Department of Anesthesia and Critical Care, Harvard Medical School, and Anesthesia Services, Massachusetts General Hospital and Shriners Hospital for Children, Boston, Massachusetts 02114
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ABSTRACT |
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Prolonged immobilization depresses insulin-induced glucose
transport in skeletal muscle and leads to a catabolic state in the
affected areas, with resultant muscle wasting. To elucidate the altered
intracellular mechanisms involved in the insulin resistance, we
examined insulin-stimulated tyrosine phosphorylation of the insulin
receptor -subunit (IR-
) and insulin receptor substrate (IRS)-1
and activation of its further downstream molecule, phosphatidylinositol 3-kinase (PI 3-K), after unilateral hindlimb immobilization in the rat.
The contralateral hindlimb served as control. After 7 days of
immobilization of the rat, insulin was injected into the portal vein,
and tibialis anterior muscles on both sides were extracted.
Immobilization reduced insulin-stimulated tyrosine phosphorylation of
IR-
and IRS-1. Insulin-stimulated binding of IRS-1 to p85, the
regulatory subunit of PI 3-K, and IRS-1-associated PI 3-K activity were
also decreased in the immobilized hindlimb. Although IR-
and p85
protein levels were unchanged, IRS-1 protein expression was
downregulated by immobilization. Thus prolonged immobilization may
cause depression of insulin-stimulated glucose transport in skeletal
muscle by altering insulin action at multiple points, including the
tyrosine phosphorylation, protein expression, and activation of
essential components of insulin signaling pathways.
insulin receptor; insulin resistance; insulin receptor substrate-1; muscle wasting; phosphatidylinositol 3-kinase
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INTRODUCTION |
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INSULIN ACTIVATES
MULTIPLE signaling pathways, leading to diverse effects on
cellular metabolism and mitogenesis. These actions of insulin are
initiated by autophosphorylation of specific tyrosine residues of the
intracellular portion of the insulin receptor -subunit (IR-
).
Activated IR-
transduces the signal to downstream components by
phosphorylating endogenous substrate proteins, such as insulin receptor
substrates (IRSs) and Shc (22). A further downstream key
molecule of IRS-1 is phosphatidylinositol 3-kinase (PI 3-K), which
consists of regulatory (p85) and catalytic (p110) subunits. After
phosphorylation by IR-
, IRS proteins bind to the p85 regulatory
subunit of PI 3-K, leading to the activation of PI 3-K
(34). This induces a diverse range of cellular responses, including glucose transporter translocation, cell growth and
proliferation, and synthesis of carbohydrates, lipids, and proteins
(8, 18, 25, 27). Thus dimerization and autophosphorylation
of IR, tyrosine phosphorylation of IRSs by IR, and subsequent binding of PI 3-K to IRSs are essential initial steps for the metabolic action
of insulin (42).
Chronic muscle disuse, produced by the conditions of prolonged bed
rest, casting or pinning of limbs, and microgravity, induces insulin
resistance and a catabolic state in the affected skeletal muscle in
humans (9, 32, 35, 37). Animal studies, including those in
rats, have confirmed that immobilization is associated with resistance
to insulin-induced glucose uptake and protein synthesis (4,
29). Insulin resistance is also a major problem in critically
ill patients, who are invariably physically inactive and/or
immobilized. These critical conditions include sepsis (26, 44), burn injury (17), and surgical trauma/stress
(26, 36, 40). In all of these conditions, including
immobilization and critical illness, the effect of insulin on potassium
uptake by the cell seems unaltered. Exercise, in contrast to
immobilization, increases insulin sensitivity in humans and rodents
(12, 21). Although short-term muscle contraction or
electrical stimulation (for ~60 min) increases glucose uptake through
insulin-independent mechanisms and has no effect on basal and
insulin-stimulated tyrosine phosphorylation of IR- and IRS-1
(12, 43), longer-term exercise is associated with improved
insulin sensitivity (7, 14). One week of exercise leads to
increased insulin sensitivity with enhanced PI 3-K activity in humans
(14), and 6 h of swimming per day for 1 or 5 days
results in increases in insulin-stimulated IRS-1 phosphorylation and PI
3-K activation in rats (7). Exercise for 9 wk increases
protein or mRNA levels of IR, IRS-1, and the p85 regulatory subunit of
PI 3-K in rats (21). It is unclear, however, whether or
how the converse, namely, chronic muscle disuse and/or immobilization,
alters insulin signaling, although immobilization and muscle disuse are
known to decrease insulin-stimulated glucose uptake.
IRS-1, in particular, is a key molecule for insulin action in skeletal muscle (20). Gene knockout of IRS-1 leads to peripheral insulin resistance in mice (2). It is reported that IRS-1 is the major tyrosine-phosphorylated protein bound to the regulatory subunit of PI 3-K (p85) in skeletal muscle in mice, whereas IRS-2 is only weakly associated with PI 3-K (39). Accordingly, IRS-2 is not necessary for insulin-stimulated glucose transport in skeletal muscle (13). IRS-3 and IRS-4 are not expressed in skeletal muscle (23, 24). Therefore, in the present study, we investigated the effect of immobilization on insulin-stimulated activation of IR, IRS-1, and PI 3-K.
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MATERIALS AND METHODS |
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Animals. Adult male Sprague-Dawley rats (175-200 g), purchased from Taconic Farms (Germantown, NY), were used for this study. The study was approved by the Institutional Animal Care Committee. The animal care facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The rats were housed in mesh cages in a room maintained at 25°C and illuminated by 12:12-h light-dark cycles. The rats were provided with standard rodent chow and water ad libitum. All surgical procedures were performed under anesthesia with 70 mg/kg pentobarbital sodium injected intraperitoneally.
Immobilization and insulin injection. The left hindlimb was immobilized by pinning the knee at 90° flexion and ankle at 90° dorsiflexion. The ankle and knee joints were immobilized, respectively, by inserting 25-gauge hypodermic needles through the calcaneus into the distal tibia, and through the proximal tibia into the distal femur, as described previously (15, 16). On the sham-immobilized contrateral side, the limb was subjected to the same manipulations, including boring of a hole through the joints, but a pin was not inserted to immobilize the joint. Thus pain was probably similar on both sides. We had previously shown that sham-immobilization of the contralateral knee and ankle joints did not alter muscle function relative to unimmobilized hindlimbs of naive animals (15, 16). In these studies (15, 16), body weight changes and muscle morphology, acetylcholine receptor changes, wet weight, and contraction in the tibialis muscle were not different between the unimmobilized contralateral side and naive separate sham-immobilized animals, indicating that the contralateral side does not undergo compensatory exercise-induced hypertrophy. In the present study, therefore, the contralateral unimmobilized hindlimb served as control.
At 7 days of immobilization, food was withdrawn for 18 h, the rat was then anesthetized, and 2.5 mU/g body weight of human insulin (Humulin R, Eli Lilly, Indianapolis, IN) diluted with saline, or saline alone, was injected into the portal vein, as described previously (10, 17). The tibialis anterior muscles of both hindlimbs were removed at 4 min after insulin or saline injection and then frozen in liquid nitrogen.Detection of tyrosine phosphorylation of IR- and IRS-1.
The frozen muscle tissue was minced with surgical scissors for 1 min in
ice-cold lysis buffer [50 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 2 mM
EDTA, 1% Nonidet P-40, 10% glycerol, 10 mM sodium fluoride, 2 mM
sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM
sodium pyrophosphate, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 0.5 µg/ml pepstatin], and thereafter was homogenized using a Polytron
PT-MR 3000 (KINEMATIKA AG, Littau, Switzerland) at maximum speed for
30 s. The homogenates were kept on ice for 30 min. The insoluble
material was removed by centrifugation at 12,000 rpm for 30 min.
Aliquots of the supernatants containing equal amounts of protein, as
determined by the Bradford protein assay, were subjected to
immunoprecipitation for 1 h at 4°C with anti-IR-
mouse
monoclonal (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-IRS-1
rabbit polyclonal antibody (Upstate Biotechnology, Lake Placid, NY).
After the addition of protein A-Sepharose CL-4B (Pharmacia Biotech,
Piscataway, NJ), the immunoprecipitates were washed three times in 50 mM HEPES-NaOH (pH 7.5) with 150 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40,
10% glycerol, 10 mM sodium fluoride, 2 mM sodium vanadate, 1 mM PMSF,
10 mM sodium pyrophosphate, 5 µg/ml aprotinin, 5 µg/ml leupeptin,
and 0.5 µg/ml pepstatin. The samples were prepared for SDS-PAGE by
addition of Laemmli sample buffer (Boston Bioproducts, Ashland, MA) and
boiling for 5 min.
Protein expression of IRS-1 and p85. The changes in protein expression of IRS-1 and p85 regulatory subunit of PI 3-K after immobilization were then investigated. Aliquots of the muscle homogenates containing equal amounts of protein were subjected to SDS-PAGE and were immunoblotted with anti-IRS-1 rabbit polyclonal antibody or anti-p85 rabbit polyclonal antibody, respectively (Upstate Biotechnology). The bands of interest were scanned as described above.
Detection of p85 associated with IRS-1. The frozen muscle tissue was minced with surgical scissors for 1 min in ice-cold lysis buffer and was then homogenized, as described previously. After sonication (Sonic dismembrator, MODEL300, Fisher, Pittsburgh, PA), the samples were kept on ice for 30 min. Insoluble material was removed by centrifugation at 12,000 rpm for 30 min. Aliquots of the supernatants containing equal amounts of protein were subjected to immunoprecipitation for 1 h at 4°C with anti-IRS-1 rabbit polyclonal antibody, provided by Drs. K. Yonezawa and K. Hara (see Ref. 46). The immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose membrane. After blocking in 5% dried milk in PBS-Tween, the membranes were incubated with anti-PI 3-K p85 rabbit polyclonal antibody (Upstate Biotechnology) followed by incubation with anti-rabbit IgG antibody conjugated with horseradish peroxidase and visualization by chemiluminescence. The bands of interest were scanned as described above.
PI 3-K activity assay.
PI 3-K activity in the immunoprecipitates obtained using anti-IRS-1
rabbit polyclonal antibody (46) was measured in vitro by
its ability to phosphorylate exogenous phosphatidylinositol (Sigma, St.
Louis, MO) to phosphatidylinositol monophosphate, as described
previously (17), with minor modifications. Briefly, 10 µl of 100 mM MgCl2 and 10 µl of PI (2 mg/ml) dissolved
in 10 mM Tris · HCl (pH 7.5) containing 1 mM EGTA were added to
the immunoprecipitates. PI 3-K reaction was started by the addition of
10 µl of 440 µM ATP containing 20 µCi of
[-32P]ATP. After 10 min at 37°C with constant
shaking, the reaction was stopped by adding 20 µl of 8 N HCl and 160 µl of CHCl3-methanol (1:1). The samples were centrifuged
(at 13,000 rpm for 10 min), and the lower organic phase was applied to
a silica gel TLC plate (Whatman) that had been prebaked for 1 h.
The plate was developed in
CHCl3-CH3OH-H2O-NH4OH
(60:47:11:3.2), dried, and visualized by autoradiography. The bands of
interest were scanned as described above.
Statistical analysis.
The level of tyrosine phosphorylation of IR- and IRS-1, the protein
expression of IR-
, IRS-1, and p85, the binding of p85 subunit of PI
3-K to IRS-1, and the IRS-1-associated PI 3-K activity in each muscle
were expressed as percentages of the corresponding levels in the
insulin-stimulated control muscle. Thus the bands of interest in the
autoradiograms were normalized to the levels in the insulin-stimulated
contralateral control muscles, denoted as 100%. The values of each of
these parameters before (basal) and after insulin stimulation were
compared between the immobilized and control limbs using the
Mann-Whitney U-test. The null hypothesis was rejected when
P < 0.05. All values are expressed as means ± SE.
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RESULTS |
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The weight of the tibialis anterior muscle was significantly
(n = 8, P < 0.01) smaller in the
7-day-immobilized hindlimb (366 ± 30 mg) compared with the
contralateral control hindlimb (531 ± 27 mg). To assess tyrosine
phosphorylation of IR- or IRS-1, muscle homogenates from immobilized
and contralateral limbs were immunoprecipitated with anti-IR-
or
anti-IRS-1 antibodies followed by immunoblotting with
anti-phosphotyrosine antibody. The basal level of tyrosine
phosphorylation of IR-
was higher in immobilized muscle than in
control muscle (Fig. 1A;
P < 0.05). By contrast, insulin-stimulated tyrosine
phosphorylation of IR-
was attenuated in immobilized muscle (Fig.
1A; P < 0.02). IR protein level did not
differ between the immobilized and control muscles (Fig.
1B). This indicates that the quantitative decrease in
tyrosine phosphorylation of IR-
after insulin treatment in the
immobilized muscle was not due to decreased IR-
protein expression
but resulted from the impaired activation of IR.
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Consistent with the attenuation of phosphorylation of IR-, tyrosine
phosphorylation of IRS-1 after insulin treatment was also decreased in
the immobilized hindlimb (Fig.
2A; P < 0.02). However, in contrast to IR-
, the recovery of IRS-1 protein
after immunoprecipitation was also decreased significantly in the
immobilized hindlimb compared with the contralateral control hindlimb
(Fig. 2B; P < 0.02). Simple Western
blotting of the homogenates with anti-IRS-1 antibody also confirmed the
reduced expression of IRS-1 in immobilized hindlimb (Fig.
3A; P < 0.01). Thus the apparent decrease in phosphorylation of IRS-1 may be
due to decreased protein expression and/or to decreased activation of
IR-
, the activation of IR-
being a crucial initial step for
subsequent phosphorylation of IRS-1 (22).
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To assess the relative contributions of the decrease in IRS-1 protein
expression and the decrease in the kinase activation of IR- to the
decrease in phosphorylation of IRS-1, the percent decline in tyrosine
phosphorylation of each protein was normalized to the level of the
recovery of the corresponding protein after immunoprecipitation.
Insulin-stimulated tyrosine phosphorylation of IR-
and IRS-1 was
reduced in immobilized muscle to 54.7% (P < 0.02) and
35.3% (P < 0.02), respectively. Although IR-
protein level did not change significantly in immobilized muscle
(90.0% of contralateral control muscle), IRS-1 protein level decreased to 66.3% (P < 0.02) after immobilization. Thus the
insulin-stimulated percent tyrosine phosphorylation of IR-
and IRS-1
in the tibialis anterior muscle, when normalized to the IR-
and
IRS-1 protein levels, was downregulated to 61.9% (Fig. 1,
P < 0.02) and 53.1% (Fig. 2; P < 0.02), respectively. These results indicate that the apparent reduction
of IRS-1 tyrosine phosphorylation derives from a decline in both
protein expression of IRS-1 and tyrosine phosphorylation of the
remaining IRS-1 protein, the latter probably resulting from impaired
kinase activation of IR.
To assess the consequence of the hypophosphorylation of IRS-1 on the
further downstream signal transduction components, the association of
IRS-1 with PI 3-K and IRS-1-associated PI 3-K activity was examined.
The immunoprecipitates obtained with anti-IRS-1 antibody were subjected
to immunoblotting with anti-PI 3-K p85 antibody. In the contralateral
control hindlimb, insulin stimulation resulted in a marked increase in
the amount of p85 bound to IRS-1. However, insulin-stimulated binding
of p85 to IRS-1 was reduced to 49.8% (P < 0.02) in
immobilized muscle (Fig. 4). In
accordance with the attenuated binding of PI 3-K to IRS-1,
insulin-stimulated PI 3-K activity was impaired in immobilized muscle
compared with the contralateral control (Fig.
5, P < 0.02). This
decrease in binding of IRS-1 to p85 and in PI 3-K activity was not due
to decreased protein expression of p85; the protein expression assessed by simple Western blotting was not different between immobilized and
contralateral sides (Fig. 3B).
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DISCUSSION |
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The present study clearly demonstrates that immobilization per se
attenuated insulin-stimulated tyrosine phosphorylation of IR- and
IRS-1, the binding of IRS-1 to PI 3-K, and IRS-1-associated PI 3-K
activation in skeletal muscle. IRS-1 protein expression was also
downregulated in immobilized muscle (Fig. 2), whereas IR-
and p85
protein levels were not altered (Figs. 1B and
3B). Not only was the total amount of phosphorylated IRS-1
decreased to 35.3%, but also the percent phosphorylation of IRS-1,
normalized to IRS-1 protein level, was reduced to 53.1% in the
immobilized limb relative to contralateral control muscle. This
indicates that reduction in insulin-stimulated IRS-1 phosphorylation in immobilized muscle is attributable to the decreases in both protein expression of IRS-1 (Fig. 2) and insulin-stimulated tyrosine kinase activation of IR (Fig. 1). It is possible that the basal increase in
IR-
phosphorylation on the immobilized side (Fig. 1) may explain the
trend toward increased basal glucose uptake in immobilized muscles in
vivo (33). However, it is not clear whether this is of
biological relevance, because there are no significant increases in
downstream insulin signaling (Figs. 2, 4, and 5). It is important to
note that insulin-independent (basal) glucose uptake is considered to
be independent of the PI 3-K pathway.
The binding of the p85 regulatory subunit of PI 3-K to IRS-1 is a key event for insulin-stimulated PI 3-K activation, and this binding is dependent on tyrosine phosphorylation of IRS-1. A decrease in tyrosine phosphorylation of IRS-1 may thus account for reductions in insulin-stimulated binding of p85 to IRS-1 (Fig. 4) and also IRS-1-associated PI 3-K activation (Fig. 5). A decreased protein expression of p85 was not observed and therefore cannot account for the decreased binding of IRS-1 to PI 3-K and decreased activation of PI 3-K. The finding of unaltered expression of p85 is consistent with previous studies of insulin resistance with burn (17). Altered expression of the p110 catalytic subunit of PI 3-K seems unlikely, as this has not been observed previously in any pathophysiological state with insulin resistance. These data together, therefore, indicate that immobilization attenuates insulin-stimulated intracellular signal transduction by attenuating IR activation and IRS-1 protein expression, and they suggest that impaired activation of IR and its downstream molecules, including IRS-1 phosphorylation and PI 3-K activation, may play important roles in immobilization-induced insulin resistance.
The decreased protein expression of IRS-1, phosphorylation of IRS-1, and activation of PI 3-K after immobilization, therefore, contrast with the decreases seen in exercise (12, 21). Long-term exercise was associated with increases in protein or mRNA levels of IR, IRS-1, and p85 (21), together with increases in insulin-stimulated IRS-1 phosphorylation and PI 3-K activation (7). Increased expression of p110, however, has also not been observed after prolonged exercise. In our study, the differences between the immobilized and contralateral sides cannot be attributed to overuse (exercise) of the contralateral side; previous studies of function and morphology, which included measurements of fiber size, contraction, fade with tetanus, wet weights, and receptor expression, did not show any differences between the contralateral sham-immobilized side and separate naive controls (15, 16). Pain in the immobilized and contralateral sides was probably the same, because all procedures, including boring of holes and excepting maintenance of pins, were similar on both sides.
A decrease in the phosphorylation capability of IR and a reduction in
IRS-1 protein expression have been reported previously in patients with
type 2 diabetes (31) and in genetically obese diabetic
(ob/ob) mice (19). We have also shown that the
insulin resistance after thermal injury was associated with attenuated tyrosine phosphorylation of IR- and IRS-1 (17). A
previous study also revealed that mice with heterozygous knockouts of
both IR and IRS-1 exhibit insulin resistance and diabetes, whereas heterozygous targeting of either one of these genes was associated with
no obvious phenotype of diabetes (5). Taken together, these findings are supportive of the notion that the combination of
functional defects in IR and IRS-1 may be important in the pathogenesis
of insulin resistance. They also suggest that a common molecular
mechanism, involving IRS-1-mediated signaling, may underlie the insulin
resistance of obesity, burn injury, and immobilization. Recent studies
have also suggested that other IRS-1-like docking proteins, including
Gab1 and p62 (Dok), may play an important role in insulin signaling
(2, 22, 42). These molecules, when tyrosine phosphorylated
in response to insulin, bind to PI 3-K. Thus it would be of interest
also to analyze, in future studies, the roles of these signaling
molecules in the altered insulin signaling after immobilization (and exercise).
It is important to reiterate that protein expression of the insulin
receptor, as assessed by immunoblots, was not altered (Fig.
1B). Thus the decreased phosphorylation of IR- seems to be related to an intrinsic mechanism inhibiting IR-
phosphorylation. Numerous factors, including epinephrine and cytokines, particularly tumor necrosis factor (TNF), are potential candidates that may play an
inhibitory role on insulin signaling (30, 34). Stress of
immobilization itself can result in release of catecholamines and
steroidal hormones. Because these mediators are released systemically, the effects would have been evident not only on the immobilized side
but also on the contralateral control side. The differences observed in
our study between the immobilized and contralateral sides, in the same
animals, are therefore not consistent with a systemic effect.
Recently, TNF has been shown to play a central role in insulin
resistance (34). Neutralization of TNF alleviated insulin resistance. Interestingly, this effect was attributed to TNF produced locally in muscle and adipose tissues, because serum concentrations of
TNF in both lean and obese diabetics are low. This suggests that TNF
acts in a paracrine and/or an autocrine manner. TNF can convert IRS-1
to hyperserine-phosphorylated form and render it an inhibitory molecule
to insulin receptor kinase (34). Increased levels of TNF
are also associated with concomitant upregulation of inducible nitric
oxide synthase (iNOS) and insulin resistance. Furthermore, the iNOS
inhibitor aminoguanidine reversed the TNF-induced impaired
insulin-stimulated glucose transport in cultured muscle cells
(3). Evidence for local expression of TNF in muscle after immobilization has not been demonstrated previously. However, in
hindlimb unloading, a form of immobilization, the administration of the
iNOS inhibitor N-nitro-L-arginine
(L-NAME) decreased the inflammatory response associated
with muscle disuse (28), suggesting that iNOS expression may be increased in immobilization. Thus the relationship between local
expression of TNF, iNOS, and insulin resistance after immobilization deserves further study.
Among the effects of insulin in muscle, uptake of glucose and protein synthesis are cardinal. Increased glucose uptake and glycogen synthesis occur through translocation of the insulin-sensitive glucose transporter GLUT-4 and activation of glycogen synthase (34). Protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 4E-binding proteins. Many of the metabolic actions of insulin, except its effect on potassium, have been documented to be mediated by PI 3-K via the activation of its downstream serine/threonine protein kinases, Akt/protein kinase B (PKB), and an atypical isoform of protein kinase C. Hence, wortmannin, a specific inhibitor of PI 3-K, inhibits insulin-induced protein synthesis. Furthermore, PI 3-K and Akt/PKB are pivotal in a pathway that conveys survival signals. Specific inhibition of PI 3-K by wortmannin or LY-294002 enhances apoptosis (6). Thus activation of PI 3-K by insulin and/or insulin-like growth factor I enhances protein synthesis and blocks apoptosis (6, 11).
The overall rate of protein synthesis and degradation in tissue tightly controls muscle mass (8, 25, 27). Insulin is known not only to stimulate protein synthesis but also to inhibit protein degradation in skeletal muscle (18). Many conditions associated with muscle wasting, including immobilization, burns, sepsis, and autoimmune deficiency syndrome (AIDS), are also associated with decreased insulin signaling (insulin resistance), despite normal or elevated plasma insulin levels (29, 33, 36, 37, 40). In these instances, protein catabolism outweighs protein synthesis, regardless of the fact that protein synthesis itself can sometimes be enhanced in these conditions (32, 33, 44). Furthermore, apoptosis is associated with muscle wasting during hindlimb unweighting (1) and also after burn injury (45). As indicated previously, PI 3-K activation is a pivotal antiapoptotic signaling molecule (6, 11, 41). Taken together, the decreased glucose uptake, decreased protein synthesis, increased protein degradation, muscle atrophy, and apoptosis previously observed after muscle disuse or immobilization might be related to decreased insulin action and defective insulin signaling via PI 3-K. Therefore, correction of insulin resistance may retard the immobilization-induced muscle wasting, including apoptosis and the associated muscle weakness.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. K. Yonezawa and K. Hara for the generous gift of anti-IRS-1 antibody and to Drs. J. Avruch and K. Ueki for helpful discussion.
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FOOTNOTES |
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This work was supported by National Institute of General Medical Sciences Grants GM-31569-18, GM-55082-4, and GM-61411-01 to J. A. J. Martyn.
Address for reprint requests and other correspondence: J. A. J. Martyn, Dept. of Anesthesia and Critical Care, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114 (E-mail: jmartyn{at}etherdome.mgh.harvard.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 25 February 2000; accepted in final form 12 July 2000.
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