vivo.
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ACKNOWLEDGEMENTS |
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We thank Susan L. Rook (Joslin Diabetes Center, Boston, MA) for technical advice, Raj K. Krishnan for helpful discussions and critical reading of the manuscript, and Dr. Joseph G. Cannon for access to laboratory facilities.
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FOOTNOTES |
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This research was partially supported by National Institute on Aging Grant AG-12834 to J.P. Kirwan and General Clinical Research Center Grant RR-10732 to The Pennsylvania State University.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. P. Kirwan, 105 Noll Physiological Research Center, The Pennsylvania State Univ., Univ. Park, PA 16802 (E-mail: jpk3{at}psu.edu).
Received 1 July 1998; accepted in final form 13 January 1999.
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