Endocrinology and Metabolism

The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb

Andrew R. Kelleher, Scot R. Kimball, Michael D. Dennis, Rudolf J. Schilder, Leonard S. Jefferson

Abstract

Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. To acquire new knowledge that will assist in the development of therapeutic strategies for minimizing disuse atrophy, the present study was undertaken with the aim of identifying molecular mechanisms that mediate control of protein synthesis and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Male Sprague-Dawley rats were subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days or served as nonimmobilized controls. Following an overnight fast, rats received either saline or l-leucine by oral gavage as a nutrient stimulus. Hindlimb skeletal muscles were extracted 30 min postgavage and analyzed for the rate of protein synthesis, mRNA expression, phosphorylation state of key proteins in the mTORC1 signaling pathway, and mTORC1 signaling repressors. In the basal state, mTORC1 signaling and protein synthesis were repressed within 24 h in the soleus of an immobilized compared with a nonimmobilized hindlimb. These responses were accompanied by a concomitant induction in expression of the mTORC1 repressors regulated in development and DNA damage responses (REDD) 1/2. The nutrient stimulus produced an elevation of similar magnitude in mTORC1 signaling in both the immobilized and nonimmobilized muscle. In contrast, phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) on Thr229 and Thr389 in response to the nutrient stimulus was severely blunted. Phosphorylation of Thr229 by PDK1 is a prerequisite for phosphorylation of Thr389 by mTORC1, suggesting that signaling through PDK1 is impaired in response to immobilization. In conclusion, the results show an immobilization-induced attenuation of mTORC1 signaling mediated by induction of REDD1/2 and defective p70S6K1 phosphorylation.

  • casting
  • anabolic resistance
  • 70-kilodalton ribosomal protein S6 kinase 1
  • 4E-binding protein 1
  • puromycin
  • mechanistic target of rapamycin complex 1
  • regulated in development and DNA damage responses 1/2

profound wasting of skeletal muscle, referred to as disuse atrophy, occurs in response to limb immobilization (10, 15, 16), limb suspension (40), and chronic bed rest (4, 12, 14). These conditions are associated with marked losses of skeletal muscle strength and function (21, 32), premature physical frailty (51), elevated health care costs, and an increased risk of mortality (36). Development of effective therapeutic strategies aimed at minimizing or preventing disuse atrophy will require a better understanding of the underlying molecular mechanisms that contribute to the loss of skeletal muscle mass. Maintenance of skeletal muscle mass is mainly dependent on the balance between rates of protein synthesis and protein degradation (39). Attenuated rates of skeletal muscle protein synthesis in the basal-fasted condition have been observed in both human (15, 17) and animal (7, 18) models of disuse atrophy induced by limb immobilization. Elevated rates of protein degradation have been observed in skeletal muscle of animal models of hindlimb immobilization (29, 33); however, its role in human disuse atrophy is unclear (39). In addition to attenuated rates of protein synthesis in the basal-fasted condition, limb immobilization creates resistance to nutrient-induced stimulation of protein synthesis in human skeletal muscle (17), a phenomenon termed “anabolic resistance” (41). According to protein balance calculations in humans, the immobilization-induced loss of muscle mass can be accounted for solely by attenuated rates of protein synthesis (15, 34, 39). Attenuated rates of protein synthesis in the basal-fasted condition and anabolic resistance have also been observed in human skeletal muscle following chronic bed rest (4, 12, 14). Taken together, the evidence suggests that attenuated rates of protein synthesis and anabolic resistance are responsible for the imbalance in protein turnover leading to disuse atrophy in humans (39, 42).

The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway plays a critical role in regulating rates of protein synthesis in skeletal muscle. (For a review of mTORC1 and skeletal muscle protein synthesis, see Ref. 27.) The mTORC1 signaling pathway regulates cap-dependent mRNA translation via phosphorylation of substrates, including the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The pathway is sensitive to nutrient and mitogenic stimuli and regulates nutrient- and exercise-induced stimulation of protein synthesis in skeletal muscle (1, 27). Meal feeding increases plasma insulin and amino acid concentrations, leading to the full activation of mTORC1 through the protein kinase B (Akt)-tuberous sclerosis complex (TSC) 1/2-Rheb signaling pathway and interaction with Rag GTPases, respectively (27, 45, 55). Inhibitory signals mediated by p53, Sestrins (8), AMP-activated protein kinase (AMPK) (6), regulated in development and DNA damage response (REDD) 1 (35), and REDD2 (37) attenuate mTORC1 signaling and rates of protein synthesis. Despite evidence of attenuated mTORC1 signaling in skeletal muscle in response to immobilization (38, 56), suspension (3, 22), and bed rest (12), no study to our knowledge has investigated the role of these repressors of mTORC1 signaling in disuse atrophy.

The purpose of the present study was to 1) characterize a model of immobilization-induced disuse atrophy that exhibits attenuated rates of protein synthesis and attenuated mTORC1 signaling and 2) assess known repressors of mTORC1 signaling to gain a better understanding of the molecular mechanism(s) responsible for immobilization-induced disuse atrophy. We hypothesized that hindlimb immobilization would attenuate rates of protein synthesis in rat hindlimb skeletal muscle due to increased expression and/or activation of one or more repressors of the mTORC1 signaling pathway.

METHODS

Animals.

Adult (8–9 wk; 230–350 g) male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MD) were housed in solid-bottom wire cages in a temperature (25°C)- and light-controlled environment. Rats were provided rodent chow (Harlan-Teklad 8604, Indianapolis, IN) and water ad libitum. Before immobilization experiments, rats were adapted to a reversed 12:12-h light-dark cycle (lights off at 0900) for a week. Animal facilities and experimental protocols were submitted to, and approved by, the Institutional Animal Care and Use Committee of the Pennsylvania State University College of Medicine.

Experimental design.

After a 1-wk adaptation period, 98 rats were anesthetized with isoflurane inhalation (2.5%) and subjected to unilateral hindlimb immobilization as described previously (29). Immobilization was performed 1, 2, 3, or 7 days before the day of the experiment. Control rats (n = 36) were prepared for immobilization but were not immobilized. An equal number of rats from each experimental group was processed on experimental days. All rats were fasted overnight (21 h) but allowed free access to water. On the day of the experiment, the rats were randomly divided into groups that received either saline (0.155 M) or 1.35 g l-leucine/kg body wt by oral gavage as a nutrient stimulus as described previously (2). Fifteen minutes after oral gavage, the rats were anesthetized using isoflurane and remained anesthetized for the remainder of the experiment.

Administration of puromycin and sample collection.

After 5 min of anesthesia, all rats were injected intravenously with 0.040 μmol puromycin/g body wt using a 10 mg/ml solution of puromycin dihydrochloride (AG Scientific, San Diego, CA) in saline. Casts were removed using a Stryker saw (Stryker Instruments, Kalamazoo, MI) after puromycin injection. Ten minutes following administration of puromycin (30 min postgavage), the soleus, gastrocnemius, and plantaris muscles were individually excised, cleared of visible fascia, weighed, and either homogenized or snap-frozen in liquid nitrogen. Soleus muscles were homogenized and processed for immunoblots as described previously (9) with minor modifications. Briefly, the muscle was homogenized in 10 volumes of homogenization buffer followed by centrifugation at 2,000 g for 3 min at 4°C. An aliquot (200 μl) of the supernatant fraction was added to an equal volume of 2X Laemmli buffer, and a separate aliquot (10 μl) was used to measure protein concentration by Bio-Rad Protein Assay. An aliquot (0.5 ml) of the homogenate was reserved for the measurement of mRNA expression as described below. Blood was extracted (1 ml) by syringe from the inferior vena cava and immediately mixed with EDTA (final concentration 7.5 mM; Sigma) to prevent clotting. The blood samples were centrifuged at 1,500 g for 10 min at 4°C, and plasma was removed and stored at −20°C until analyzed. Rats were killed by opening the chest cavity while under isoflurane anesthesia.

SDS-PAGE and immunoblot procedure.

Muscle samples in 2X Laemmli sample buffer were diluted with 1X Laemmli sample buffer to equal protein concentrations and then subjected to protein immunoblot analysis as described previously (52). Protein phosphorylation and expression were assessed by immunoblot analysis using 4–20% Bio-Rad Criterion gels. Hyperphosphorylation of p70S6K1 and 4E-BP1 were assessed by immunoblot analysis using 7.5 and 15% polyacrylamide gels, respectively, with 0.19% bisacrylamide to permit resolution of p70S6K1 and 4E-BP1 into multiple electrophoretic forms (1, 28). Polyvinylidene difluoride membranes were incubated with primary antibodies recognizing proteins phosphorylated on specific residues, including: p70S6K1 Thr389, 4E-BP1 Thr37/46, 4E-BP1 Ser65, AMPK Thr172, eukaryotic initiation factor 2α (eIF2α) Ser51, or p44/42 Thr202Tyr204 [extracellular signal-regulated kinase (ERK) 1/2], all of which were from Cell Signaling Technology (Danvers, MA), or anti-phospho-p70S6K1 Thr229 from Abcam (Cambridge, MA). Alternatively, blots were probed with antibodies against AMPK or ERK1/2 (Cell Signaling Technology); p70S6K1 or 4E-BP1 from Bethyl Laboratories (Montgomery, TX); α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA); hypoxia-inducing factor 1 α-subunit (HIF1-α) from Novus Biologicals (Littleton, CO); or eIF2α (mouse monoclonal antibody; hybridoma cells provided by the late Dr. Edgar Henshaw), which was produced in-house. Activating transcription factor 4 (ATF4) antibody was generously provided by Dr. Michael Kilberg (University of Florida). Blots were developed using a FluorChem M Multifluor System (ProteinSimple, San Jose, CA) and analyzed using AlphaView (ProteinSimple) and Genetools (Syngene, Cambridge, MA) software.

Measurement of skeletal muscle protein synthesis.

Skeletal muscle protein synthesis was measured by the incorporation of puromycin into peptide chains as described (20). Briefly, immunoblot membranes were incubated and rocked overnight at 4°C with a mouse monoclonal anti-puromycin antibody generated in-house (1 μg/ml in Tris-buffered saline). Immunoblots were prepared and developed as described above. Puromycin incorporation was assessed by summating the immunoblot intensity of all protein bands and subtracting background. Soleus muscle samples from rats not injected with puromycin were included in immunoblot analysis, and values obtained using these samples were subtracted from values obtained for the other samples.

Measurement of mRNA expression.

RNA was isolated from skeletal muscle homogenates following a standard TRIzol protocol (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. Total RNA was reverse transcribed using an ABI High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). For quantitation of specific mRNA, reverse transcriptase-polymerase chain reaction was performed using Taqman gene expression assays as previously described (25). Primers were purchased from Applied Biosystems, including: Ddit4 (REDD1) (assay ID: Rn01433735_g1); Ddit4l (REDD2) (Rn00589659_g1); β-actin (Rn00667869_m1); Sesn1 (Rn01440906_m1); RGD1566319 (Sesn2) (Rn01520467_m1); and ATF4 (Rn00824644_g1). β-Actin mRNA expression across a 7-day period of immobilization was verified as a control against Rpl32 (Rn00820748_g1) and Hprt (Rn01527838_g1).

Statistical analysis.

Results from individual experiments (n = 5/group) were replicated in two or more independent experiments and are presented as means ± SE calculated from pooled data. Outliers were determined using Grubb's test (α-level 0.05) and removed. One-way analysis of variance and Student's t-test were used to compare differences among groups. All comparisons were analyzed using GraphPad Software. Differences between groups were considered significant at P < 0.05.

RESULTS

Effect of immobilization on muscle mass.

Initially, experiments were designed to examine the time course of changes in muscle mass and protein synthesis following unilateral hindlimb immobilization. Illustrated in Fig. 1 is the muscle mass-to-body mass ratio of three muscles of the plantarflexor group, the soleus, gastrocnemius, and plantaris. Following 1 and 2 days of immobilization, there was no detectable change in mass for any of the three muscles. By 3 days, however, the mass for each muscle was significantly (P < 0.05) reduced, and the reduction in mass was greater 7 days following immobilization. No differences in protein content (mg/g) were observed in muscles from immobilized compared with nonimmobilized hindlimbs across the 3-day period (data not shown). Because the reduction in mass was greatest for the soleus it was selected for further investigation into the mechanism(s) contributing to disuse atrophy.

Fig. 1.

Time course changes in muscle weight following immobilization. Muscle mass is expressed as a ratio of muscle-to-body weight for soleus (A), gastrocnemius (B), and plantaris (C) muscle. Immobilized rats had one hindlimb immobilized for 1, 2, 3, or 7 days (Imm, immobilized; N leg, nonimmobilized limb). Control rats were not immobilized. Data are means ± SE; n = 6–26 rats/group. ‡P < 0.05 vs. nonimmobilized limb.

Effect of immobilization on muscle protein synthesis.

To investigate the cause of the observed loss of muscle mass, rates of protein synthesis were measured in the soleus after 1, 2, 3, and 7 days of immobilization. As illustrated in Fig. 2, immobilization resulted in an attenuation of the rate of protein synthesis of ∼40–50% on each of the days studied compared with nonimmobilized hindlimbs, and the effect was maximal after only a single day of immobilization.

Fig. 2.

Effect of immobilization on rate of protein synthesis in the soleus muscle. Bars represent the amount of puromycin incorporated into protein as assessed by immunoblot analysis. Immobilized rats had one hindlimb immobilized for 1, 2, 3, or 7 days. Control rats were not immobilized. Data are means ± SE; n = 5–10 rats/group. *P < 0.05 vs. equivalent fasted condition.

Effect of immobilization on mTORC1 signaling.

To gain an understanding of potential events that cause attenuated protein synthesis following hindlimb immobilization, mTORC1 signaling was assessed across the first 3 days of immobilization in both a fasted condition (saline gavage) and in response to an oral leucine gavage as a nutrient stimulus. A gel-shift analysis of the phosphorylation state of p70S6K1 (Fig. 3A) and 4E-BP1 (Fig. 3B) demonstrated attenuated mTORC1 signaling in soleus muscle of the immobilized compared with the nonimmobilized hindlimb across the 3-day time period. This analysis also showed that mTORC1 signaling was elevated in response to the nutrient stimulus with the magnitude of increase in the phosphorylation state of p70S6K1 and 4E-BP1 being similar in the soleus of both the immobilized and nonimmobilized hindlimbs. Similar results were obtained when the relative phosphorylation of 4E-BP1 at Ser65 (i.e., a site phosphorylated by mTORC1) was assessed. Figure 3C shows that relative phosphorylation of 4E-BP1 Ser65 was attenuated in the soleus of the immobilized compared with the nonimmobilized hindlimb across the 3-day time period. Moreover, the nutrient stimulus produced an elevation of similar magnitude in 4E-BP1 Ser65 phosphorylation in the soleus of both the immobilized and nonimmobilized hindlimb. Overall, these results point to an immobilization-induced attenuation of mTORC1 signaling that is nonetheless responsive to stimulation by leucine administration.

Fig. 3.

Effect of immobilization on 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) hyperphosphorylation and 4E-binding protein 1 (4E-BP1) phosphorylation in the soleus muscle. Both basal and leucine-induced stimulation of p70S6K1 and 4E-BP1 phosphorylation were attenuated in the soleus of a hindlimb immobilized for 1, 2, and 3 days. A: hyperphosphorylation of p70S6K1 was assessed by protein immunoblot analysis; bars represent the proportion of the protein present in the top three bands (bands 2–4) relative to all four bands. B: hyperphosphorylation of 4E-BP1 was assessed by protein immunoblot analysis; bars represent the proportion of the protein present in the hyperphosphorylated γ-form relative to all three bands. C: phosphorylation of 4E-BP1 at Ser65 was assessed by protein immunoblot analysis; bars represent the mean phospho-to-total protein ratio. Phosphorylation of 4E-BP1 at Ser65 is expressed relative to total 4E-BP1 expression (blot not pictured). We observed no changes in 4E-BP1 protein expression across 3 days of hindlimb immobilization. Samples were run on the same blot, but not in contiguous lanes. Noncontiguous lanes are denoted by a white line. Immobilized rats had one hindlimb immobilized for 1, 2, or 3 days. Control rats were not immobilized. Rats were administered either 1.35 g/kg body wt l-leucine (L) or an equal volume of saline (S) by oral gavage. Data are means ± SE; n = 4–10 rats/group. ‡P < 0.05 vs. nonimmobilized Limb; *P < 0.05 vs. equivalent saline-gavaged condition.

Effect of immobilization on repressors of mTORC1 signaling.

A number of upstream regulatory inputs were investigated as potential mediators of the attenuated mTORC1 signaling observed in soleus muscle of immobilized compared with nonimmobilized hindlimbs (Table 1). Sestrin 1 and Sestrin 2, whose expression is regulated by the transcription factor p53, act through AMPK to repress mTORC1 signaling (8). However, examination of mRNA expression for Sestrin 1 and Sestrin 2 revealed no differences between immobilized and nonimmobilized hindlimbs. Moreover, no differences were observed in protein expression of Sestrin 1 or p53 between immobilized and nonimmobilized hindlimbs. Finally, immobilization did not alter AMPK phosphorylation at Thr172.

View this table:
Table 1.

End points not associated with immobilization-induced repression of mTORC1 signaling

In contrast, two regulatory inputs were observed to change in parallel with the attenuation of mTORC1 signaling following immobilization. As illustrated in Fig. 4, expression of mRNA for REDD1 and REDD2, both of which repress mTORC1 signaling through activation of TSC1/2 (37, 50), was induced as early as 1 day following immobilization, and their expression increased further following 2 and 3 days. Hypoxia was considered as a potential mediator of the induced expression of REDD1 and REDD2 mRNA. We next examined protein expression of HIF1-α, a known inducer of REDD1 mRNA expression (11, 24), as a potential mediator of the responses of REDD1 and REDD2 mRNA to immobilization. However, its expression was not elevated with immobilization (Table 1). Stress in the endoplasmic reticulum (ER stress) was also considered as being responsible for the elevated expression of REDD1 and REDD2. However, no significant differences were observed in eIF2α phosphorylation at Ser51 (a marker of ER stress) or ATF mRNA and protein expression (a known inducer of REDD1) (54) between immobilized and nonimmobilized hindlimbs (Table 1). Finally, bloodborne regulatory inputs such as glucocorticoids, which are known to induce REDD1 expression in muscle (35, 53), were also considered but were not pursued due to the lack of a systemic effect on mTORC1 signaling in the nonimmobilized hindlimb.

Fig. 4.

Effect of immobilization on regulated in development and DNA damage response (REDD) mRNA expression in soleus muscle. REDD1 and REDD2 mRNA expression were elevated in the soleus of a hindlimb immobilized for 1, 2, and 3 days compared with control. Bars represent the mean REDD1 mRNA (A) or REDD2 mRNA (B)-to-β-actin mRNA ratio assessed using Taqman gene expression assay. Immobilized rats had one hindlimb immobilized for 1, 2, or 3 days. Control rats were not immobilized. Rats were administered either 1.35 g/kg body wt l-leucine or an equal volume of saline by oral gavage. Data are means ± SE; n = 9–10 rats/group. ‡P < 0.05 vs. nonimmobilized limb; *P < 0.05 vs. equivalent saline-gavaged condition.

Effect of immobilization on p70S6K1 phosphorylation at Thr389.

Intriguingly, although leucine-induced phosphorylation of 4E-BP1 at Ser65 appeared to be unaffected by immobilization, a different pattern of response was observed when relative phosphorylation of p70S6K1 at Thr389 was assessed as a marker of mTORC1 signaling (Fig. 5A). Thus, although relative phosphorylation at this site was likewise attenuated across the 3-day time period, the response to the leucine-induced stimulus was markedly different between the two conditions. In the soleus of the immobilized hindlimb, phosphorylation at this site was elevated 3- to 4-fold following leucine administration, whereas in the nonimmobilized hindlimb the elevation was 7- to 14-fold compared with the saline-administered control. Given that phosphorylation of p70S6K at Thr229 (a PDK1 targeted site) is a prerequisite for mTORC1-mediated phosphorylation of Thr389 (26), its responses to immobilization and a nutrient stimulus, respectively, were assessed. As illustrated in Fig. 5B, phosphorylation of p70S6K1 at Thr229 was of similar magnitude in the soleus of both the immobilized and nonimmobilized hindlimb and for the former did not respond to the leucine-induced stimulus. In contrast, leucine administration produced a robust elevation in Thr229 phosphorylation in the soleus of the nonimmobilized hindlimb. Thus, analysis of p70S6K1 phosphorylation sites Thr389 and Thr229 supports an immobilization-induced state of anabolic resistance that has been described in other models of disuse atrophy (12, 17, 43).

Fig. 5.

Effect of immobilization on site-specific p70S6K1 phosphorylation in the soleus muscle. Both basal and leucine-induced stimulation of p70S6K1 phosphorylation were attenuated in the soleus of a hindlimb immobilized for 1, 2, and 3 days. Phosphorylation of p70S6K1 at Thr389 (A) and Thr229 (B) was assessed by protein immunoblot analysis; bars represent the mean phospho-to-total protein ratio. Samples were run on the same blot, but not in contiguous lanes. Noncontiguous lanes are denoted by a white line. Immobilized rats had one hindlimb immobilized for 1, 2, or 3 days. Control rats were not immobilized. Rats were administered either 1.35 g/kg body wt l-leucine or an equal volume of saline by oral gavage. Data are means ± SE; n = 4–10 rats/group. ‡P < 0.05 vs. nonimmobilized Limb; *P < 0.05 vs. equivalent saline-gavaged condition.

DISCUSSION

In the present study, protein synthesis was maximally attenuated in the soleus muscle within 24 h of immobilization, and the attenuation was sustained for at least 7 days compared with the contralateral, nonimmobilized limb. In contrast, protein synthesis was unaltered in the nonimmobilized limb compared with control rats. This finding is in agreement with previous studies showing that immobilization-induced repression of protein synthesis begins to manifest as early as 6 h after application of a cast and is maintained throughout a 7-day period (7, 19). The magnitude of repression in response to immobilization observed in the present study (reduction to ∼50% of either the nonimmobilized or control value) also agrees with previous studies utilizing animal models of immobilization [reduction of 30–70% compared with control (7, 19)] as well as in human studies using limb suspension (10) and bed rest (14) in which protein synthesis was reduced to 53 and 50% of control values, respectively.

The repression of protein synthesis observed in the present study was accompanied by attenuated signaling through mTORC1 in the soleus muscle of immobilized compared with nonimmobilized hindlimbs. The attenuation of mTORC1 signaling occurred within 24 h of immobilization, as assessed by a reduction in the proportion of p70S6K1 and 4E-BP1 present in hyperphosphorylated forms, as well as reduced phosphorylation of p70S6K1 at Thr389 and 4E-BP1 at Ser65. Attenuated mTORC1 signaling has been observed in skeletal muscle of rats and mice subjected to hindlimb unloading (5, 22), immobilization (30), or denervation (22) but was not observed in one study in rats following 5 days of unilateral hindlimb immobilization (29). Studies in humans have failed to detect a change in mTORC1 signaling following 10 or 21 days of limb suspension, despite significant reductions in the rate of myofibrillar protein synthesis (10). The basis for these disparate results in mTORC1 signaling are not clear but could be due to different muscles being studied, e.g., unloading attenuated mTORC1 signaling in soleus muscle (5) but not in vastus lateralis (10). They could also be due to the model being employed, e.g., immobilization vs. unloading, species, or to the feeding status of the animals.

The immobilization-induced attenuation of mTORC1 signaling likely involves upstream regulatory inputs to the kinase. The best-characterized upstream regulator of mTORC1 signaling is the ras homolog enriched in brain (Rheb). Rheb is a small GTPase that is regulated by the GTPase activator protein referred to as TSC2 acting in a complex with TSC1. When Rheb is complexed with GTP it acts as a direct activator of mTORC1 signaling (23). In contrast, the Rheb·GDP complex does not activate mTORC1. Thus, by activating Rheb's GTPase activity, the TSC1·TSC2 complex promotes accumulation of Rheb·GDP and consequently attenuation of mTORC1 signaling. The TSC1·TSC2 complex acts to integrate signals from several upstream pathways, including those emanating from Akt and ERK (31, 47). Both of these kinases phosphorylate and thereby inactivate the TSC1·TSC2 complex, leading to a stimulation of mTORC1 signaling. In the present study, immobilization did not reduce phosphorylation of TSC2 at Ser939, a site directly phosphorylated by Akt (44). The possibility that immobilization promoted reduced phosphorylation of TSC2 by ERK1/2 could not be tested in the present study because of the lack of an antibody that reliably detects rat TSC2 phosphorylated at Ser644, a site phosphorylated by ERK1/2 (31). However, no difference in ERK1/2 Thr202/Tyr204 phosphorylation was observed, suggesting that ERK1/2 signaling did not mediate the immobilization-induced attenuation of mTORC1 signaling.

In addition to Akt and ERK, the TSC1·TSC2 complex is also regulated by the p53-AMPK signaling pathway. Previous studies have shown that p53 expression is induced in muscle in response to hindlimb suspension (48), and chronic activation of p53 in muscle promotes atrophy (46). Moreover, two downstream targets of p53 action, Sestrins 1 and 2, have been shown to repress mTORC1 signaling through activation of AMPK (8). However, neither Sestrin 1 or Sestrin 2 mRNA, Sestrin 1 protein expression, nor AMPK phosphorylation on the activating residue, Thr172, was observed in the present study, suggesting that the attenuation of mTORC1 signaling was not mediated by the p53-Sestrin-AMPK signaling pathway.

Like AMPK, REDD1 and REDD2 also act to attenuate mTORC1 signaling through a mechanism involving TSC1/2 (37, 50). In the present study, REDD1 and REDD2 mRNA expression was induced (100 and 300%, respectively) after 1 day of immobilization followed by a larger induction (150–250 and 300–500%) after 2 and 3 days, changes that paralleled the greater attenuation of mTORC1 signaling at the later time points. Enhanced protein expression of either REDD1 or REDD2 could not be assessed because of the lack of antibodies that reliably detect the rat proteins. Nonetheless, previous studies have demonstrated concomitant responses of REDD1 mRNA and protein expression to ER stress (54), starvation (35), and dexamethasone treatment (53). Thus, it seems reasonable to conclude that the increases in REDD1 and REDD2 mRNA expression observed in the present study are indicative of similar responses in expression of the respective proteins.

REDD1 and REDD2 mRNA expression are potently induced in response to a variety of stresses. One potential mechanism for the observed induction of REDD1/2 expression with hindlimb immobilization could be development of hypoxia (11) due to a reduction in blood flow to the immobilized hindlimb. However, HIF-1α expression was not elevated in soleus muscle from the immobilized hindlimb compared with the contralateral, nonimmobilized hindlimb. In addition, the lack of change in either eIF2α phosphorylation on Ser51 or ATF4 expression suggests that ER stress was not involved in the induction of REDD1 expression (54). REDD1 expression is also upregulated in response to other stresses, e.g., conditions that cause DNA damage (13) or decrease ATP concentrations (49), or in response to oxidative stress (13). However, the mechanism(s) through which such stresses lead to upregulation of REDD1 expression are presently undefined.

In addition to attenuated rates of protein synthesis in the basal, fasted condition, both immobilization (17) and bed rest (4, 12) are associated with development of resistance to nutrient-induced stimulation of skeletal muscle protein synthesis in humans. Moreover, during preparation of this manuscript, two studies were published showing that skeletal muscle protein synthesis is resistant to nutrient-induced stimulation during hindlimb immobilization in old rats (33) and in mice (30). The studies showed that global rates of protein synthesis in skeletal muscle were unresponsive to chow feeding or oral leucine administration, respectively, in an immobilized hindlimb compared with fasted controls. In the present study, the leucine-induced stimulus produced an elevation of similar magnitude in mTORC1 signaling in soleus muscle from both the immobilized and nonimmobilized hindlimbs, as judged by gel-shift analysis of the phosphorylation state of p70S6K1 and 4E-BP1. Assessment of 4E-BP1 Ser65, a site of mTORC1 mediated phosphorylation, produced results similar to those obtained with the gel-shift analysis. It is noteworthy that all of the mTORC1 phosphorylation sites on 4E-BP1 as well as several rapamycin-sensitive sites in the COOH-terminus of p70S6K1 are followed by a Pro residue, providing a possible explanation for the coordinated changes in 4E-BP1 phosphorylation on Ser65 and hyperphosphorylation of 4E-BP1 and p70S6K1. In contrast, p70S6K1 Thr389 is not followed by Pro but instead is followed by a Tyr residue, suggesting that its phosphorylation by mTORC1 might be differentially regulated compared with 4E-BP1. Indeed, in contrast to 4E-BP1 phosphorylation on Ser65 or hyperphosphorylation of either 4E-BP1 or p70S6K1, phosphorylation of p70S6K1 on both Thr229 and Thr389 in response to the nutrient stimulus was severely blunted in soleus muscle from immobilized compared with nonimmobilized hindlimbs. Because phosphorylation of Thr229 by PDK1 is a prerequisite for phosphorylation of Thr389 by mTORC1 (26), the results are consistent with a model in which PDK1 activation is rapidly impaired in response to immobilization. In conclusion, the results presented here demonstrate an immobilization-induced attenuation of mTORC1 signaling mediated by REDD1 and REDD2 that is nonetheless responsive to a leucine stimulus. More importantly, they suggest that phosphorylation of p70S6K1 on Thr389, which is required for full activation of the kinase and thus signaling to its downstream substrates, fails to respond appropriately to the leucine stimulus. This lack of response is apparently due to a defect in PDK1 signaling and would explain the phenomenon of anabolic resistance referred to in other models of disuse atrophy (12, 17, 43).

GRANTS

This work was supported by funds from National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-15658 (L. S. Jefferson) and DK-088416 (M. D. Dennis) and by a grant from the Pennsylvania Department of Health using Tobacco Settlement Funds.

DISCLOSURES

The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions.

AUTHOR CONTRIBUTIONS

Author contributions: A.R.K., S.R.K., R.J.S., and L.S.J. conception and design of research; A.R.K. performed experiments; A.R.K. analyzed data; A.R.K., S.R.K., M.D.D., and L.S.J. interpreted results of experiments; A.R.K. prepared figures; A.R.K. drafted manuscript; A.R.K., S.R.K., M.D.D., and L.S.J. edited and revised manuscript; A.R.K., S.R.K., M.D.D., R.J.S. and L.S.J. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Dr. Alex P. Tuckow for intellectual contributions in the initial stages of the project. We also thank Sharon Rannels, Holly Lacko, Lydia Kutzler, and all members of the Jefferson Laboratory for assistance in performing the studies. Finally, we thank Dr. Michael Kilberg for providing the ATF4 antibody.

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  56. 56.
View Abstract