FINAL ACCEPTED VERSION JAP-01233-2005.R1
Effect of Severe Short-term Malnutrition on Diaphragm Muscle
Signal Transduction Pathways Influencing Protein Turnover
Michael I. Lewis1, 2, Sue C. Bodine3, 4, Nader Kamangar1, Xuan Xu1,
Xiaoyu Da1, and Mario Fournier1, 2
1Division of Pulmonary/Critical Care Medicine
The Burns & Allen Research Institute, Cedars-Sinai Medical Center
2The David Geffen School of Medicine at the University of California Los Angeles
Los Angeles, CA 90048 and
3Section of Neurobiology, Physiology, and Behavior, College of Biological Sciences
4Department of Physiology and Membrane Biology
University of California Davis School of Medicine
Davis, CA 95616
Running Title: Diaphragm muscle signaling with malnutrition
ADDRESS CORRESPONDENCE TO:
Michael I. Lewis, M.D.
Cedars-Sinai Medical Center
8700 Beverly Boulevard, Room 6732
Los Angeles, California 90048
TEL: (310) 423-1832; FAX (310) 423-0129
E-MAIL: [email protected]
Articles in PresS. J Appl Physiol (February 16, 2006). doi:10.1152/japplphysiol.01233.2005
Copyright © 2006 by the American Physiological Society.
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ABSTRACT
The aim of this study was to evaluate the effect of nutritional deprivation (ND) on signal
transduction pathways impinging on the translational apparatus in the diaphragm muscle.
Male rats were divided into 2 groups: 1) ND (20% of usual food intake) for 4 days with
water provided at libitum; and 2) free-eating controls (CTL). Total protein and RNA were
extracted from the diaphragm. IGF-I mRNA was analyzed by RT-PCR. Protein analyses of
key cytoplasmic proteins for 3 signaling pathways deemed important in influencing protein
turnover were performed by Western blot. Body weight was reduced 30% with ND while
CTL gained 17%. Diaphragm mass decreased by 29% with ND. Muscle IGF-I mRNA
abundance was reduced by 63% in ND animals. PI3 kinase/Akt/mTOR pathway: ND
resulted in a 55% reduction in phosphorylated (Ser473) Akt. The phosphorylation of mTOR
at Ser2448 was reduced by 85% with ND. Downstream effectors important in translation
initiation were also impacted by ND. Phosphorylated (Thr389) p70S6K was significantly
reduced (35%) with ND. The translational repressor 4E-BP1 was also significantly
dephosphorylated with ND. PI3 kinase/Akt/Glycogen Synthase Kinase (GSK)-3 pathway:
The phosphorylation (Ser21/9) of both GSK-3α/β was increased (55/45%) with ND. MAP
kinase/ERK pathway: The phosphorylation (Thr202/Tyr204) of both ERK1/2 (p44/p42) was
reduced (64/55%) with ND. The concentrations of total protein for all signaling
intermediates of the 3 pathways were 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.
Key Words: Nutritional deprivation; muscle fiber atrophy; cachexia; mRNA translation
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INTRODUCTION
Protein turnover refers to the normal remodeling process whereby protein stores are
continually undergoing removal of old proteins (degradation) and replacement with new
ones (synthesis) (40). Disordered protein turnover if severe, disturbs this delicate balance,
resulting in significant loss of protein, particularly in skeletal muscles, the major protein
reservoir of the body (40). Protein metabolism is very sensitive to nutritional state, with
several studies demonstrating reductions in whole mixed muscle protein synthesis and
enhanced degradation in skeletal muscles (including the diaphragm) following acute
nutritional deprivation (ND; 16,18,22,34).
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 have 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). Further, we reported significant
attenuation of the diaphragm fiber atrophy in rats following 3 days of complete ND with
the concomitant administration of IGF-I by constant infusion (32). In addition, IGF-I
infusion in rats provided with 50% of caloric and protein needs completely prevented
atrophy of any diaphragm fiber type (31). While 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.
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It has recently been reported that the PI3K/Akt/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 with acute ND-induced muscle atrophy.
The aim of this study was therefore to evaluate the effects of short-term malnutrition on 3
important signal transduction pathways (Fig. 1) involved in protein turnover in skeletal
muscle. The signaling pathways chosen are those in which IGF-I (e.g., 5,46), insulin (e.g.,
23,41,45), amino acids (e.g., 1,23,41,58) and corticosteroids (e.g., 37,51,52) have been
shown to impact either directly or indirectly. Our rationale for comprehensive evaluation
of 3 signal transduction pathways important in protein turnover was to provide a firm basis
for future mechanistic studies geared at improving our understanding of potential
molecular targets in the management of disordered protein turnover and diaphragm fiber
atrophy with short-term ND.
The studies were performed on the diaphragm muscle, one of the major primary
inspiratory muscles. Reduced muscle bulk with ND may have important consequences on
the diaphragm with regard to functional force reserve and endurance capacity, which under
conditions of increase demand or excessive loading can lead to task failure (i.e., ventilatory
failure) or prolong or prevent weaning from mechanical ventilation. These issues are of
major importance in patients with both acute and chronic lung disorders (29).
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METHODS
Animal Groups and Nutritional Paradigm
Young adult male Sprague Dawley rats (initial body weight: 167 ± 5 g) were
studied. The animals were divided into 2 groups: 1) a free-eating control group (CTL; n =
10) who had ad libitum access to food and water, and 2) a nutritionally deprived group
(ND; n = 9) who were fed 20% of their normal food intake (Purina rat chow) all at once
with water provided ad libitum. Specifically, food was withdrawn from free-eating CTL
animals 6 to 8 hours prior to terminal experiments. Terminal experiments were performed
in ND animals 18 hours following food provision, which resulted in at least 14 to 16 hours
of fasting (see Critique of Methods in Discussion for methodologic rationale in both
groups). The experimental period lasted 4 days. Animals were individually housed with a
dark: light cycle of 12 hours each and ambient temperature 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 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 based on published rat cDNA sequences. Primers sequences for
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IGF-I (53; GenBank accession no. X06107 [gi:56424]) were the following: upstream (5’ to
3’) AAG CCT ACA AAG TCA GCT CG (bp 595–614) and downstream (5’ to 3’) GGT
CTT GTT TCC TGC ACT TC (bp 760-741). Primers sequences for GAPDH (42;
GenBank accession no. X02231 [gi:56187]) were the following: upstream (5’ to 3’) CAT
CAA CGA CCC CTT CAT TGA (bp 161-181) and downstream (5’ to 3’) ATG ATG TTC
TGG GCT GCC CCA (bp 685-665). The expected lengths of the reverse transcriptase-
polymerase chain reaction (RT-PCR) products were 114 bp for IGF-I and 525 bp for
GAPDH. GAPDH is a valid housekeeping gene since it is not affected in catabolic states
such malnutrition and corticosteroid treatments.
Semiquantitative RT-PCR: Two µg total RNA was reverse transcribed using oligo-dt
primer (Invitrogen) and Omniscript RT kit (Qiagen, Valencia, CA) and reactions yielded
20 µl of cDNA. RT-generated cDNA for both 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 sec, 60°C for 45 sec
and 72°C for 2 min). Ten µl from each PCR products 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 a 1:10 ratio of cold cell lysis buffer (Cell Signaling Technologies, Beverly,
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MA) according to manufacturer’s protocol. For mTOR analysis only, 0.4% CHAPS was
also added to the lysis buffer. Homogenization was performed with a Polytron
homogenizer and homogenates were centrifuged at 14,000 RPM. The supernatant was
aliquoted in microcentrifuge tubes. Protein concentration was determined using a
commercial protein assay kit (Bio-Rad) based on the Bradford (6) method and measured
with a spectrophotometer (SmartSpec 3000, Bio-Rad). For mTOR analysis only,
immunoprecipitation was performed by incubating the lysate with the primary antibody
overnight at 4°C and by adding protein A agarose beads (Sigma Chemicals, Saint Louis,
MO) for a further 3 hours incubation, washing and resuspending the pellet in SDS sample
buffer prior to electrophoresis.
SDS-PAGE and Western blotting: Samples were boiled and cooled prior to being used for
electrophoresis. Protein extracts were loaded on 4-20% linear gradient gels, except for 4E-
BP1 and mTOR, which utilized 10% polyacrylamide gels. 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 hour. The blots were visualized following development
with enhanced chemiluminescence reagents (ECL streptavidin-HRP, Amersham,
Piscataway, NJ) according to manufacturer’s protocol. In a few instances blots were re-
used by exposing them to stripping buffer (Restore , Pierce Biotechnology, Rockford, IL)
and re-probed with a different antibody. Blots were exposed to X-ray film in a cassette, the
films scanned, and identified bands 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.
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Primary antibodies: Blots were incubated with the following antibodies obtained from
Cell Signaling Technologies: Total Akt, phosphorylated Akt at the C-terminus (Ser473),
total mTOR, phosphorylated mTOR at the regulatory domain (Ser2448), phosphorylated
p70S6K (Thr389), the site most closely linked to growth factor stimulation, phosphorylated
GSK-3α/β (Ser21/9), ERK1/2 (p44/p42) and phosphorylated ERK1/2 (Thr202/Tyr204).
Antibodies for total p70S6K, total GSK-3α/β and total 4E-BP1 were obtained from Santa
Cruz Biotechnologies (Santa Cruz, CA). Phosphospecific 4E-BP1 (PHAS-I) antibody was
from Zymed Laboratories (South San Francisco, CA).
Statistical Analysis
The distribution of all data was tested for normality and statistical analysis was then
performed using a one-way ANOVA (SigmaStat v. 2.0, Jandel, Richmond, CA) to
compare differences between the independent groups. An α level of 0.05 was used to
determine significance. Values are means ± SEM.
RESULTS
Body and muscle weights
In ND rats, daily body weights declined progressively, with a significant decrement
(by 30%; P < 0.01) after 4 days (Fig. 2A). By contrast, the body weight of CTL animals
increased (by 17%; P < 0.05; Fig. 2A). Thus after 4 days, the body weight of ND rats was
47% below expected (P < 0.001). In ND rats, diaphragm muscle weight was reduced by
29% compared to CTL (P < 0.0001; Fig. 2B). In ND animals, muscle weights for the
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tibialis anterior, medial gastrocnemius and extensor digitorum longus were reduced by
26.4 to 28.7% compared to CTL (P < 0.001; Fig. 2B & C), while that of the soleus was
reduced by 19.4% (P < 0.05; Fig. 2C).
Diaphragm muscle IGF-I mRNA
A marked reduction in the abundance of IGF-I mRNA was observed in the
diaphragm of ND rats after 4 days of severe ND (decreased 63% compared to CTL; P <
0.001; Fig. 3).
Signal transduction pathways: Diaphragm muscle protein analysis
PI3 Kinase/Akt/mTOR pathway. ND resulted in a significant reduction (55%) in
phosphorylated (Ser473) Akt in diaphragm muscle compared to CTL (P < 0.01; Fig. 4A).
By contrast, total Akt was not significantly different between the groups (Fig. 4A). The
impact of ND on the phosphorylation of mTOR at Ser2448 was even more striking
compared to CTL (reduced by 85%; P < 0.01; Fig. 4B). Total mTOR was not significantly
different between the groups. ND also impacted downstream effectors important in mRNA
translation. With ND, a 35% reduction in phosphorylated (Thr389) p70S6K was observed in
the diaphragm of ND rats compared to CTL (P < 0.05; Fig. 4C), while no change was
noted in total p70S6K between the groups. Further, the phosphorylation state of the
translational repressor, 4E-BP1 (PHAS-I) was also evaluated (Fig. 4D). With ND, the fully
phosphorylated γ isoform of 4E-BP1 was essentially non-existent (P < 0.0001) and the
partially phosphorylated β isoform was significantly reduced (56%; P < 0.01), while the
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unphosphorylated α form of 4E-BP1 was significantly increased (greater than 2-fold; P <
0.001).
PI3 Kinase/Akt/GSK-3 pathway. The levels of phosphorylated (Ser21) GSK-3α were
increased (by 55%; P < 0.05; Fig. 5A). Similarly, levels of phosphorylated (Ser9) GSK-3β
were also significantly increased in the ND diaphragm (by 45%; P < 0.05; Fig. 5B). By
contrast, levels of both total GSK-3α and GSK-3β were unchanged by ND (Fig. 5A & B).
MAP Kinase/ERK pathway. A significant reduction in phosphorylated (Thr202) ERK1
(p44) was observed in the ND diaphragm (by 64%; P < 0.001; Fig. 5C). Levels of
phosphorylated (Tyr204) ERK2 (p42) were also significantly decreased in the diaphragm of
ND animals (by 45%; P < 0.01; Fig. 5D). By contrast, levels of both total ERK1/2
(p44/p42) were not significantly different from CTL following short-term ND (Fig. 5C &
D).
DISCUSSION
Severe short-term ND resulted in significant loss of diaphragm muscle mass. While
the concentration of total protein for signaling intermediates was preserved with ND, there
were altered phosphorylation states of several key proteins of the 3 signal transduction
pathways (important in muscle protein turnover) that were comprehensively evaluated.
Critique of Methods
Nutritional Paradigm: In developing our nutritional paradigm, our intent was to avoid a
post-prandial state while still evaluating the muscle signaling milieu that would be most
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representative of continued muscle fiber growth in free-eating CTL animals and inhibition
of muscle fiber growth/atrophy in the ND animals. To achieve this, food was withdrawn
from free-eating CTL rats 6 to 8 hours prior to terminal experiments. The rationale behind
this is the fact that greater that 80% of food intake in normal rats occurs during the night
(i.e., darkness; both our own observations and those reported in literature, e.g., 3,23). Thus
our approach simulates a free-eating state, while avoiding phosphorylation changes from
recent food intake (~1 hour post-prandial as commonly reported in the literature, e.g.,
1,2,27,57), and affords sufficient time for gastric emptying and food absorption. In the ND
animals, experiments were performed 18 hours following food provision. While it is likely
that most, if not all, of the food was consumed fairly rapidly, some residual food was
observed on several occasions a few hours following provision. Thus the ND animals were
subjected to at least 14 to 16 hours 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, one of the major primary inspiratory muscles constituting the vital air pump of
the chest, which 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.
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Animal Model of Severe Short-term ND: Clinical Justification: It has recently been
emphasized that early on with acute critical illness, there is a low rate of adequate nutrition
(13,19,38,44), including the development of malnutrition in both pre- (20) and post-
operative patients (26) and acute protein/energy malnutrition in children (8,9). Reasons for
this include both 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 patients kept nil per mouth. In such cases,
resuscitative maintenance fluids promote only ~20-25% of caloric support. Further,
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, etc. (7,11,12,39).
While previously healthy patients can tolerate short-term ND without any
problems, patients with co-morbidities and nutritionally depleted of cachectic states (e.g.,
chronic congestive heart failure, chronic obstructive pulmonary disease, etc.) 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 weaning attempts 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
While the phosphorylation states of several key proteins of the 3 signal
transduction pathways evaluated in the diaphragm were altered following 4 days of ND, it
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is interesting to note that the concentration of total protein for all signaling intermediates
was preserved in its entirety as muscle atrophy was taking place. We postulate that, as total
muscle protein decreased with muscle mass loss, there was simply a proportional decrease
in those signaling protein intermediates. Thus, relative to muscle protein concentration
following ND, these signaling proteins would be maintained. However, the proportions of
many phosphorylated signaling proteins may have been altered subsequent to the impact of
the ND paradigm on the energy production system, resulting in a shortage of phosphagen
rich nucleotides, which ultimately impaired the muscle capacity to maintain the active
process of phosphorylation of these signaling intermediates.
PI3K/Akt/mTOR pathway (Fig. 1). Ligand (e.g., insulin, 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. For example,
PI3K can be activated by direct association with tyrosine phosphorylated IGF-I receptor or
in association with IRS-1. Activated PI3K facilitates through a number of steps 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, the initiation factor 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
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augmented binding capacity and stimulate translation of mRNAs, which encode
components of the protein synthesis apparatus (15).
Recent data by Bodine and co-workers (5) in vivo 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. Of interest, 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
either 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. Further, reduced phosphorylation of Akt may also impact significantly on
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 factors (50). Further,
mTOR is sensitive to glucose, amino acids and energy balance, all of which would be
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expected to be impacted by the severe ND imposed in the current study (15,41,50). With
regard to amino acids, recent studies show 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, which can influence muscle protein
synthesis. This needs to be further explored, if one is to fully elucidate potential molecular
targets, redundancy of pathways and adaptive changes to severe nutritional insults.
PI3K/Akt/GSK-3 pathways (Fig. 1). GSK-3 is a known substrate of Akt and is involved in
numerous processes including glycogen synthesis, protein synthesis and transcription
factor activity (see review 17). Akt phosphorylates and inactivates GSK-3, which relieves
its inhibitory effect on the eukaryotic initiation factor (eIF)2B. 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). Of interest, inhibition of GSK-3β
produces hypertrophy of skeletal myotubes in culture (55). In the present study, the
significant increments in both phosphorylated GSK-3α and β were therefore unexpected as
our model was characterized by decreased phosphorylation of Akt and prominent muscle
fiber atrophy. Possible explanations include the following: 1) Inactivation of GSK-3 could
occur by Akt-independent mechanisms. Indeed other kinases have been reported to serine
phosphorylate and inactivate GSK-3 such as PKA, PKCδ, MAPK-activated protein kinase
1 (also known as p90RSK) and p70S6K (17). 2) Proteolytic release of amino acids could
inhibit GSK-3 possibly via influences on p70S6K (17). 3) The inhibition of GSK-3 may
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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 to other functions of GSK-3 unrelated to glycogen or protein synthesis. It should also be
stressed that the increased phosphorylation of GSK-3α and β in the present study reflects
one point in time at 4 days. Indeed preliminary data from our laboratory show that one day
after acute ND GSK-3β is twice as elevated compared to levels at day 4 (Fournier et al.,
unpublished observations). Thus, the level of GSK-3β inactivation appears to decrease
with time, reflecting a dynamic state.
MAPK/ERK pathway (Fig. 1). 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 impact on mRNA translation (56). Thus reduced
phosphorylated ERK1 may negatively impact on translational factors and the translational
apparatus important in muscle fiber protein synthesis and maintenance of growth.
Signal Transduction Pathways and Protein Degradation
Recently, an increased appreciation of signal transduction events mediating muscle
fiber atrophy or hypertrophy, which appear to highlight and hinge on Akt have been
emphasized (24,47,48,54). The importance of this is that it links and bridges the synthesis
and degradation arms of protein turnover. Thus activation of Akt1 can influence signaling
pathways, as described above and in Fig. 1, that mediate muscle protein synthesis, while at
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the same time phosphorylating and inhibiting forkhead box o (Foxo) transcription factors,
thus blocking their induction of muscle specific E3 ligases (e.g., MAFbx [atrogin-1],
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 be appear intuitively obvious that
influencing downstream targets would engender more specific therapeutic effects 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). Further, it is not known if downstream targets (e.g., 4E-BP1)
have muscle/tissue specificity. This would be important to address if 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
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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 protein from the 3 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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ACKNOWLEDGEMENT
This work was supported by National Institutes of Health grant HL 071227 and by
University of California-Tobacco-Related Disease Research Program grant 7RT-0161.
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LEGENDS
Figure 1. Simplified schematic of signal transduction pathways impinging on the
translational apparatus. Arrows indicate an activation event; blunt-ended symbols indicate
inhibition. RECEPTOR refers to tyrosine kinase family of growth factor receptors (e.g.,
insulin, IGF-I). Abbreviations: PI3K: phosphatidylinositol 3-kinase; Akt: serine/threonine
kinase also known as protein kinase B; mTOR: mammalian target of rapamycin (also known
as FK506-binding protein 12-rapamycin-associated protein, FRAP); 4E-BP1: eukaryotic
initiation factor 4E (eIF4E)-binding protein 1, also known as PHAS-I; p70S6K: 70-kDa
ribosomal protein S6 kinase; ERK1/2: extracellular signal-regulated kinases 1 and 2, also
known as mitogen-activated protein kinases, MAPKs, p44 and p42; GSK3: glycogen
synthase kinase-3; Sos: Son of sevenless guanine nucleotide exchange factor, GRB2: Growth
factor receptor-bound protein 2.
Figure 2. Daily body weights (A), final diaphragm (DIA; B), tibialis anterior (TA; B),
medial gastrocnemius (MG; B), extensor digitorum longus (EDL; C), and soleus (SOL; C)
muscle weights in control (CTL) and nutritionally deprived (ND) animals. Values are
means ± SEM. Note a gradual significant decrement (-30%) in body weight in ND rats (A)
while there was a gain (17%) in body weight in CTL animals. Also note a significant
reduction in the mass of mixed muscles (-26 to -29%; B & C) as well as that of the soleus
(-19%; C) after 4 days of severe short-term ND.
Figure 3. Diaphragm muscle IGF-I mRNA by PCR analysis relative to GAPDH mRNA in
control (CTL) and nutritionally deprived (ND) animals. Values are means ± SEM of the
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ratios. Note a 63% decrease in diaphragm IGF-I abundance after 4 days of severe short-
term ND compared to controls.
Figure 4. Total and phosphorylated (P) (Ser473) Akt (A), total and phosphorylated (P)
(Ser2448) mTOR (B), total and phosphorylated (P) (Thr389) p70S6K (C), and phosphorylation
state of 4E-BP1 (PHAS-I) (D) in the diaphragm of control (CTL) and nutritionally
deprived (ND) animals. Values are means ± SEM relative to mean CTL values, except for
4E-BP1 (D) depicted in mean grey level intensities to allow comparison across isoforms.
Note a significant reduction in phosphorylated Akt (A), mTOR (B), and p70S6K (C)
following 4 days of severe short-term ND while the concentration of total protein for the 3
signaling intermediates was preserved with ND. Also note a significantly increased non-
phosphorylated (α) isoform and significantly reduced fully phosphorylated (γ) and partially
phosphorylated (β) isoforms of 4E-BP1 (D) following 4 days of severe short-term ND
compared to controls.
Figure 5. Total and phosphorylated (P) (Ser21/9) GSK-3α (A) and β (B), and
(Thr202/Tyr204) ERK1 (p44; C) and 2 (p42; D) in the diaphragm of control (CTL) and
nutritionally deprived (ND) animals. Values are means ± SEM relative to mean CTL
values. Note a significant increase in both phosphorylated GSK-3α (A) and β (B)
following 4 days of severe short-term ND compared to controls. In contrast, note a
significant reduction in both phosphorylated ERK1 (p44) (C) and 2 (p42) (D) with ND
compared to controls. Also note that the concentration of total protein for the 4 signaling
intermediates was preserved with ND.
JAP-01233-2005.R1
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Figure 1
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0 1 2 3 4100
125
150
175
200
CTLND
A
Days
Dai
ly B
ody
Wei
ght
(g)
DIA TA MG0
100
200
300
400
500
600B
*
**
CTLND
Mus
cle
Wei
ght
(mg)
EDL SOL0
10
20
30
40
50
60
70
80C
* *
Mus
cle
Wei
ght
(g)
Figure 2
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CTL ND
GAPDH
IGF-I
CTL ND0.00
0.25
0.50
0.75
1.00
*
Rat
io o
f IG
F-I
/GA
PD
H
Figure 3
JAP-01233-2005.R1
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Akt P-Akt0.0
0.2
0.4
0.6
0.8
1.0
1.2 CTLND
*
A
Fol
d C
han
ge
p70 P-p700.0
0.2
0.4
0.6
0.8
1.0
1.2
*
C
Fol
d C
hang
e
mTOR P-mTOR0.0
0.5
1.0
1.5
*
B
Fol
d C
hang
e
0
50
100
150
200
250
*
*
*
D
αααα ββββ γγγγ
Gre
y L
evel
Int
ensi
ty (
x10
3 )
γγγγββββαααα
CTL ND
Akt
P-Akt
CTL ND
P-p70p70
CTL ND
P-mTOR
mTOR CTL ND
Figure 4
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GSK3-αααα P-GSK3-αααα0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
CTL
ND
*A
Fol
d C
han
ge
p44 P-p440.00
0.25
0.50
0.75
1.00
1.25C
*Fol
d C
hang
e
GSK3-ββββ P-GSK3-ββββ0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75*
B
Fol
d C
hang
e
p42 P-p420.00
0.25
0.50
0.75
1.00
1.25D
*
Fol
d C
hang
e
GSK3-ααααGSK3-ββββ
P-GSK3-ααααP-GSK3-ββββ
p44p42
P-p44P-p42
CTL ND CTL ND
Figure 5
