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1Division of Pulmonary/Critical Care Medicine, Burns and Allen Research Institute, Cedars-Sinai Medical Center, 2David Geffen School of Medicine, University of California Los Angeles, Los Angeles; 3Section of Neurobiology, Physiology, and Behavior, College of Biological Sciences, and 4Department of Physiology and Membrane Biology, University of California Davis School of Medicine, Davis, California
Submitted 28 September 2005 ; accepted in final form 6 February 2006
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ABSTRACT |
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(Ser21) and GSK-3
(Ser9) was increased 55 and 45%, respectively, with ND. Phosphorylation of ERK1 (Thr202) and ERK2 (Tyr204), p44 and p42, respectively, was reduced 64 and 55%, respectively, with ND. Total protein concentration for all signaling intermediates of the three pathways was preserved. We conclude that short-term ND altered the phosphorylation states of key proteins of several pathways involved in protein turnover. This forms the framework for future studies aimed at identifying therapeutic targets in the management of short-term nutritionally induced cachectic states. nutritional deprivation; muscle fiber atrophy; cachexia; mRNA translation
The major signals mediating disordered protein turnover with ND are complex and not fully understood. However, reduced levels of insulin-like growth factor I (IGF-I), insulin, amino acids, and enhanced elaboration of corticosteroids are likely important candidates. We previously reported reduced serum levels of IGF-I with acute ND (33) and reduced IGF-I protein levels in the diaphragm muscle of rats subjected to varying degrees of food restriction and body weight loss (32). Furthermore, we reported significant attenuation of diaphragm fiber atrophy in rats after 3 days of complete ND with the concomitant administration of IGF-I by constant infusion (32). In addition, in rats provided with 50% of caloric and protein needs, IGF-I infusion completely prevented atrophy of any diaphragm fiber type (31). Although it would thus appear that IGF-I is a major signal in the context of acute ND, this does not preclude other important influences, such as those mediated by insulin, amino acids, and enhanced endogenous corticosteroid production.
It has recently been reported that the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway (Fig. 1) is a major regulator of skeletal muscle hypertrophy and atrophy (5). We hypothesize that additional important signaling pathways are also involved in acute ND-induced muscle atrophy. The aim of this study was therefore to evaluate the effects of short-term malnutrition on three important signal transduction pathways (Fig. 1) involved in protein turnover in skeletal muscle. The signaling pathways are those in which IGF-I (5, 46), insulin (23, 41, 45), amino acids (1, 23, 41, 58), and corticosteroids (37, 51, 52) have direct or indirect effects. Our rationale for comprehensive evaluation of three signal transduction pathways important in protein turnover was to provide a firm basis for future mechanistic studies to improve our understanding of potential molecular targets in the management of disordered protein turnover and diaphragm fiber atrophy with short-term ND.
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METHODS |
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14–16 h of fasting (see Critique of Methods for methodological rationale for both groups). The experimental period lasted 4 days. The animals were individually housed with a 12:12-h dark-light cycle, and ambient temperature was maintained at 22°C. The research protocol was approved by the Burns and Allen Research Institute Animal Care and Use Committee of Cedars-Sinai Medical Center. IGF-I mRNA Analysis Total RNA extraction. Total RNA was extracted from 50-mg samples of the left costal diaphragm with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Quality and concentrations of total RNA were determined with a spectrophotometer (SmartSpec 3000, Bio-Rad, Hercules, CA). Samples were stored at –80°C in RNase-free water until analysis.
Oligonucleotides. The primers for IGF-I and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were designed on the basis of published rat cDNA sequences. Primer sequences for IGF-I (53) [GenBank accession no. X06107 (gi:56424)] were as follows: upstream (5' to 3'), AAG CCT ACA AAG TCA GCT CG (bp 595–614); downstream (5' to 3'), GGT CTT GTT TCC TGC ACT TC (bp 760–741). Primer sequences for GAPDH (42) [GenBank accession no. X02231 (gi:56187)] were as follows: upstream (5' to 3'), CAT CAA CGA CCC CTT CAT TGA (bp 161–181); downstream (5' to 3'), ATG ATG TTC TGG GCT GCC CCA (bp 685–665). The expected lengths of the RT-PCR products were 114 bp for IGF-I and 525 bp for GAPDH. GAPDH is a valid housekeeping gene, because it is not affected in catabolic states such as malnutrition and corticosteroid treatment.
Semiquantitative RT-PCR. Oligo(dT) primer (Invitrogen) and Omniscript RT kit (Qiagen, Valencia, CA) were used for RT of 2 µg of total RNA, which yielded 20 µl of cDNA. RT-generated cDNA for IGF-I and GAPDH were amplified using PCR (MJ Research thermal cycler) with the following experimental conditions: initial denaturation at 95°C for 3 min followed by 30 cycles (95°C for 30 s, 60°C for 45 s, and 72°C for 2 min). From each PCR product, 10 µl were loaded on 4% agarose gels and electrophoresed for separation using ethidium bromide for visualization under ultraviolet light. The relative amounts of the PCR products were measured by densitometry (Kodak Electrophoresis Documentation and Analysis System 120) and normalized to levels of GAPDH.
Protein Analyses Protein extraction. Soluble protein was extracted from 50-mg samples of the right costal diaphragm in cold cell lysis buffer at a 1:10 ratio (Cell Signaling Technologies, Beverly, MA) according to the manufacturer's protocol. For analysis of mTOR only, 0.4% CHAPS was added to the lysis buffer. Homogenization was performed with a Polytron homogenizer, and homogenates were centrifuged at 14,000 rpm. The supernatant was separated into aliquots in microcentrifuge tubes. Protein concentration was determined using a commercial protein assay kit (Bio-Rad) on the basis of the Bradford (6) method and measured with a spectrophotometer (SmartSpec 3000). For mTOR analysis only, the lysate was immunoprecipitated with the primary antibody overnight at 4°C, protein A-agarose beads (Sigma Chemical, St. Louis, MO) were added, and the sample was incubated for another 3 h and washed before the pellet was resuspended in SDS sample buffer and subjected to electrophoresis.
SDS-PAGE and Western blotting. Samples were boiled and cooled before they were used for electrophoresis. Protein extracts were loaded on 4–20% linear gradient gels, except initiation factor 4E-binding protein 1 (4E-BP1) and mTOR, for which 10% polyacrylamide gels were used. Proteins were then electrophoretically transferred to nitrocellulose membranes. Blots were incubated with primary antibodies at 4°C overnight, washed, and incubated with an appropriate secondary antibody at room temperature for 1 h. The blots were visualized after development with enhanced chemiluminescence reagents (streptavidin-horseradish peroxidase; Amersham, Piscataway, NJ) according to the manufacturer's protocol. In a few instances, the blots were reused: they were exposed to stripping buffer (Restore, Pierce Biotechnology, Rockford, IL) and reprobed with a different antibody. Blots were exposed to X-ray film in a cassette, the films were scanned, and identified bands were analyzed by densitometry using a Kodak Analysis System. Western blot data from the ND group were expressed relative to measured mean values from the Ctl group.
Primary antibodies.
Blots were incubated with antibodies for total Akt, phosphorylated Akt at the COOH terminus (Ser473), total mTOR, phosphorylated mTOR at the regulatory domain (Ser2448), phosphorylated (Thr389) 70-kDa ribosomal protein S6 kinase (p70S6K), the site most closely linked to growth factor stimulation, phosphorylated glycogen synthase kinase (GSK)-3 (Ser21) and GSK-3 (Ser9), ERK1 and ERK2 (p44 and p42, respectively), and phosphorylated ERK1 (Thr202) and ERK2 (Tyr204), obtained from Cell Signaling Technologies. Antibodies for total p70S6K, total GSK-3 and GSK-3, and total 4E-BP1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific 4E-BP1 (PHAS-I) antibody was obtained from Zymed Laboratories (South San Francisco, CA).
Statistical Analysis
The distribution of all data was tested for normality, and statistical analysis was performed using a one-way ANOVA (SigmaStat version 2.0, Jandel, Richmond, CA) to compare differences between the independent groups. An level of 0.05 was used to determine significance. Values are means ± SE.
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RESULTS |
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-isoform of 4E-BP1 was essentially nonexistent (P < 0.0001), the partially phosphorylated -isoform was significantly reduced (56%, P < 0.01), and the unphosphorylated -isoform was significantly increased (>2-fold, P < 0.001).
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were increased (by 55%, P < 0.05; Fig. 5A) and the levels of phosphorylated (Ser9) GSK-3 were similarly significantly increased (by 45%, P < 0.05; Fig. 5B) in the ND diaphragm. By contrast, levels of total GSK-3 and GSK-3 were unchanged by ND (Fig. 5, A and B).
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DISCUSSION
Severe short-term ND resulted in significant loss of diaphragm muscle mass. Although the concentration of total protein for signaling intermediates was preserved with ND, altered phosphorylation states of several key proteins of the three signal transduction pathways (important in muscle protein turnover) were comprehensively evaluated.
Critique of Methods
Nutritional paradigm.
In developing our nutritional paradigm, our intent was to avoid a postprandial state while still evaluating the muscle signaling milieu that would be most representative of continued muscle fiber growth in free-eating Ctl animals and inhibition of muscle fiber growth/atrophy in ND animals. To achieve this, food was withdrawn from free-eating Ctl rats 6–8 h before terminal experiments. The rationale for this is that >80% of food intake in normal rats occurs during the night (i.e., darkness) (unpublished observations; Refs. 3, 23). Thus our approach simulates a free-eating state while avoiding phosphorylation changes from recent food intake [
1 h postprandial, as commonly reported in the literature (1, 2, 27, 57)] and affords sufficient time for gastric emptying and food absorption. In the ND animals, experiments were performed 18 h after food was provided. Although it is likely that most, if not all, of the food was consumed fairly rapidly, on several occasions, some residual food was observed after a few hours. Thus the ND animals were subjected to 14–16 h of fasting. We believe that this condition best represents the muscle signaling milieu responsible for muscle fiber atrophy and an appropriate contrast with the free-eating Ctl animals.
Choice of diaphragm muscle. There is a strong rationale for pursuing studies on the diaphragm, which, as one of the major primary inspiratory muscles constituting the vital air pump of the chest, enables adequate ventilation under resting conditions or with increased demand, as may occur with acute or chronic lung disorders. Clinically, diaphragm dysfunction has a significant impact on the morbidity and/or mortality of patients with acute or chronic lung diseases (29). Our intent was not to generalize our findings to limb muscles, which exhibit very different activation histories (i.e., not phasically recruited throughout life, like the diaphragm, and often quiescent for long periods), loading conditions, fiber type proportions, muscle architecture, and biochemical properties.
Animal model of severe short-term ND: clinical justification.
It has recently been emphasized that in the early stage of acute critical illness, there is a low rate of adequate nutrition (13, 19, 38, 44), including the development of malnutrition in pre- (20) and postoperative patients (26) and acute protein/energy malnutrition in children (8, 9). Reasons for this include underprescription and suboptimal delivery of nutrients (13, 38). Further acute disease processes often preclude giving adequate nutritional support in the short term (13) because of hemodynamic instability and/or restriction of intake by mouth. In such cases, resuscitative maintenance fluids promote only 20–25% of caloric support. Furthermore, patients may not tolerate or absorb enteral supplements because of adynamic ileus or other gastrointestinal problems, bowel wall edema and villous atrophy, abdominal bloating, and discomfort (7, 11, 12, 39).
Although previously healthy patients can tolerate short-term ND without any problems, patients with comorbidities and nutritional depletion of cachectic states (e.g., chronic congestive heart failure and chronic obstructive pulmonary disease) can suffer a further stepped reduction in respiratory and limb muscle mass that may be difficult to reverse or prevent. A further critical reduction in respiratory muscle mass may significantly prolong attempts to wean patients from mechanical ventilation (25). By contrast, even small improvements in respiratory muscle bulk and strength may have profound influences in the ability to liberate patients from mechanical ventilation (28, 43).
Signal Transduction Pathways and Downstream Effectors Although the phosphorylation states of several key proteins of the three signal transduction pathways evaluated in the diaphragm were altered after 4 days of ND, the total protein concentration for all signaling intermediates was preserved in its entirety during muscle atrophy. We postulate that, as total muscle protein decreased with loss of muscle mass, there was simply a proportional decrease in those signaling protein intermediates. Thus, relative to muscle protein concentration after ND, these signaling proteins would be maintained. However, the proportions of many phosphorylated signaling proteins may have been altered by the impact of the ND paradigm on the energy production system, resulting in a shortage of phosphagen-rich nucleotides, which ultimately impaired the ability of the muscle to maintain the active process of phosphorylation of these signaling intermediates.
PI3K-Akt-mTOR pathway. Ligand (e.g., insulin and IGF-I) binding to members of the receptor tyrosine kinase family of growth factor receptors results in a conformational change, which leads to autophosphorylation of multiple tyrosine residues (Fig. 1). For example, PI3K can be activated by direct association with tyrosine-phosphorylated IGF-I receptor or association with insulin receptor substrate 1. Through a number of steps, activated PI3K facilitates the phosphorylation of the serine-threonine kinase Akt (10). Recent data (in muscle cell cultures and intact rat limb muscle) support the hypothesis that enhanced Akt activity results in phosphorylation of a key regulatory domain of mTOR at Ser2448 (41, 45, 49). Downstream targets of mTOR include 4E-BP1 and p70S6K. With a low demand for mRNA translation, eukaryotic initiation factor 4E (eIF4E) is sequestered by 4E-BP1, preventing interaction with other initiation factors. Phosphorylation of 4E-BP1 destabilizes the eIF4E·4E-BP1 complex with release of eIF4E and the start of translation initiation (45). p70S6K phosphorylates S6, a 40S ribosomal protein. Ribosomes phosphorylated on S6 have augmented binding capacity and stimulate translation of mRNAs, which encode components of the protein synthesis apparatus (15).
Recent in vivo data reported by Bodine and co-workers (5) highlight the importance of the Akt-mTOR pathway as a crucial regulator of muscle fiber size. A compensatory hypertrophy model in rat limb muscles demonstrated phosphorylation of Akt and downstream targets 4E-BP1 and p70S6K. Rapamycin (an mTOR inhibitor) inhibited 95% of the hypertrophic response. By contrast, with hindlimb suspension, muscle atrophy was accompanied by decreased phosphorylation of Akt and downstream effectors. Genetic manipulation of the Akt-mTOR pathway confirms its importance in the genesis of muscle fiber hypertrophy or prevention of atrophy (5). In the present study, reduced phosphorylation of Akt, mTOR, and downstream effectors 4E-BP1 and p70S6K was observed in our model of severe short-term ND. This is also the first demonstration of significantly reduced phosphorylation of mTOR at Ser2448 with malnutrition in skeletal muscle. Thus a significant impact on impaired muscle mRNA translation initiation and translational efficiency is likely mediated by this pathway to impair muscle protein synthesis. Furthermore, reduced phosphorylation of Akt may also significantly impact pathways mediating muscle protein degradation (see below).
Key signaling molecules of this pathway, including downstream effectors, can be influenced by reduced circulating and/or local muscle levels of insulin (23, 41, 45), IGF-I (5, 47), or amino acids (1, 23, 41, 58), as well as enhanced endogenous levels of corticosteroids (37, 51, 52), all of which may be evident with acute severe ND. For example, p70S6K and/or 4E-BP1 can be affected by all the above-mentioned factors (50). Furthermore, mTOR is sensitive to glucose, amino acids, and energy balance, all of which would be expected to be impacted by the severe ND imposed in the present study (15, 41, 50). Recent studies have shown that leucine is the most potent of the branched-chain amino acids in enhancing muscle mRNA translation and that the regulatory role of leucine may be specific for muscle (2).
Our study confirms that the PI3K-Akt-mTOR pathway is significantly affected by severe short-term ND at multiple levels. However, there is paucity of literature on the impact on other signal transduction pathways that can influence muscle protein synthesis. This needs to be further explored, if potential molecular targets, redundancy of pathways, and adaptive changes to severe nutritional insults are to be fully elucidated.
PI3K-Akt-GSK-3 pathway.
GSK-3, a known substrate of Akt, is involved in numerous processes, including glycogen synthesis, protein synthesis, and transcription factor activity (for review, see Ref. 17). Akt phosphorylates and inactivates GSK-3, which diminishes its inhibitory effect on eIF2B. The latter regulates the binding state of eIF2, which acts to shuttle the initiator methionyl tRNA to the 40S ribosome, thus promoting protein synthesis (50). Inhibition of GSK-3 produces hypertrophy of skeletal myotubes in culture (55). In the present study, the significant increments in phosphorylated GSK-3 and GSK-3 were therefore unexpected, inasmuch as our model was characterized by decreased phosphorylation of Akt and prominent muscle fiber atrophy. The unexpected increase in GSK could be explained as follows: 1) GSK-3 could be inactivated by Akt-independent mechanisms. Indeed, phosphorylation by other kinases, such as PKA, PKC-
, MAPK- 1 (also known as p90RSK), and p70S6K (17), has been reported to occur at serine and to inactivate GSK-3. 2) Proteolytic release of amino acids could inhibit GSK-3, possibly via influences on p70S6K (17). 3) The inhibition of GSK-3 may reflect adaptive responses to severe short-term ND. Depletion of glycogen stores is itself a stimulus to promote greater activation of glycogen synthase (presumably through inhibition of GSK-3). One can also speculate that our results regarding GSK-3 reflect adaptations to offset disordered protein turnover and promotion of muscle protein synthesis or other functions of GSK-3 unrelated to glycogen or protein synthesis. It should also be stressed that the increased phosphorylation of GSK-3 and GSK-3 in the present study reflects one point in time at 4 days. Indeed, preliminary data from our laboratory show that at 1 day after acute ND GSK-3 is twice as elevated as at 4 days after acute ND (unpublished observations). Thus the level of GSK-3 inactivation appears to decrease with time, reflecting a dynamic state.
MAPK-ERK pathway. In the present study, a reduction in phosphorylated ERK1 was observed with ND. In vitro, ERK has been shown to phosphorylate 4E-BP1 independent of any PI3K-Akt signaling (14, 35). In addition, further signaling through ERK-interacting kinase (MNK) may also affect mRNA translation (56). Thus reduced phosphorylated ERK1 may negatively affect translational factors and the translational apparatus important in muscle fiber protein synthesis and maintenance of growth.
Signal Transduction Pathways and Protein Degradation Recently, there has been an increased appreciation of signal transduction events mediating muscle fiber atrophy or hypertrophy that appear to highlight and depend on Akt (24, 47, 48, 54). The importance of these events is that they link and bridge the synthesis and degradation arms of protein turnover. Thus activation of Akt1 can influence signaling pathways, as described above and in Fig. 1, which mediate muscle protein synthesis, while at the same time phosphorylating and inhibiting forkhead box o (Foxo) transcription factors and, thus, blocking their induction of muscle-specific E3 ligases [e.g., MAFbx (atrogin-1) and MuRF1], which are important in ubiquitin-proteasome-mediated proteolysis (4, 21). By contrast, dephosphorylation of Akt1 (as described in our study) would be expected to activate Foxo transcription factors with the subsequent transcription of MAFbx, MuRF1, and other atrophy-related genes to promote muscle protein degradation and wasting. In our recent studies with acute ND, this indeed appears to be the case (30).
Conclusions and Future Directions The present study presents a comprehensive descriptive analysis of aberrations in the muscle protein synthetic pathways, with strong inference for involvement of proteolytic pathways (as suggested above). This forms the framework on which key questions can be addressed (in a cause-and-effect manner) in the genesis of appropriate therapeutic molecular targets. For example, it would appear to be intuitively obvious that influencing downstream targets would engender more specific therapeutic effects, inasmuch as upstream proteins have protean functions, some of which are key to cell survival or impact on other cellular systems affecting function (e.g., effect of rapamycin, an mTOR inhibitor, on cellular immune function). Furthermore, it is not known whether downstream targets (e.g., 4E-BP1) have muscle/tissue specificity. This would be important to address whether the goal of therapy is to increase lean muscle mass in cachectic states and not other compartments, such as fat mass. Other useful insights might include research into redundancy or adaptability of the various signal transduction pathways, which could serve to limit or augment therapeutic influences or another signal transduction pathway. Lastly, the bridging of both arms of protein turnover by Akt needs to be further explored.
In conclusion, severe diaphragm muscle wasting induced by short-term ND was accompanied by significant alterations of key proteins from the three major signal transduction pathways involved in protein turnover. This forms the framework for future studies aimed at identifying appropriate therapeutic targets in the management of short-term nutritionally induced cachectic states.
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FOOTNOTES |
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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.
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negatively regulates myotube hypertrophy. Am J Physiol Cell Physiol 283: C545–C551, 2002.This article has been cited by other articles:
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  M. I. Lewis Mechanisms underlying myogenesis: complex and likely to become more so! J Appl Physiol, December 1, 2006; 101(6): 1539 - 1540. [Full Text] [PDF]
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