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*Burns
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*LITHIUM CHLORIDE
*LITHIUM COMPOUNDS
Endocrinology Vol. 146, No. 7 3141-3149
Copyright © 2005 by The Endocrine Society

Protein Breakdown in Muscle from Burned Rats Is Blocked by Insulin-Like Growth Factor I and Glycogen Synthase Kinase-3ß Inhibitors

Cheng-Hui Fang, Bing-Guo Li, J. Howard James, Jy-Kung King, Amy R. Evenson, Glenn D. Warden and Per-Olof Hasselgren

Shriners Hospital for Children (C.-H.F., B.-G.L., J.H.J., J.-K.K., G.D.W.), Cincinnati, Ohio 45229; Department of Surgery, University of Cincinnati (J.H.J., J.-K.K., G.D.W.), Cincinnati, Ohio 45229; and Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School (A.R.E., P.-O.H.), Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Dr. Cheng-Hui Fang, Shriners Hospital for Children, 3229 Burnet Avenue, Cincinnati, Ohio 45229. E-mail: cfang{at}shrinenet.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We reported previously that IGF-I inhibits burn-induced muscle proteolysis. Recent studies suggest that activation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway with downstream phosphorylation of Forkhead box O transcription factors is an important mechanism of IGF-I-induced anabolic effects in skeletal muscle. The potential roles of other mechanisms in the anabolic effects of IGF-I are less well understood. In this study we tested the roles of mammalian target of rapamycin and glycogen synthase kinase-3ß (GSK-3ß) phosphorylation as well as MAPK- and calcineurin-dependent signaling pathways in the anticatabolic effects of IGF-I by incubating extensor digitorum longus muscles from burned rats in the presence of IGF-I and specific signaling pathway inhibitors. Surprisingly, the PI3K inhibitors LY294002 and wortmannin reduced basal protein breakdown. No additional inhibition by IGF-I was noticed in the presence of LY294002 or wortmannin. Inhibition of proteolysis by IGF-I was associated with phosphorylation (inactivation) of GSK-3ß. In addition, the GSK-3ß inhibitors, lithium chloride and thiadiazolidinone-8, reduced protein breakdown in a similar fashion as IGF-I. Lithium chloride, but not thiadiazolidinone-8, increased the levels of phosphorylated Foxo 1 in incubated muscles from burned rats. Inhibitors of mammalian target of rapamycin, MAPK, and calcineurin did not prevent the IGF-I-induced inhibition of muscle proteolysis. Our results suggest that IGF-I inhibits protein breakdown at least in part through a PI3K/Akt/GSK3ß-dependent mechanism. Additional experiments showed that similar mechanisms were responsible for the effect of IGF-I in muscle from nonburned rats. Taken together with recent reports in the literature, the present results suggest that IGF-I inhibits protein breakdown in skeletal muscle by multiple mechanisms, including PI3K/Akt-mediated inactivation of GSK-3ß and Foxo transcription factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BURN INJURY IS associated with a pronounced catabolic response in skeletal muscle, mainly reflecting accelerated ubiquitin-proteasome-dependent breakdown of myofibrillar proteins (1, 2, 3). Muscle wasting in burn patients has significant clinical consequences (4), including delayed ambulation with increased risk for thromboembolic complications and need for prolonged ventilatory support and care in the intensive care unit when respiratory muscles are affected. In addition, skeletal muscle is the major source of whole body protein loss after thermal injury. Therefore, treatments that reduce burn-induced muscle wasting can have important clinical implications (5, 6).

In recent studies, we found that IGF-I inhibited burn-induced muscle protein degradation, both in vitro, when exposing incubated muscles from burned rats to the hormone (7, 8), and in vivo, when treating burned rats with IGF-I (9). Anticatabolic effects of IGF-I in patients with thermal injury have been reported as well (5, 10, 11). Although the anticatabolic effects of IGF-I in skeletal muscle after burn are well established, the mechanisms by which the hormone exerts these effects on burn-induced muscle catabolism are not fully understood. Results from other studies suggest that activation of phosphotidylinositol 3-kinase (PI3K)/Akt signaling is essential for the anabolic effects of IGF-I in skeletal muscle (12). Activation of PI3K/Akt results in downstream phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), mammalian target of rapamycin (mTOR), and Forkhead box O (Foxo) transcription factors. Recent studies suggest that among these mechanisms, phosphorylation (inactivation) of Foxo transcription factors is particularly important for the anabolic effects of IGF-I in skeletal muscle (13, 14). In contrast, the roles of GSK-3ß and mTOR in IGF-I-induced inhibition of protein breakdown in skeletal muscle are not well understood, but the fact that inhibition of Foxo transcription factors plays an important role does not rule out the possibility that other mechanisms are involved as well.

In the present study we tested the involvement of GSK-3ß and mTOR in IGF-I-induced inhibition of protein breakdown in muscles from burned rats. Because other studies suggest that IGF-I may exert some of its metabolic effects through MAPK- and calcineurin-dependent cell signaling (15, 16), these mechanisms were also examined. Our results suggest that activation of PI3K/Akt with downstream phosphorylation (inactivation) of GSK-3ß at least in part regulates the IGF-I-induced inhibition of protein breakdown in muscles from burned rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
LY294002, wortmannin, rapamycin, thiadiazolidinone-8 (TDZD-8), cyclosporin A (CsA), PD98059, SB203580, and SB202190 were purchased from Calbiochem (San Diego, CA). Lithium chloride (LiCl) was obtained from Sigma-Aldrich Corp. (St. Louis, MO). Western blotting reagents were purchased from Bio-Rad Laboratories (Hercules, CA). IGF-I was a gift from Genentech (South San Francisco, CA). Antibodies (rabbit polyclonal) were purchased from Cell Signaling Technology (Beverly, MA): against mouse Akt and phospho-(Ser473)-Akt, against human p70S6K and phospho-(Thr389)-p70S6K, against human GSK-3ß and phospho-(Ser9)-GSK-3ß, against rat p44/42 MAPK and human phospho-(Thr202/Tyr204)-p44/42 MAPK, against human p38 MAPK and phospho-(Thr180/Tyr182)-p38 MAPK, and against human Foxo 1 and phospho-(Ser256)-Foxo 1 and 4. Horseradish peroxidase-conjugated antirabbit IgG was also obtained from Cell Signaling Technology.

Experimental animals
A 30% total body surface area full-thickness burn injury was inflicted on the backs of male Sprague Dawley rats, weighing 50–60 g, as described in detail previously (3, 7, 8, 9). Other rats underwent sham procedure, i.e. general anesthesia was induced, and the back was shaved, but no burn injury was inflicted. The rats had free access to drinking water, and the sham-burned rats were pair-fed with the burned rats. The animals were cared for in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the institutional animal care and use committee at University of Cincinnati.

Muscle incubations
Twenty-four hours after burn injury, extensor digitourum longus muscles were harvested from rats under pentobarbital anesthesia and incubated for 2 h for determination of protein breakdown rates as described previously (3, 7, 8, 9). Protein breakdown rates were determined by measuring the net release of tyrosine in the absence or presence of IGF-I (1 µg/ml). This concentration of IGF-I caused maximal inhibition of protein degradation in vitro in muscles from burned rats in previous experiments (7). Although it may be argued that 1 µg/ml is a high, unphysiological concentration of IGF-I, it should be noted that in the present study, mechanisms involved in the effects of IGF-I treatment were examined rather than mechanisms involved in the effects of endogenous IGF-I. Similar, and even higher, concentrations of IGF-I have been used for studies in incubated rat muscles by other researchers as well (17). One concern when relatively high concentrations of IGF-I are used is whether the effects are secondary to binding to the insulin receptor, rather than to the IGF-I receptor. We therefore performed a control experiment in which the increase in protein synthesis in incubated muscles from burned rats caused by 1 µg/ml IGF-I was blocked by IGF-I receptor antibody (control, 85 ± 4; IGF-I, 136 ± 13; IGF-I plus IGF-I receptor antibody, 83 ± 4 nmol phenylalanine/g·2 h), whereas the insulin-induced increase in protein synthesis was not affected by this antibody (control, 98 ± 6; 1 mU/ml insulin, 155 ± 12; insulin plus IGF-I receptor antibody, 174 ± 15 nmol phenylalanine/g·2 h). The results suggest that the effects of 1 µg/ml IGF-I in incubated rat muscles is mainly caused by IGF-I receptor binding.

When the roles of different signaling pathways in IGF-I-induced inhibition of protein degradation were tested, muscles were incubated in the presence of different inhibitors of the signaling pathways, as outlined in Results. To allow time for the uptake of the various inhibitors, muscles were exposed to inhibitors for 15 min before addition of IGF-I. When the effects of LiCl were tested, equimolar concentrations of NaCl were added to control muscles, as indicated in the figure legends.

Muscle ATP levels
ATP concentrations were determined in muscles incubated for 2 h in the absence or presence of LY294002. After the 2-h incubation, muscles were immediately frozen in liquid nitrogen and stored at –80 C until analysis. The frozen muscles were pulverized in plastic tubes that had been cooled in liquid nitrogen, and after homogenization, ATP was measured as ethenopurine derivatives (18) by a modification of the HPLC method described by Kawamoto et al. (19). Elution buffer was maintained at a flow rate of 1 ml/min through a 3.9 x 150-mm NovaPak C18 column (Waters Corp., Milford, MA). Pure solvent A (100 mM KH2PO4 and 5 mM tetrabutylammonium bromide with 2% acetonitrile, pH 3.3) was delivered to the column for the first 2.9 min after injection, then a mixture of 90% A/10% B (50% acetonitrile in water) was delivered from 2.9–13 min. Ethenopurine derivatives were prepared in autosampler vials using 15 µl neutralized supernatant to which were added 435 µl citrate-phosphate buffer (620 ml 0.1 M citric acid plus 380 ml 0.2 N Na2HPO4, pH 4.0), followed by 40 µl 1.4 M chloroacetyl-dehyde. The capped samples were heated to 80 C for 40 min, and the reaction was stopped by cooling the vials on ice.

Western blot analysis
Muscles were homogenized in ice-cold lysis buffer (10 µl/mg muscle weight) containing 9.1 mM Na2HPO4, 1.7 mM NaH2PO4 (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.5 mM phenylmethylsulfonylfluoride, 50 µg/ml aprotinin, and 1 mM sodium orthovanadate. After homogenization, the samples were centrifuged at 10,000 x g for 10 min at 4 C. Protein concentrations in the supernatants were determined according to the method described by Bradford (20) using the Bio-Rad protein assay. Muscle extracts containing 50 µg protein were boiled in an equal volume of Laemmli sample buffer with 5% 2-mercaptoethanol. Proteins were separated by electrophoresis on a 4–20% gradient gel (Bio-Rad Laboratories, Richmond, CA) and transferred to nitrocellulose membranes (Immobilon P., Millipore Corp., Bedford, MA) in a transfer buffer consisting of 25 mM Tris-HCl, 192 mM glycine, and 20% methanol. The membranes were blocked for 60 min with 5% nonfat dried milk in Tris-buffered saline containing 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween 20 (TBST) and incubated overnight in TBST at 4 C with the following rabbit polyclonal antibodies as primary antibodies: anti-Akt and anti phospho-Akt, anti-ERK1/2 (p44/42) and antiphospho-ERK1/2, anti-p38 and antiphospho-p38, anti-p70S6K and antiphospho-p70S6K, anti-GSK-3ß and antiphospho-GSK-3ß, and anti-Foxo1 and antiphospho-Foxo 1 and 4. After washing in TBST, the blots were incubated with horseradish peroxidase-conjugated antirabbit IgG as secondary antibody for 60 min at room temperature. After three rinses in TBST, immunoreactive bands were detected using the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) and exposed on radiographic film (Eastman Kodak Co., Rochester, NY).

Real-time PCR
Atrogin-1 mRNA levels were determined by real-time PCR as described in detail recently (21). The extracted RNA was treated with deoxyribonuclease (DNA-free kit, Ambion, Inc., Austin, TX), and a PCR was also performed on total RNA that had not been reverse transcribed to control for the absence of genomic DNA in the RNA preparation. The sequences of the forward, reverse, and double-labeled oligonucleotides for atrogin-1 were as follows, respectively: 5'-CTT TCA ACA GAC TGG ACT TCT CGA-3', 5'-CAG CTC CAA CAG CCT TAC TAC GT-3', and 5'-TGC CAT CCT GGA TTC CAG AAG ATT CAA C-3'. Amplification of 18S RNA was performed in the same reaction tubes as an internal standard with an alternatively labeled probe (VIC-labeled probe) to distinguish its product from that derived from atrogin-1 RNA. Atrogin-1 mRNA concentrations were normalized to the 18S mRNA levels. Experiments were performed in triplicate for each standard and muscle sample.

Statistics
Results are presented as the mean ± SEM. ANOVA, followed by Tukey’s test, were used for statistical analysis; P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We examined the influence of IGF-I on activation of PI3K signaling by determining tissue levels of phosphorylated Akt (p-Akt). When muscles from burned rats were incubated in the presence of IGF-I (1 µg/ml), the levels of p-Akt increased (Fig. 1Go, A and B). To test whether PI3K activation was needed for the anticatabolic effect of IGF-I, muscles were treated with the PI3K inhibitors LY294002 and wortmannin. As expected, the IGF-I-induced increase in p-Akt levels was blocked by LY294002 and wortmannin, with a maximal inhibition noted at 50–100 µM LY294002 and 0.5 µM wortmannin (Fig. 1Go, A and B).



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  		FIG. 1. Effects of IGF-I and LY294002 (A) or wortmannin (B) on total Akt and p-Akt levels in muscles from burned rats, incubated for 2 h in the absence or presence of 1 µg/ml IGF-I with the indicated concentrations of LY294002 or wortmannin. Total Akt and p-Akt levels were determined by Western blotting. The blots are representative of four repeated blots. The effects of LY294002 (C) and wortmannin (D) on IGF-I-induced inhibition of protein degradation in muscles from burned rats, incubated for 2 h in the absence or presence of 1 µg/ml IGF-I with 100 µM LY294002 or 0.5 µM wortmannin. Results are the mean ± SEM (n ≥ 6 for each group). *, P < 0.05 vs. control (no IGF-I).

 
When muscles were incubated with IGF-I, protein breakdown was reduced by approximately 30% (Fig. 1Go, C and D). LY294002 and wortmannin inhibited basal protein breakdown rates (Fig. 1Go, C and D). This effect of the PI3K inhibitors was not accompanied by increased phosphorylation of Akt (Fig. 1Go, A and B) or GSK-3ß (Fig. 2AGo), suggesting that a different mechanism is involved in the inhibition of protein degradation by LY294002 and wortmannin than that by IGF-I. Of importance for the present study, no additional inhibition of proteolysis was noticed when IGF-I was added to muscles incubated in the presence of LY294002 or wortmannin, suggesting that the mechanism by which IGF-I inhibits protein degradation was blocked by the PI3K/Akt inhibitors.



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  		FIG. 2. A, The effects of IGF-I and LY294002 on total GSK-3ß and p-GSK-3ß levels in muscles from burned rats, incubated for 2 h in the absence or presence of 1 µg/ml IGF-I with the indicated concentrations of LY294002. Total GSK-3ß and p-GSK-3ß levels were determined by Western blotting. The blots are representative of four repeated blots. B, The effects of IGF-I and LiCl on protein degradation in muscles from burned rats, incubated for 2 h in the absence or presence of 1 µg/ml IGF-I or various concentrations of LiCl. NaCl (50 mM) was added to the incubation medium of control and IGF-I-treated muscles. C, The effects of IGF-I and TDZD-8 on protein degradation in muscles from burned rats, incubated for 2 h in the absence or presence of 1 µg/ml IGF-I or various concentrations of TDZD-8. For both B and C, results are the mean ± SEM (n = 6 for each group). a, P < 0.05 vs. control; b, P < 0.05 vs. IGF-I.

 
The inhibition of protein breakdown by LY294002 and wortmannin was surprising and seemingly contradicts previous reports demonstrating that PI3K is a negative regulator of muscle protein breakdown (12). It should be noted, however, that inhibition of muscle protein breakdown by wortmannin was observed in another study as well (22). To examine whether the inhibition by LY294002 and wortmannin was the consequences of cell injury and reduced energy levels caused by the inhibitors, ATP concentrations were determined in paired muscles incubated for 2 h in the presence of 100 µM LY294002. ATP levels were not influenced by LY294002 in these experiments, but were 6.77 ± 0.62 and 6.84 ± 0.52 µmol/g (mean ± SEM; n = 9 paired muscles) after incubation in the absence and presence of LY294002, respectively (not significant). The ATP levels reported here are in line with previous reports of ATP levels in rat muscles (23). Thus, it is not likely that the reduced protein breakdown observed in muscles incubated in the presence of LY294002 reflected reduced tissue energy levels.

GSK-3ß provides a signaling pathway downstream of Akt (24). In this study we found (Fig. 2AGo) that treatment of muscles from burned rats with IGF-I resulted in increased tissue levels of phosphorylated (inactivated) GSK-3ß. As expected, LY294002 inhibited the IGF-I-induced phosphorylation of GSK-3ß in a dose-dependent fashion. To also test the relationship between GSK-3ß inhibition and reduced protein degradation, muscles were treated with LiCl, an inhibitor of GSK-3ß (24). Treatment of muscles from burned rats with LiCl (5–50 mM) in the absence of IGF-I resulted in a dose-dependent inhibition of protein degradation, and the effect of LiCl was similar to that of IGF-I (Fig. 2BGo).

We also examined the role of GSK-3ß by using a new, non-ATP-competitive, highly specific GSK-3ß inhibitor, TDZD-8 (25). Treatment of incubated muscles with TDZD-8 (6.25–50 µM) in the absence of IGF-I resulted in a dose-dependent inhibition of protein breakdown (Fig. 2CGo). TDZD-8 at 50 µM reduced protein breakdown to 35% of the rate in untreated, control muscles, significantly below that caused by IGF-I alone (Fig. 2CGo).

In a recent study (25), the IC50 for the inhibitory effect of TDZD-8 on GSK-3ß activity was 2 µM. It should be noted, however, that TDZD-8 was tested in a cell-free system in that study. In the present experiments, the concentrations of TDZD-8 needed to inhibit protein degradation were relatively high; the need for higher concentrations of TDZD-8 to inhibit protein breakdown probably reflects the fact that the drug was used to treat incubated, intact muscles rather than a cell-free system. It should be noted that TDZD-8 at high concentrations may influence other kinases as well (in addition to GSK-3ß), but the IC50 for those kinases was greater than 100 µM in a cell-free system (25) and may be even higher when used in incubated intact muscles. Thus, it is reasonable to assume that the effects of TDZD-8 noted here (Fig. 2CGo), at least at concentrations up to 25 µM, reflected inhibition of GSK-3ß. There is a possibility that the pronounced reduction of protein degradation caused by 50 µM TDZD-8 (to levels below those seen in IGF-I-treated muscles) may reflect nonspecific effects of TDZD-8.

Considering recent reports suggesting that phosphorylation (inactivation) of Foxo transcription factors is an essential mechanism of IGF-I-induced anabolic effects in skeletal muscle (13, 14), we next examined the effects of LiCl and TDZD-8 on Foxo phosphorylation. When muscles from burned rats were incubated in the presence of 25 µM TDZD-8, tissue levels of phosphorylated Foxo 1 and 4 were unchanged (Fig. 3AGo). Unchanged levels of phosphorylated Foxo 1 and 4 do not necessarily rule out the possibility that TDZD-8 reduced Foxo activity independently of phosphorylation, although, to our knowledge, such a mechanism of Foxo inactivation has not been reported. Incubation of muscles in the presence of LiCl resulted in increased levels of phosphorylated Foxo 1, probably reflecting the fact that LiCl can activate PI3K/Akt (26) in addition to having a direct inhibitory effect on GSK-3ß (24).



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  		FIG. 3. The effects of LiCl and TDZD-8 on phosphorylated Foxo 1 and 4 and total Foxo 1 levels (A) and atrogin-1 mRNA levels (B) in incubated extensor digitourum longus muscles from burned rats. Muscles were incubated for 2 h in the absence or presence of 25 µM TDZD-8 or 25 mM LiCl. Foxo levels were determined by Western blotting, and atrogin-1 mRNA levels were measured by real-time PCR.

 
To also test the effects of TDZD-8 and LiCl on catabolic factors in muscle from burned rats, atrogin-1 mRNA levels were determined. Atrogin-1 is a ubiquitin ligase that has been found in recent studies to be substantially up-regulated in various conditions characterized by muscle wasting (27, 28). When muscles from burned rats were treated with TDZD-8 or LiCl, atrogin-1 mRNA levels were not significantly altered (Fig. 3BGo), suggesting either that the time of treatment (2 h) was too short to induce significant changes in mRNA levels or that LiCl and TDZD-8 reduce protein degradation independently of atrogin-1 mRNA expression. Unchanged atrogin-1 mRNA expression, of course, does not preclude changes in atrogin-1 activity.

In addition to phosphorylating GSK-3ß, activated Akt phosphorylates (activates) mTOR, which, in turn, phosphorylates and activates p70S6K (29). Treatment of incubated muscles from burned rats with IGF-I resulted in increased levels of phosphorylated p70S6K (p-p70S6K; Fig. 4AGo), suggesting that IGF-I activated the mTOR/p70S6K signaling pathway. The mTOR inhibitor rapamycin inhibited the IGF-I-induced increase in p-p70S6K, with a maximal effect seen at a rapamycin concentration of 400 nM (Fig. 4AGo). The same concentration of rapamycin did not prevent the IGF-I-induced decrease in protein breakdown (Fig. 4BGo), suggesting that the inhibition of protein degradation by IGF-I was not regulated by mTOR under the present experimental conditions. Although p70S6K activity may be regulated by multiple phosphorylation sites, an anti-p-p70S6K (Thr389) antibody was used in the present experiments, because previous studies suggest that phosphorylation of Thr389 may be particularly important for the regulation of p70S6K activity in vivo (30).



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  		FIG. 4. A, The effects of IGF-I and rapamycin on total p70S6K and p-p70S6K levels in muscles from burned rats. Muscles were incubated for 2 h in the absence or presence of 1 µg/ml IGF-I and/or the indicated concentrations of rapamycin. Total p70S6K and p-p70S6K levels were determined by Western blotting as described in the text. The blots are representative of four repeated blots. B, The effects of rapamycin on IGF-I-induced inhibition of protein degradation in muscles from burned rats, incubated for 2 h in the absence or presence of 1 µg/ml IGF-I with 400 nM rapamycin. Results are the mean ± SEM (n > 6 for each group). *, P < 0.05 vs. no IGF-I.

 
IGF-I may exert some of its metabolic effects by activating MAPK signaling pathways (15, 16). In the present study, treatment of muscles from burned rats with IGF-I resulted in increased tissue levels of the phosphorylated forms of ERK1/2 (p44/42) MAPKs (Fig. 5AGo). As expected, treatment with the MAPK kinase inhibitor, PD98059, blocked the activation of ERK1/2 MAPK, with maximal inhibition observed at 50 µM PD98059. Incubation of muscles in the presence of 50 µM PD98059 did not prevent the IGF-I-induced inhibition of protein breakdown (Fig. 5CGo).



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  		FIG. 5. A, The effects of IGF-I and PD98059 on total ERK1/2 MAPK and p-ERK MAPK levels in muscles from burned rats. Muscles were incubated for 2 h in the absence or presence of 1 µg/ml IGF-I with the indicated concentrations of PD98059. Total ERK1/2 MAPK and p-ERK MAPK levels were determined by Western blotting. The blots are representative of four repeated blots. B, The effects of IGF-I on total p38 MAPK and p-p38 MAPK levels in muscles from burned rats. Muscles were incubated for 2 h in the absence or presence of 1 µg/ml IGF-I. Total p38 and p-p38 MAPK levels were determined by Western blotting. C, The effects of IGF-I (1 µg/ml), PD98059 (50 µM), and IGF-I plus PD98059. D, The effects IGF-I (1 µg/ml), SB203580 (20 µM), and IGF-I plus SB203580. E, The effects of IGF-I, SB202190 (50 µM), and IGF-I plus SB202190 on protein degradation in incubated muscles from burned rats. Protein degradation rates were measured as net release of tyrosine during incubation for 2 h. Results are the mean ± SEM (n ≥ 6 for each group). *, P < 0.05 vs. control and inhibitor alone.

 
We next tested whether p38 MAPK is involved in IGF-I-induced inhibition of muscle protein degradation. IGF-I had no effect on p38 phosphorylation (Fig. 5BGo), and incubation of muscles in the presence of the p38 inhibitor SB203580 (20 µM) did not prevent the IGF-I-induced inhibition of muscle protein breakdown (Fig. 5DGo). Because SB203580 does not inhibit p38{gamma}, the p38 isoform primarily expressed in skeletal muscle (31), muscles were incubated in the presence of 50 µM SB202190. This concentration of SB202190 was shown in a previous study to completely block p38{gamma} activity (31), but did not influence the IGF-I-induced inhibition of muscle protein degradation in the current study (Fig. 5EGo). Taken together, the results shown in Fig. 5Go suggest that IGF-I-induced inhibition of protein breakdown in muscle from burned rats is not regulated by MAPK signaling.

Previous studies suggested that IGF-I activates the phosphatase calcineurin in cultured myotubes (32, 33). In the present study, incubation of muscles from burned rats in the presence of 10 or 25 µM of the calcineurin inhibitor, CsA, concentrations higher than that previously shown to inhibit calcineurin activity in cultured myotubes (12, 32), did not prevent the inhibitory effect of IGF-I on protein degradation (Fig. 6Go). These observations suggest that calcineurin activity is not involved in the effects of IGF-I on protein breakdown in muscle from burned rats.



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  		FIG. 6. The effects of IGF-I (1 µg/ml), CsA (10 and 25 µM), and their combination on protein degradation in incubated muscles from burned rats. Protein degradation rates were measured as net release of tyrosine during the 2-h incubation. Results are the mean ± SEM (n = 6 for each group). *, P < 0.05 vs. control and CsA (10 and 25 µM).

 
Although the present study was mainly focused on mechanisms regulating the anabolic effects of IGF-I in muscle from burned rats, it was important to test whether the mechanisms were specific for burn muscles. Experiments similar to those described above were therefore performed in muscles from pair-fed, sham-burned rats. Confirming previous reports of increased muscle protein breakdown after burn injury (1, 2, 3), protein breakdown rates were approximately 25% lower in muscles from sham-burned rats than in muscles from burned rats (Fig. 7Go; compare with Figs. 1Go, 2Go, and 4–6GoGoGo). Also, in muscles from sham-burned rats, LY294002 inhibited protein breakdown, and no additional inhibition of protein breakdown was noticed when IGF-I (1 µg/ml) was added in the presence of LY294002 (Fig. 7AGo). The inhibition of protein breakdown by LY294002 was approximately 25% and 35% in muscles from sham-burned and burned rats, respectively. Treatment of muscles from sham-burned rats with LiCl or TDZD-8 reduced protein breakdown to the same extent as IGF-I (Fig. 7BGo). The inhibition of protein breakdown by LiCl was approximately 40% in muscles from both sham-burned and burned rats. The corresponding values for TDZD-8 were 32% and 40%, respectively. Rapamycin alone did not influence protein degradation and did not prevent the effect of IGF-I in muscles from sham-burned rats (Fig. 7CGo). Inhibition of ERK1/2 and p38 MAPKs with PD98059 and SB202190, respectively, did not influence the inhibitory effect of IGF-I on protein degradation in muscles from sham-burned rats (Fig. 7Go, D and E). Finally, CsA alone did not significantly alter protein degradation and did not prevent the effect of IGF-I in muscles from sham-burned rats (Fig. 7FGo).



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  		FIG. 7. A, The effects of IGF-I (1 µg/ml) and LY294002 (100 µM) on protein degradation in incubated muscles from nonburned rats. B, The effects of IGF-I (1 µg/ml), LiCl (50 mM), and TDZD-8 (25 µM) on protein degradation in incubated muscles from nonburned rats. NaCl (50 mM) was added to the incubation medium of control, IGF-I-treated, and TDZD-8-treated muscles. C, The effects of IGF-I (1 µg/ml) and rapamycin (400 nM) on protein degradation in incubated muscles from nonburned rats. D, The effects of IGF-I (1 µg/ml) and PD98059 (50 µM) on protein degradation in incubated muscles from nonburned rats. E, The effects of IGF-I (1 µg/ml) and SB202190 (50 µM) on protein degradation in incubated muscles from nonburned rats. F, The effects of IGF-I (1 µg/ml) and CsA (25 µM) on protein degradation in incubated muscles from nonburned rats. Results are the mean ± SEM (n > 6 for each group). *, P < 0.05 vs. control for A and B; *, P < 0.05 vs. control and the respective inhibitors for C–F.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study support the concept that IGF-I inhibits muscle protein breakdown at least in part through a PI3K/GSK-3ß-dependent mechanism. The results do not rule out the possibility that other mechanisms, including inhibition of Foxo transcription factors, may also be involved in IGF-I-induced inhibition of muscle protein degradation.

The present experiments were mainly focused on the effects of IGF-I on protein degradation in atrophying muscle after burn injury. Most previous studies of the signaling pathways involved in the anabolic effects of IGF-I in skeletal muscle were performed in cultured muscle cells (12, 15, 16, 34, 35). To our knowledge, only one study has been published previously in which both protein breakdown and IGF-I-induced cell signaling were examined in incubated intact muscles from normal rats (22). The authors of that report concluded that although PI3K seemed to be essential for the anabolic effect of IGF-I in incubated intact rat muscles, the downstream mechanisms remained to be determined. The present report extends the previous observations by defining a likely downstream mechanism of PI3K in IGF-I-induced inhibition of muscle protein breakdown, i.e. phosphorylation (inactivation) of GSK-3ß. This adds to more recent studies in which evidence was found that phosphorylation (inactivation) of Foxo transcription factors is an important mechanism of IGF-I-induced anabolic effects in cultured myotubes (13, 14). Taken together, the present results and previous studies (13, 14) suggest that multiple mechanisms, downstream of PI3K/Akt, may be involved in the effects of IGF-I in skeletal muscle.

The finding in the present study that LY294002 and wortmannin reduced protein degradation in incubated muscles in the absence of IGF-I was surprising and may seem contradictory to previous reports that increased PI3K/Akt activity is associated with inhibition of muscle catabolism. Interestingly, inhibition of protein degradation by wortmannin and LY294002 was observed in other studies as well. For example, in a recent study by Dardavet et al. (22), wortmannin reduced basal protein breakdown rates in incubated rat muscles. A similar effect of wortmannin and LY294002 was reported in isolated hepatocytes (36). The authors of that report interpreted this inhibition as evidence that PI3K activity is required for intracellular autophagy, an important pathway of intracellular protein degradation. Additional evidence for a role of PI3K signaling in the regulation of autophagy was reported in yeast (37) and hepatocytes (38). It is conceivable that in the present study, the same mechanism (inhibition of autophagy) may be involved in the reduction of muscle protein breakdown caused by LY294002 and wortmannin. Thus, it may be speculated that different proteolytic pathways are inhibited by wortmannin and LY294002, on one hand, and by IGF-I, on the other hand; wortmannin and LY294002 may block autophagy, whereas IGF-I may mainly inhibit ubiquitin-proteasome-dependent proteolysis (8, 9). If that is the case, the effects of the PI3K inhibitors will be different when they are tested alone (inhibiting autophagy-related protein degradation) than when they are tested together with IGF-I (blocking the IGF-I-induced, PI3K/Akt-dependent inhibition of ubiquitin-proteasome-dependent proteolysis). Another potential mechanism by which LY294002 and wortmannin inhibit protein degradation could be by causing cell injury and reducing energy levels in the incubated muscles. The results in the present study showing unaltered ATP levels in muscles incubated in the presence of LY294002 argue against that mechanism. Interestingly, in recent experiments we observed that LY294002 inhibited protein breakdown in muscles from rats with other catabolic conditions as well, including fasting, sepsis, and dexamethasone treatment (our unpublished observations). Thus, the inhibitory effects of LY294002 and wortmannin do not seem to be specific for the present experimental conditions.

It is obvious that more studies need to be performed to define mechanisms by which wortmannin and LY294002 inhibit muscle protein breakdown. Regardless of those mechanisms, the present results clearly showed that IGF-I did not further reduce protein degradation in the presence of wortmannin or LY294002, supporting the concept that IGF-I inhibited protein degradation by a PI3K/Akt-dependent mechanism.

The potential role of GSK-3ß inhibition in the reduction of protein breakdown was supported by the observations in the present study that treatment of muscles with IGF-I resulted in phosphorylation (inactivation) of GSK-3ß, and that the GSK-3ß inhibitors, LiCl and TDZD-8, reduced protein breakdown in the muscles. These observations support the concept that inhibition of GSK-3ß activity may explain at least in part why IGF-I reduces protein degradation in skeletal muscle. It is possible that in the current experiments, IGF-I and the GSK-3ß inhibitors, LiCl and TDZD-8 (and possibly LY294002 and wortmannin as well), inhibited protein breakdown through the same downstream signaling mechanism of PI3K/Akt, i.e. inhibition of GSK-3ß. Recent studies suggest that inhibition of Foxo transcription factors is also involved in the effects of IGF-I on protein degradation in skeletal muscle and may actually be the most important mechanism (13, 14). Indeed, our findings of increased Foxo 1 phosphorylation in muscles incubated in the presence of LiCl suggest that inhibition of Foxo activity may have been involved in the present experiments as well. In contrast, our results suggest that mTOR, although activated by IGF-I as suggested by increased phosphorylation of p70S6K, was not involved in IGF-I-induced inhibition of muscle protein breakdown. This conclusion is in agreement with a previous report (22).

The present results, indicating a role for the PI3K/GSK-3ß signaling pathway in the anticatabolic effects of IGF-I, support recent studies in cultured myotubes in which both pharmacological and genetic evidence of this mechanism was provided (12, 34, 39). It should be noted, however, that in those studies the anabolic effects of IGF-I were mainly assessed as muscle cell hypertrophy (increased myotube diameter), and although muscle cell protein content was measured, no measurements of protein breakdown rates were performed. In the present study, protein breakdown rates were determined because muscle wasting induced by burn injury mainly reflects accelerated protein degradation (1, 2, 3).

The mechanism(s) by which GSK-3ß inactivation inhibits muscle protein breakdown is not known from the present or previous studies. Maintained nuclear levels of the transcription factor nuclear factor AT has been proposed to be one mechanism by which GSK-3ß phosphorylation may mediate IGF-I-induced muscle hypertrophy (34). It should be noted that a role for phosphorylated (inactivated) GSK-3ß in the anabolic effects of IGF-I raises the possibility that dephosphorylation (activation) of GSK-3ß might be a mechanism of increased protein breakdown in atrophying muscle. Indeed, results supporting that concept were reported recently (12, 34).

Previous studies provided evidence that IGF-I activates MAPK-dependent signaling in myotubes (15, 16) and that the MAPK signaling plays a primary role in myoblast proliferation by IGF-I (40). The role of MAPK in IGF-I-induced inhibition of protein degradation, however, has not been defined. The results of the present study suggest that although ERK MAPK signaling was activated by IGF-I, it was not involved in the inhibition of protein degradation caused by the hormone. p38, another member of the MAPK family, is classically known as a stress-induced MAPK, responding to bacterial endotoxins, proinflammatory cytokines, and physical-chemical stimuli such as UV light and increased extracellular osmolarity (31, 41, 42). The present observations that IGF-I did not phosphorylate p38 and that SB202190 did not block the inhibition of protein breakdown by IGF-I suggest that the p38 MAPK signaling pathway does not mediate the inhibitory effect of IGF-I on muscle protein degradation.

The finding in the present study that treatment of muscles with the calcineurin inhibitor, CsA, did not block the anticatabolic effect of IGF-I is in line with recent studies by Rommel et al. (12). In those studies, CsA did not prevent the IGF-I-induced hypertrophy of cultured myotubes, indicating that calcineurin-dependent cell signaling is not involved in the IGF-I-mediated anabolic effects in muscle. Those and our results contrast with previous reports in which evidence for a role of calcineurin was found in IGF-I-induced effects in cultured muscle cell (32, 33). One possible reason for these apparently contradictory results may be that IGF-I-mediated cell differentiation, rather than hypertrophy, influenced the results in studies in which evidence for a role of calcineurin was found (32, 33).

In addition to IGF-I-induced inhibition of protein breakdown in muscles from burned rats, the present study suggests that IGF-I inhibits protein degradation in muscles from sham-burned rats as well, consistent with our previous reports (7, 43), and that PI3K/GSK-3ß signaling may also be involved in the inhibitory effect of IGF-I on protein breakdown under normal conditions.

Thus, the results reported here suggest that IGF-I inhibits muscle protein degradation at least in part through a PI3K/GKS-3ß-dependent mechanism. This study is important because it examines for the first time signaling pathways involved in IGF-I-induced inhibition of protein breakdown in skeletal muscle after burn injury. The report extends previous observations by implicating GSK-3ß phosphorylation as a likely downstream mechanism of PI3K-mediated inhibition of protein breakdown in IGF-I-treated muscles. A role for GSK-3ß in the anticatabolic effect of IGF-I suggests that GSK-3ß may become an important target to inhibit muscle wasting in the future. Indeed, our results in the experiments in which the new specific GSK-3ß inhibitor, TDZD-8, was used would support that concept.

It should be noted that the present results do not rule out the possibility that downstream targets of the PI3K/Akt pathway, other than or in addition to GSK-3ß, may be involved in the regulation of muscle mass. Indeed, recent studies published after completion of the present work support that possibility. For example, Sandri et al. (13) reported data showing that activation (by dephosphorylation) and inactivation (by phosphorylation) of Foxo transcription factors play important roles in the catabolic effects of dexamethasone and the anabolic effects of IGF-I, respectively, in cultured myotubes. In a concurrent report by Stitt et al. (14), additional evidence for a role for Foxo transcription factors was reported in cultured myotubes treated with dexamethasone and IGF-I. The authors of that report concluded that although activation of Foxo1 was not sufficient to induce transcription of the muscle wasting-associated genes, atrogin-1 and MuRF1, inactivation of Foxo transcription factors was a necessary step in the IGF-I-induced inhibition of muscle atrophy caused by dexamethasone.

Interestingly, in the study by Sandri et al. (13), overexpression of constitutively active GSK-3ß in the myotubes resulted in increased atrogin-1 expression and activation of the atrogin-1 promoter, although the changes induced by GSK-3ß were less pronounced than those induced by overexpression of Foxo 3. Thus, it is possible that multiple downstream targets of the PI3K/Akt pathway, including GSK-3ß and Foxo transcription factors, are responsible for the anabolic effects of IGF-I. A potential cross-talk between GSK-3ß and Foxo transcription factors may also need to be considered. It is possible that the relative roles of these mechanisms may be different in different experimental and clinical conditions. The results of the present study, showing a pronounced phosphorylation of GSK-3ß by IGF-I and inhibition of protein degradation to the same extent by IGF-I and the GSK-3ß inhibitors, LiCl and TDZD-8, in muscles from burned rats, provide at least circumstantial evidence that IGF-I may inhibit protein breakdown in muscle after burn injury by inactivating GSK-3ß. Our results, of course, do not exclude the possibility that inactivation of Foxo transcription factors played a role in the effects of IGF-I observed in this study. It will be important in future experiments to determine to what degree Foxo transcription factors are involved in the regulation of muscle mass in various clinical conditions characterized by muscle wasting, such as burn injury, sepsis, and cancer, in addition to their involvement in the regulation of atrophy in cultured myotubes.

A final important implication of the present results is that muscle remains sensitive to IGF-I after burn injury, at least with regard to the effects on protein breakdown and signaling pathways, thus confirming previous studies in which we found that treatment of incubated muscles from burned rats or treatment of burned rats in vivo with IGF-I reduced protein breakdown (7, 8, 9, 43). This contrasts with some of the effects of insulin that are impaired after burn injury (44). In a recent study, insulin-induced activation of the PI3K/Akt signaling pathway was attenuated in skeletal muscle from burned rats (45). In the same study, insulin-stimulated activation of ERK was not affected by burn injury, indicating that the insulin resistance with regard to the PI3K/Akt signaling pathway was at the postreceptor level. Because in the present study, IGF-I-induced PI3K/Akt signaling was intact in muscle from burned rats, it is possible that burn-induced insulin resistance was caused by a postreceptor mechanism upstream of PI3K/Akt and specific for insulin signaling (46). Insulin resistance and maintained IGF-I sensitivity in skeletal muscle after burn injury have important clinical implications, suggesting that IGF-I may be more effective than insulin in the treatment of burn-induced muscle wasting.


   Footnotes
 
This work was supported in part by Grant 8610 from the Shriners of North America, Tampa, FL (to C.-H.F.); and by National Institutes of Health Grants R01-DK-37908 (to P.-O.H.) and R01-NR-008545 (to P.-O.H.). A.R.E. was supported by a National Research Service Award from the National Institutes of Health.

First Published Online March 31, 2005

Abbreviations: CsA, Cyclosporin A; Foxo, Forkhead box O; GSK, glycogen synthase kinase; mTOR, mammalian target of rapamycin; p-, phosphorylated; PI3K, phosphotidylinositol 3-kinase; TBST, Tris-buffered saline containing Tween 20; TDZD-8, thiadiazolidinone-8.

Received July 8, 2004.

Accepted for publication March 21, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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