Immobilization depresses insulin signaling in skeletal muscle

Munetaka Hirose, Masao Kaneki, Hiroki Sugita, Shingo Yasuhara, and J. A. Jeevendra Martyn

Department of Anesthesia and Critical Care, Harvard Medical School, and Anesthesia Services, Massachusetts General Hospital and Shriners Hospital for Children, Boston, Massachusetts 02114


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -subunit (IR-beta ) 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-beta 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-beta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -subunit (IR-beta ). Activated IR-beta 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-beta , 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-beta 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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.

The immunoprecipitates were subjected to SDS-PAGE in 7.5% acrylamide resolving gels and transferred electrophoretically to nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were then blocked in 5% dried milk in PBS containing 0.1% Tween 20 (PBS-Tween) for 2 h at room temperature and immunoblotted with anti-phosphotyrosine (PY99; Santa Cruz Biotechnology), anti-IR-beta , or anti-IRS-1 antibody for 1 h at room temperature. After three washes with PBS-Tween, the membrane was incubated for 30 min with anti-mouse or -rabbit IgG antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology). The antigen-antibody complexes were visualized using chemiluminescence luminol reagent (Santa Cruz Biotechnology). Bands of interest were scanned by use of MD-4000 (Alps Electric, San Jose, CA) and were quantified with National Institutes of Health Image 1.61 software (NTIS, Springfield, VA).

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 [gamma -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-beta and IRS-1, the protein expression of IR-beta , 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta or IRS-1, muscle homogenates from immobilized and contralateral limbs were immunoprecipitated with anti-IR-beta or anti-IRS-1 antibodies followed by immunoblotting with anti-phosphotyrosine antibody. The basal level of tyrosine phosphorylation of IR-beta was higher in immobilized muscle than in control muscle (Fig. 1A; P < 0.05). By contrast, insulin-stimulated tyrosine phosphorylation of IR-beta 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-beta after insulin treatment in the immobilized muscle was not due to decreased IR-beta protein expression but resulted from the impaired activation of IR.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Levels of tyrosine phosphorylation of insulin receptor (IR) beta -subunit and IR-beta protein in the immobilized (IMB) and contralateral (Control) hindlimbs. Equal amounts of muscle protein were immunoprecipitated (IP) and immunoblotted (IB) with anti-phosphotyrosine (PY; A) or anti-IR-beta antibody (IR; B). Solid and open bars on lower panels, basal and insulin-stimulated levels, respectively. Whereas basal tyrosine phosphorylation of IR-beta increased in IMB limbs (A), insulin-stimulated tyrosine phosphorylation of IR-beta was attenuated in IMB compared with control limbs (expressed as percent phosphorylation normalized to IR-beta protein levels). IR-beta protein content, however, was not altered after immobilization (B). Results displayed on top panels represent typical immunoblots. *P < 0.05, **P < 0.02 vs. Control, n = 4 for each group.

Consistent with the attenuation of phosphorylation of IR-beta , 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-beta , 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-beta , the activation of IR-beta being a crucial initial step for subsequent phosphorylation of IRS-1 (22).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Levels of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1, A) and IRS-1 protein (B) in the immobilized (IMB) and contralateral (Control) hindlimbs. Equal amounts of protein were subjected to immunoprecipitation with anti IRS-1 antibody (IP) and immunoblotting (IB) with anti-phosphotyrosine (PY) or anti-IRS-1 antibody (IRS). Solid and open bars on lower panel, basal and insulin-stimulated levels, respectively. Immobilization was associated with reductions in both insulin-stimulated tyrosine phosphorylation of IRS-1, expressed as percent phosphorylation normalized to IRS-1 protein levels (A), and IRS-1 protein expression (B). Results displayed on top panels represent typical immunoblots. **P < 0.02 vs. Control, n = 4 for each group.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   Protein expression of IRS-1 (A) and p85 of phosphatidylinositol 3-kinase (PI 3-K) after immobilization. Equal amounts of protein, separated by electrophoresis and subjected to Western blot analysis (IB), revealed reduced IRS-1 but not p85 after immobilization. Results displayed on top panels represent typical immunoblots. ***P < 0.01 vs. control, n = 4 for each group.

To assess the relative contributions of the decrease in IRS-1 protein expression and the decrease in the kinase activation of IR-beta 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-beta and IRS-1 was reduced in immobilized muscle to 54.7% (P < 0.02) and 35.3% (P < 0.02), respectively. Although IR-beta 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-beta and IRS-1 in the tibialis anterior muscle, when normalized to the IR-beta 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).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Insulin-stimulated association of IRS-1 with PI 3-K after immobilization. Equal amounts of protein, immunoprecipitated with IRS-1 antibody, were immunoblotted (IB) with anti-p85. Solid and open bars on lower panel, basal and insulin-stimulated association of p85 with IRS-1, respectively. Immobilization reduced the association of p85 with IRS-1. Results displayed on top panels represent a typical immunoblot. **P < 0.02 vs. Control, n = 4 for each group.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 5.   Insulin-stimulated IRS-1-associated PI 3-K activation after immobilization. Equal amounts of protein were immunoprecipitated with IRS-1 antibody, and PI 3-K activity was assessed by phosphorylation of phosphatidylinositol to phosphatidylinositol monophosphate (PIP). Solid and open bars on lower panel represent basal and insulin-stimulated IRS-associated PI 3-K activity, respectively. Immobilization caused a reduction in insulin-stimulated activation of PI 3-K. Results displayed on top panel represent a typical experiment. **P < 0.02 vs. Control, n = 4 for each group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study clearly demonstrates that immobilization per se attenuated insulin-stimulated tyrosine phosphorylation of IR-beta 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-beta 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-beta 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-beta 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-beta seems to be related to an intrinsic mechanism inhibiting IR-beta 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 Nomega -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, DL, Linderman JK, Roy RR, Bigbee AJ, Grindeland RE, Mukku V, and Edgerton VR. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am J Physiol Cell Physiol 273: C579-C587, 1997[Abstract/Free Full Text].

2.   Araki, E, Lipes MA, Patti ME, Bruning JC, Haag BR, Johnson RS, and Kahn CR. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372: 186-190, 1994[ISI][Medline].

3.   Bedard, S, Marcotte B, and Marette A. Cytokines modulate glucose transport in skeletal muscle by inducing expression of inducible nitric oxide synthase. Biochem J 325: 487-493, 1997[ISI][Medline].

4.   Booth, FW. Effect of limb immobilization on skeletal muscle. J Appl Physiol 52: 1113-1118, 1982[Abstract/Free Full Text].

5.   Bruning, JC, Winnay J, Bonner-Weir S, Taylor SI, Accili D, and Kahn CR. Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Cell 88: 561-572, 1997[ISI][Medline].

6.   Butt, AJ, Firth SM, and Baxter RC. The IGF axis and programmed cell death. Immunol Cell Biol 77: 256-262, 1999[ISI][Medline].

7.   Chibalin, AV, Yu M, Ryder JW, Song XM, Galuska D., Krook A, Wallberg-Henriksson H, and Zierath JR. Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: differential effects on insulin-receptor substrates 1 and 2. Proc Natl Acad Sci USA 97: 38-43, 2000[Abstract/Free Full Text].

8.   Dardevet, D, Sornet C, Vary T, and Grizard J. Phosphatidylinositol 3-kinase and p70 s6 kinase participate in the regulation of protein turnover in skeletal muscle by insulin and insulin-like growth factor-1. Endocrinology 137: 4087-4094, 1996[Abstract].

9.   Ferrando, AA, Lane HW, Stuart CA, Davis-Street J, and Wolfe RR. Prolonged bed rest decreases skeletal muscle and whole body protein synthesis. Am J Physiol Endocrinol Metab 270: E627-E633, 1996[Abstract/Free Full Text].

10.   Folli, F, Saad MJ, Backer JM, and Kahn CR. Insulin stimulation of phosphatidylinositol 3-kinase activity and association with insulin receptor substrate 1 in liver and muscle of the intact rat. J Biol Chem 267: 22171-22177, 1992[Abstract/Free Full Text].

11.   Franke, TF, Kaplan DR, and Cantley LC. PI3K: downstream AKTion blocks apoptosis. Cell 88: 435-437, 1997[ISI][Medline].

12.   Hayashi, T, Wojtaszewski JF, and Goodyear LJ. Exercise regulation of glucose transport in skeletal muscle. Am J Physiol Endocrinol Metab 273: E1039-E1051, 1997[ISI][Medline].

13.   Higaki, Y, Wojtaszewski JF, Hirshman MF, Withers DJ, Towery H, White MF, and Goodyear LJ. Insulin receptor substrate-2 is not necessary for insulin- and exercise-stimulated glucose transport in skeletal muscle. J Biol Chem 274: 20791-20795, 1999[Abstract/Free Full Text].

14.   Houmard, JA, Shaw CD, Hickey MSMS, and Tanner CJ. Effect of short-term exercise training on insulin-stimulated PI 3-kinase activity in human skeletal muscle. Am J Physiol Endocrinol Metab 277: E1055-E1060, 1999[Abstract/Free Full Text].

15.   Ibebunjo, C, and Martyn JAJ Fiber atrophy, but not changes in acetylcholine receptor expression, contributes to the muscle dysfunction after immobilization. Crit Care Med 27: 275-285, 1999[ISI][Medline].

16.   Ibebunjo, C, Nosek MT, Itani MS, and Martyn JAJ Mechanisms for the paradoxical resistance to D-tubocurarine during immobilization-induced muscle atrophy. J Pharmacol Exp Ther 283: 443-451, 1997[Abstract/Free Full Text].

17.   Ikezu, T, Okamoto T, Yonezawa K, Tompkins RG, and Martyn JAJ Analysis of thermal injury-induced insulin resistance in rodents. Implication of postreceptor mechanisms. J Biol Chem 272: 25289-25295, 1997[Abstract/Free Full Text].

18.   Jefferson, LS, Li JB, and Rannels SR. Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. J Biol Chem 252: 1476-1483, 1977[Abstract].

19.   Kerouz, NJ, Horsch D, Pons S, and Kahn CR. Differential regulation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) and phosphatidylinositol 3-kinase isoforms in liver and muscle of the obese diabetic (ob/ob) mouse. J Clin Invest 100: 3164-3172, 1997[Abstract/Free Full Text].

20.   Kido, Y, Burks DJ, Withers D, Bruning JC, Kahn CR, White MF, and Accili D. Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2. J Clin Invest 105: 199-205, 2000[Abstract/Free Full Text].

21.   Kim, Y, Inoue T, Nakajima R, Nakae K, Tamura T, Tokuyama K, and Suzuki M. Effects of endurance training on gene expression of insulin signal transduction pathway. Biochem Biophys Res Commun 210: 766-773, 1995[ISI][Medline].

22.   Lamothe, B, Baudry A, Desbois P, Lamotte L, Bucchini D, De Meyts P, and Joshi RL. Genetic engineering in mice: impact on insulin signalling and action. Biochem J 335: 193-204, 1998[ISI][Medline].

23.   Lavan, BE, Fantin VR, Chang ET, Lane WS, Keller SR, and Lienhard GE. A novel 160-kDa phosphotyrosine protein in insulin-treated embryonic kidney cells is a new member of the insulin receptor substrate family. J Biol Chem 272: 21403-21407, 1997[Abstract/Free Full Text].

24.   Lavan, BE, Lane WS, and Lienhard GE. The 60-kDa phosphotyrosine protein in insulin-treated adipocytes is a new member of the insulin receptor substrate family. J Biol Chem 272: 11439-11443, 1997[Abstract/Free Full Text].

25.   Mitch, WE, and Goldberg AL. Mechanisms of muscle wasting. The role of the ubiquitin-proteasome pathway. N Engl J Med 335: 1897-1905, 1996[Free Full Text].

26.   Mizock, BA. Alterations in carbohydrate metabolism during stress: a review of the literature. Am J Med 98: 75-84, 1995[ISI][Medline].

27.   Palmer, RM, Thompson MG, Knott RM, Canbell CP, Thom A, and Morrison KS. Insulin and insulin-like growth factor-1 responsiveness and signaling mechanisms in C2C12 satellite cells: effect of differentiation and fusion. Biochim Biophys Acta 1355: 167-176, 1997[ISI][Medline].

28.   Pizza, FX, Hernandez IJ, and Tidball JG. Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use. J Leukoc Biol 64: 427-433, 1998[Abstract].

29.   Ploug, T, Ohkuwa T, Handberg A, Vissing J, and Galbo H. Effect of immobilization on glucose transport and glucose transporter expression in rat skeletal muscle. Am J Physiol Endocrinol Metab 268: E980-E986, 1995[Abstract/Free Full Text].

30.   Reavean, GM, Lithell H, and Landsberg L. Hypertension and associated metabolic abnormalities: the role of insulin resistance and the sympathadrenal system. N Engl J Med 334: 374-384, 1996[Free Full Text].

31.   Rondinone, CM, Wang LM, Lonnroth P, Wesslau C, Pierce JH, and Smith U. Insulin receptor substrate (IRS) 1 is reduced and IRS-2 is the main docking protein for phosphatidylinositol 3-kinase in adipocytes from subjects with non-insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 94: 4171-4175, 1997[Abstract/Free Full Text].

32.   Shangraw, RE, Stuart CA, Prince MJ, Peters EJ, and Wolfe RR. Insulin responsiveness of protein metabolism in vivo following bedrest in humans. Am J Physiol Endocrinol Metab 255: E548-E558, 1988[Abstract/Free Full Text].

33.   Shangraw, RE, and Turinsky J. Effect of disuse and thermal injury on protein turnover in skeletal muscle. J Surg Res 33: 345-355, 1982[ISI][Medline].

34.   Shephard, PR, and Kahn BB. Glucose transporters and insulin action. N Engl J Med 341: 248-257, 1999[Free Full Text].

35.   Stein, TP, Schulter MD, and Boden G. Development of insulin resistance by astronauts during spaceflight. Aviat Space Environ Med 65: 1091-1096, 1994[ISI][Medline].

36.   Strommer, L, Permert J, Arnelo U, Koehler C, Isaksson B, Larsson J, Lundkvist I, Bjornholm M, Kawano Y, Wallberg-Henriksson H, and Zierath JR. Skeletal muscle insulin resistance after trauma: insulin signaling and glucose transport. Am J Physiol Endocrinol Metab 275: E351-E358, 1998[Abstract].

37.   Stuart, CA, Shangraw RE, Prince MJ, Peters EJ, and Wolfe RR. Bed-rest-induced insulin resistance occurs primarily in muscle. Metabolism 37: 802-806, 1988[ISI][Medline].

38.   Tawa, NE, Jr, Odessey R, and Goldberg AL. Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles. J Clin Invest 100: 197-203, 1997[Abstract/Free Full Text].

39.   Terauchi, Y, Tsuji Y, Satoh S, Minoura H, Murakami K, Okuno A, Inukai K, Asano T, Kaburagi Y, Ueki K, Nakajima H, Hanafusa T, Matsuzawa Y, Sekihara H, Yin Y, Barrett JC, Oda H, Ishikawa T, Akanuma Y, Komuro I, Suzuki M, Yamamura K, Kodama T, Suzuki H, Koyasu S, Aizawa S, Tobe K, Fukui Y, Yazaki Y, and Kadowaki T. Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase. Nat Genet 21: 230-235, 1999[ISI][Medline].

40.   Thorell, A, Nygren J, and Ljungqvist O. Insulin resistance: a marker of surgical stress. Curr Opin Clin Nutr Metab Care 2: 69-78, 1999[Medline].

41.   Ueki, K, Yamamoto-Honda R, Kaburagi Y, Yamauchi T, Tobe K, Burgering BM, Coffer PJ, Komuro I, Akanuma Y, Yazaki Y, and Kadowaki T. Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem 273: 5315-5322, 1998[Abstract/Free Full Text].

42.   White, MF. The IRS-signaling system: a network of docking proteins that mediate insulin action and cytokine action. Recent Prog Horm Res 53: 119-133, 1998[Medline].

43.   Wojtaszewski, JF, Hansen BF, Kiens B, and Richter EA. Insulin signaling in human skeletal muscle: time course and effect of exercise. Diabetes 46: 1775-1781, 1997[Abstract].

44.   Wolfe, RR. Substrate utilization/insulin resistance in sepsis/trauma. Baillieres Clin Endocrinol Metab 11: 645-657, 1997[ISI][Medline].

45.   Yasuhara, S, Kanakubo E, Perez ME, Kaneki M, Fujita T, Okamoto T, and Martyn JAJ The 1999 Moyer award. Burn injury induces skeletal muscle apoptosis and the activation of caspase pathways in rats. J Burn Care Rehabil 20: 462-470, 1999[ISI][Medline].

46.   Yonezawa, K, Ando AY, Kaburagi R, Yamamoto-Honda T, Kitamura T, Hara K, Nakafuku M, Okabayashi Y, Kadowaki T, Kaziro Y, and Kasuga M. Signal transduction pathways from insulin receptors to Ras. Analysis by mutant insulin receptors. J Biol Chem 269: 4634-4640, 1994[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 279(6):E1235-E1241
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society