Am J Physiol Endocrinol Metab
281: E676–E682, 2001.
Elevated IGF-II mRNA and phosphorylation of 4E-BP1 and
p70S6k in muscle showing clenbuterol-induced anabolism
A. A. SNEDDON, M. I. DELDAY, J. STEVEN, AND C. A. MALTIN
The Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom
Received 28 February 2001; accepted in final form 30 May 2001
hypertrophy; ␤-agonist; growth factor; protein translation;
insulin-like growth factor II; eukaryotic initiation factor 4E
binding protein-1
more than 3 million people per
annum in Europe and the United States. At present,
there are few therapeutic approaches to the reversal of
this condition, but advances toward new drug development are being made by identifying the precise cellular
signaling mechanism(s) that drives muscle hypertrophy. Many current strategies involve subjecting normal muscle to stimuli that induce a hypertrophic response, so that the underlying changes in gene
expression may be examined. For example, increased
mechanical or electrical activity in muscle provokes
changes in transcript levels for a number of genes (7).
Similar changes in gene expression (22) occur in muscles treated with the anabolic ␤2-adrenoceptor agonist
clenbuterol, which promotes muscle-specific hypertrophy in both normal and pathological states of muscle
and has clinical utility to treat muscle wasting diseases
in humans (23). However, presently, the precise mechMUSCLE WASTING AFFECTS
Address for reprint requests and other correspondence: A. Sneddon, Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, Scotland, UK (E-mail: [email protected]).
E676
anism whereby clenbuterol mediates its effects is unknown.
Evidence suggests that skeletal muscle hypertrophy
is associated with transient activation of insulin-like
growth factor (IGF) gene transcripts; the hypertrophic
stimuli of work overload and passive stretch are both
associated with increased levels of IGF-I transcripts in
muscle (39). A similar role has been proposed for IGFII, but in this instance it has been suggested that the
production of IGF-II may be autocrine or paracrine (36)
and that innervation status (12) together with muscle
activity (6) may operate or cooperate to influence the
level of IGF-II gene transcripts. Because it has been
suggested that the action of clenbuterol may be dependent on both innervation status (22) and activity level
(20), it was speculated that one possible mechanism
through which the drug could elicit the observed effects
on muscle mass and protein metabolism was via the
IGF-II axis. Consequently, the present study was designed to examine the potential role for IGF-II in clenbuterol-induced muscle protein anabolism. In addition,
the expression of H19 was also examined, since IGF-II
and H19 are assumed to be functionally coupled because of their reciprocal imprinting and identical spatiotemporal expression (11, 13). Furthermore, H19 has
been implicated as exhibiting a muscle-specific function (28), and recent evidence suggests it may regulate
IGF-II expression (40); hence, its potential role in hypertrophy was investigated. Because clenbuterol-induced protein accretion appears to be initially mediated via a stimulation of muscle protein synthesis (24),
the effect and timing of clenbuterol action on eukaryotic initiation factor (eIF)4E binding protein-1 (4EBP1) and p70S6k, proteins involved in IGF-induced
activation of protein translation, were also investigated. 4E-BP1 binds to and regulates the availability of
eIF4E, which is thought to be one of the rate-limiting
factors for translational initiation (29). When phosphorylated at the appropriate site(s), 4E-BP1 dissociates from eIF4E, allowing the factor to participate in
translational initiation. p70S6k mediates the phosphorylation of the 40S ribosomal S6 protein leading to an
upregulation of translation of mRNAs encoding ribosomal proteins and elongation factors (15). By investiThe 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.
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
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Sneddon, A. A., M. I. Delday, J. Steven, and C. A.
Maltin. Elevated IGF-II mRNA and phosphorylation of 4EBP1 and p70S6k in muscle showing clenbuterol-induced
anabolism. Am J Physiol Endocrinol Metab 281: E676–E682,
2001.—Muscle wasting affects large numbers of people, but
few therapeutic approaches exist to treat and/or reverse this
condition. The ␤2-adrenoceptor agonist clenbuterol produces
a muscle-specific protein anabolism in both normal and catabolic muscle and has been used to limit muscle wasting in
humans. Because clenbuterol appears to interact with or
mimic innervation, its effect on the expression of the neurotrophic agents insulin-like growth factor (IGF)-II and H19
and their putative pathways was examined in normal rat
plantaris muscle. The results showed that the well-documented early effects of clenbuterol on protein metabolism
were preceded by elevated levels of IGF-II and H19 transcripts together with increased phosphorylation of eukaryotic
initiation factor (eIF)4E binding protein-1 (4E-BP1) and
p70S6k. By 3 days, transcript levels for IGF-II and H19 and
4E-BP1 and p70S6k phosphorylation had returned to control
values. These novel findings indicate that clenbuterol-induced muscle anabolism is potentially mediated, at least in
part, by an IGF-II-induced activation of 4E-BP1 and p70S6k.
CLENBUTEROL ACTIVATION OF 4E-BP1, P70S6K, AND IGF-II
gation of the effect of clenbuterol on the temporal
expression of IGF-II/H19 in muscle and nonmuscle
tissues, and the activation of 4E-BP1 and p70S6k in
relation to the increase in the fractional rate of protein
synthesis, a further insight into the mechanism of
action of the drug would be attained.
METHODS
AJP-Endocrinol Metab • VOL
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the following DNA templates: a rat H19 cDNA fragment
(nucleotides 483–1098) (31), a rat IGF-II cDNA fragment
(nucleotides 978–1434) (8), and a 1.2-kb EcoR1-Pst1 fragment of the rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA (30). Each Northern blot was probed sequentially with IGF-II, H19, and GAPDH probes with stripping in
between. The intensities of the 3.8-kb IGF-II and the 2.5-kb
H19 mRNAs were quantified (Instant Imager, Packard Instrument) after loading variations were corrected for by
GAPDH expression levels. Results were expressed on a within-blot basis as a percentage of the 1-day control. Data were
analyzed using Student’s t-test, and statistical significance
was set at the 5% probability level.
Immunocytochemical detection of IGF-II. Plantaris muscles from control and 1-day clenbuterol-treated animals
(groups 1 and 2; 4 animals/group) were prepared for cryostat
sectioning before being stored in liquid nitrogen. Serial
transverse sections (6 ␮m) were cut, air dried for 30 min at
room temperature, and fixed in fresh ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). The sections were washed 3 ⫻ 10 min in PBS, treated for 5 min with
0.1% Saponin in PBS, washed 2 ⫻ 10 min in PBS, and
incubated overnight at 4°C with primary antibody (1:50 dilution; R&D Systems). After 3 ⫻ 10-min washes with PBS,
secondary antibody (anti-goat-FITC, 1:1,000; Sigma, St.
Louis, MO) was applied, and the sections were incubated for
60 min at room temperature in a humidified chamber. The
slides were then washed as before, treated with 4,6-diamidino-2-phenylindole (DAPI, 1 ng/ml; Sigma) for 5 min,
washed thoroughly in PBS, and mounted in Vectashield
(Vecta Labs). For negative controls, the primary antibody
was incubated with a 1:10 molar ratio excess of IGF-II protein (R&D Systems) for 1 h at room temperature before being
added to the section.
Western immunoblotting. Samples of plantaris muscle (n ⫽
4/treatment) from 1, 2, 3, and 7 days were rapidly crushed
between aluminum blocks that had been precooled to ⫺80°C
and then homogenized on ice for 30 s in 20 mM Tris, pH 7.0,
0.27 M sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100,
1 mM sodium orthovanadate, 50 mM sodium ␤-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride,
1 mM 1,4-dithiothreitol (DTT), 22 ␮M leupeptin, 8 ␮M aprotinin, 16 ␮M trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), 40 ␮M bestatin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and 15 ␮M pepstatin A. The
protein concentrations were assayed [bicinchoninic acid
(BCA) assay kit; Pierce, Rockford, IL], and 150 ␮g (for 4EBP1) and 20 ␮g (for p70S6k) of total protein from each treatment group were combined with Laemmli sample buffer and
subjected to SDS-PAGE analysis. Gels were electrotransferred onto Immobilon-P membrane (Millipore, Bedford, MA)
and blocked for 1 h at room temperature in TBST (Trisbuffered saline, pH 7.6, with 0.1% Tween 20) containing 5%
(wt/vol) dried milk (Marvel, Stafford, UK). Primary antibody
[anti-phospho-4E-BP1 (Thr70), Cell Signaling Technology,
Beverly, MA], p70S6k (Santa Cruz Biotechnology, Santa
Cruz, CA), or anti-phospho-p70S6k [(Thr412) Upstate Biotechnology, Lake Placid, NY] was added in TBST ⫹ 5% Marvel for
1 h at room temperature. Membranes were washed (6 ⫻ 5
min) in TBST and then incubated for 30 min in secondary
antibody conjugated to horseradish peroxidase (Chemicon
International, Harrow, UK). After an additional six washes
(see above), the membranes were left in Tris-buffered saline
(TBS), and chemiluminescent detection was carried out
(Pierce). Specificity of the bands was assessed by the inclusion of an appropriate positive control from the manufacturer.
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Animals and treatments. Male Rowett hooded Lister rats
were used throughout. All procedures and animal husbandry
complied with the Animals (Scientific Procedures) Act 1986.
At 22 days of age, rats were introduced to a semisynthetic,
nutritionally complete control diet (PW-3) (32) for 4 days and
were then divided into 16 groups of equal mean weight.
Groups 1, 3, 5, 7, 9, 11, 13, and 15 were offered PW-3 as
before, whereas groups 2, 4, 6, 8, 10, 12, 14, and 16 were
offered PW-3 containing clenbuterol (3 mg/kg diet). The number of animals in each group is shown in RESULTS. Previous
similar studies have shown that the food intake of either
control or medicated diet is between 10 and 12 g/day (Ref. 33
and unpublished observations). At intervals after starting
the experimental dietary regimen, animals from the control
and clenbuterol groups were euthanized (groups 1 and 2, 1
day; groups 3 and 4, 2 days; groups 5 and 6, 3 days; and
groups 7 and 8, 7 days), and plantaris muscles were rapidly
removed bilaterally, weighed, and, after appropriate preparation, rapidly frozen in liquid nitrogen before analysis. One
muscle from each animal was used for Northern hybridization, and the other muscle was used for either Western
blotting or immunohistochemistry. Samples of gut, liver, and
heart were also removed and frozen in nitrogen before Northern analysis. In addition, after 1 day (groups 9 and 10), 2 days
(groups 11 and 12), 3 days (groups 13 and 14), and 7 days
(groups 15 and 16), plantaris muscle protein synthesis was
measured. Muscles from these animals were also used to
estimate total protein and RNA and DNA content.
Measurement of protein synthesis. Previous studies demonstrated that protein synthesis rates in clenbuterol-treated
animals increase between 1.5 and 3 days (24). Between 1 and
7 days of treatment, the fractional rate of protein synthesis in
plantaris muscle in both control and clenbuterol-treated animals (n ⫽ 6) was measured using the flooding dose technique
(10). Briefly, [2,6-3H]phenylalanine (100 ␮Ci/100 g body wt;
Amersham) combined with 150 mmol/100 g body wt of unlabeled phenylalanine was administered to each rat by the tail
vein (10). After 10 min, animals were killed (10), and the
hindlimbs were removed, stripped of skin, and rapidly
plunged into iced water. After being cooled, the plantaris
muscles were removed bilaterally, with one muscle being
used for measurement of protein synthesis and the other
being used for determination of total protein (19), RNA (27),
and DNA (3) content. Phenylalanine specific activity was
measured (10), and the fractional rate of protein synthesis
(ks; expressed as the percentage of protein synthesized per
day) was calculated using the following relationship (10):
ks ⫽ 100 ⫻ SB/SA ⫻ t, where SA and SB are the specific
radioactivities of free and protein-bound phenylalanine, respectively, and t is the incorporation time in days. For statistical comparison between groups, the data were analyzed
by analysis of variance and Student’s t-test with statistical
significance set at the 5% probability level.
RNA extraction and Northern hybridization analysis. Total
RNA was extracted from tissues (n ⫽ 5/group) using TRIzol
reagent (GIBCO-BRL). Northern blot analysis of RNA was
performed using standard techniques (34). 32P-labeled
probes were generated by random primed labeling by use of
MRNA
E678
CLENBUTEROL ACTIVATION OF 4E-BP1, P70S6K, AND IGF-II
RESULTS
Additionally, no effect of clenbuterol was detected on
the expression levels of H19 mRNA in these tissues
(data not shown).
Immunolocalization of IGF-II in plantaris muscle.
IGF-II immunopositive staining was associated with
some, but not all, myonuclei in muscles from all groups
(Fig. 3, A and B, m⫹ and m⫺). In addition, nonmuscle
cell nuclei stained positive with the DNA dye DAPI
(Fig. 3A, nm) but in general did not show IGF-II positivity (Fig. 3B, nm). The specificity of the IGF-II immunopositive staining was validated by demonstrating
that no signal was obtained by first preincubating the
primary antibody with an excess amount of recombinant IGF-II protein (Fig. 3C).
Western blot analysis of 4E-BP1 phosphorylation in
plantaris muscle. 4E-BP1 can be multiply phosphorylated, leading to reduced electrophoretic mobility, and
typically three bands (␣, ␤, and ␥) are resolved. For
skeletal muscle, the ␥- and ␤-forms appear to predominate, whereas the unphosphorylated ␣-form is very
often of low intensity or absent (37). Bands corresponding to ␥- and ␤-forms were detected in both control and
treated muscles at 1, 2, 3, and 7 days using an antiphospho-4E-BP1 antibody (Fig. 4A). The more highly
phosphorylated form of the protein, the ␥-form, does
not bind eIF4E (18). After 1 and 2 days of treatment
with clenbuterol, the abundance of the ␥-form was
significantly enhanced (Fig. 4, A and B), but by 3 and 7
days, the signal strength for both ␤- and ␥-forms was
similar. A very similar banding pattern was obtained
with an anti-4E-BP1 antibody that recognizes both
phosphorylated and nonphosphorylated forms (not
shown).
Western blot analysis of p70S6k phosphorylation in
plantaris muscle. p70S6k is activated by insulin and
growth factors through phosphorylation at multiple
sites in the protein. Phosphorylation of two sites
(Thr252 and Thr412) has been shown to contribute the
most to activation of p70S6k (38). With the use of an
anti-phospho-p70S6k (Thr412) antibody, phosphorylated
p70S6k was increased 500% after 1 day of clenbuterol
treatment (Fig. 5, A and B). By 3 days, p70S6k activation, as assessed by Thr412 phosphorylation, was down-
Table 1. Effect of clenbuterol on protein metabolism in rat plantaris muscle
Wet Wt, mg
RNA, ␮g
Protein, mg
ks, %/Day
ks 䡠 RNA⫺1 䡠 Protein⫺1 ⫻ 103
18.9 ⫾ 0.7
19.2 ⫾ 2.8
15.3 ⫾ 3.5
13.2 ⫾ 0.9
1.5 ⫾ 0.6
1.7 ⫾ 0.2
1.4 ⫾ 0.4
1.2 ⫾ 0.1
20.5 ⫾ 2.6
26.1 ⫾ 3.3†
23.0 ⫾ 2.5†
11.7 ⫾ 0.6
1.7 ⫾ 0.8
1.9 ⫾ 0.2
1.8 ⫾ 0.3
1.0 ⫾ 0.1
Control
1
2
3
7
Day
Days
Days
Days
62.2 ⫾ 1.3
57.5 ⫾ 5.7
59.5 ⫾ 5.6
79.8 ⫾ 7.5
11.5 ⫾ 0.3
11.1 ⫾ 1.2
11.6 ⫾ 0.8
13.3 ⫾ 1.1
145.2 ⫾ 7.1
122.7 ⫾ 5.0
125.3 ⫾ 9.6
148.0 ⫾ 13.4
Clenbuterol
1
2
3
7
Day
Days
Days
Days
65.3 ⫾ 3.7
58.2 ⫾ 1.8
68.5 ⫾ 4.8*
92.3 ⫾ 7.4*
12.4 ⫾ 0.8
10.8 ⫾ 0.5
13.9 ⫾ 0.9†
15.8 ⫾ 1.5†
150.0 ⫾ 9.7
145.5 ⫾ 2.9‡
173.2 ⫾ 11.4†
179.0 ⫾ 10.6‡
Values are means ⫾ SD, with 6 animals in each group. ks, Fractional rate of protein synthesis. The effect of clenbuterol treatment was
assessed by use of analysis of variance and Student’s t-test: * P ⬍ 0.05, † P ⬍ 0.01, and ‡ P ⬍ 0.001.
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Muscle composition and protein synthesis. The wellknown effects of clenbuterol on the weight of plantaris
muscles were clearly documented in the present study
and are shown in Table 1. RNA content and the fractional rate of protein synthesis were significantly increased in clenbuterol-treated muscles after 2 days
(Table 1). By 7 days of treatment, rates of protein
synthesis had returned to control values, although
RNA contents remained elevated. Changes in RNA and
protein synthesis rates preceded the increase in muscle
protein content, which was evident by 3 days of treatment and was persistent at 7 days (Table 1). No significant changes in body weight were observed (data
not shown), but muscles from the animals offered the
clenbuterol-containing diet showed a significant increase in wet weight after 3 days, concomitant with the
increase in protein content. The changes in RNA and
the fractional rate of protein synthesis occurred
roughly at the same time and preceded the increases in
protein and wet weight as might be expected.
IGF-II and H19 mRNA expression in plantaris muscle. Three major IGF-II mRNA transcripts of 4.6, 3.8,
and 1.2 kb and several minor transcripts of 3.2, 2.7,
and 2.2 kb were detected in plantaris muscle. After 1
day of clenbuterol treatment, the expression of the
most abundant 3.8-kb IGF-II message was increased
80% over control levels (Fig. 1, A and B). Concomitant
increases in the expression of the other IGF-II transcripts were also evident. The level of induction of the
3.8-kb message was increased 50% over control after 2
days, but levels fell thereafter, such that by 7 days
after clenbuterol treatment, levels were downregulated
by 60% compared with control. A single H19 mRNA
transcript of 2.5 kb was detected in plantaris muscle.
Clenbuterol treatment resulted in a 50% increase of
H19 mRNA transcript levels at 1 and 2 days, with a
return to control levels by 3 days (Fig. 1, C and D).
GAPDH transcript levels did not change (Fig. 1C).
IGF-II and H19 expression in nonskeletal muscle
tissues. The administration of clenbuterol did not induce expression of IGF-II mRNA in nonskeletal muscle
tissues such as liver, gut, or heart muscle (Fig. 2).
MRNA
CLENBUTEROL ACTIVATION OF 4E-BP1, P70S6K, AND IGF-II
MRNA
E679
regulated to control levels (Fig. 5B). As a loading control, the Western blot was probed with an antibody
that recognizes both phosphorylated and nonphosphorylated p70S6k protein (Fig. 5A).
DISCUSSION
Many studies (24, 26, 33) have indicated that clenbuterol-induced muscle anabolism is mediated by at
least two mechanisms: an initial transient increase in
the fractional rate of protein synthesis (up to 3–4 days)
followed by a more prolonged depression in the fractional rate of protein degradation. The mechanism
involved in the clenbuterol-stimulated transient increase in protein synthesis is unknown. The current
study addressed the role of IGF-II and H19 and 4EBP1 and p70S6k in this process.
In this study, clenbuterol induced muscle protein
gain at 3 and 7 days, which was preceded by an increase in total RNA content and an increase in the
fractional rate of protein synthesis, which waned after
day 3. Both IGF-II and H19 transcript levels were
elevated by clenbuterol at day 1 and 2 before the
increase in the fractional rate of protein synthesis in
innervated plantaris muscle. Additionally, concomitant elevated phosphorylation of both 4E-BP1 and
p70S6k was also observed at days 1 and 2. The increased protein gain observed at day 7, occurring at a
time when the fractional rate of protein synthesis had
AJP-Endocrinol Metab • VOL
Fig. 2. The effect of clenbuterol on IGF-II transcript levels in nonmuscle tissues. Rats were either untreated (⫺) or treated (⫹) with
clenbuterol for 1 day, and IGF-II levels were then measured by
Northern blotting of total RNA from plantaris muscle (SkM), liver
(Liv), gut, and heart (Hrt). The ethidium bromide (EtBr)-stained gel
is shown below the autoradiograph as a loading control. Representative Northern blots from 3 separate experiments are shown.
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Fig. 1. The effect of clenbuterol on insulin-like growth factor (IGF)-II and
H19 transcript levels in plantaris muscle. Rats were either untreated or
treated with clenbuterol for 1, 2, 3, or 7
days (1d, 2d, 3d, or 7d, respectively). A:
autoradiograph showing transcript
sizes and levels for IGF-II from control
(⫺) and clenbuterol-treated (⫹) rats. B:
histogram showing mean values ⫾ SE
(n ⫽ 5) for the IGF-II 3.8-kb transcript
level from control (open bars) and clenbuterol-treated (solid bars) rats expressed as a percentage of 1d control
value. C: autoradiograph showing
transcript size and levels for H19 (top)
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; bottom) from control (⫺) and clenbuterol-treated (⫹)
rats. D: histogram showing mean values ⫾ SE (n ⫽ 5) for H19 transcript
levels from control (open bars) and
clenbuterol-treated (solid bars) rats expressed as a percentage of 1d control
values. Significance of difference from
control (**P ⬍ 0.01 and *P ⬍ 0.05) was
determined by Student’s t-test.
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CLENBUTEROL ACTIVATION OF 4E-BP1, P70S6K, AND IGF-II
MRNA
Fig. 3. IGF-II immunolocalization in plantaris muscle. Control plantaris muscle (1 day) stained with 4,6-diamidino-2-phenylindole
(DAPI; A) or anti-IGF-II antibody (B). m⫹, Myonuclei showing associated immunopositive staining for IGF-II; m⫺, those that do not
stain for IGF-II but are positive with DAPI; nm, nonmuscle cell
nuclei that do not stain for IGF-II. C: anti-IGF-II antibody preincubated with excess IGF-II protein. Bar, 60 ␮m.
returned to basal level, is most likely explained by the
subsequent decrease in the fractional rate of protein
degradation induced by the drug (33).
In muscle, the anabolic actions of insulin and IGF-I
on protein metabolism are well documented (29).
Downstream signaling from insulin and IGF-I receptors involves multiple pathways leading to the phosphorylation of a number of translation initiation factors, regulatory proteins, and the 40S ribosomal
subunit. The phosphorylation of the 4E-BP1 is a key
AJP-Endocrinol Metab • VOL
Fig. 5. A: Western blot showing Thr412 phosphorylation of p70S6k in
control (⫺) and clenbuterol-treated (⫹) plantaris muscle at 1, 2, 3,
and 7 days using anti-phospho-p70S6k (Thr412; top) and anti-p70S6k
(bottom) antibodies. B: histogram showing mean values ⫾ SE (n ⫽ 4
minimum) for phospho-p70S6k levels from control (open bars) and
clenbuterol-treated (solid bars) rats. Significance of difference from
control (***P ⬍ 0.001) was determined by Student’s t-test.
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Fig. 4. A: Western blot showing abundance of phospho-eukaryotic
initiation factor (eIF) 4E binding protein-1 (4E-BP1) protein in
control (⫺) and clenbuterol-treated (⫹) plantaris muscle at 1, 2, 3,
and 7 days. B: histogram showing mean values ⫾ SE (n ⫽ 4 minimum) for phospho-4E-BP1 levels from control (open bars) and clenbuterol-treated (solid bars) rats. The amount of 4E-BP1 in the ␥-form
is expressed as a percentage of the total eIF4E binding protein-1
(4E-BP1) forms (␤- plus ␥-form). Significance of difference from
control (*P ⬍ 0.05) was determined by Student’s t-test.
CLENBUTEROL ACTIVATION OF 4E-BP1, P70S6K, AND IGF-II
AJP-Endocrinol Metab • VOL
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and inhibits protein synthesis (16, 35). Clenbuterol
increases cAMP levels in muscle, suggesting that the
observed effects on 4E-BP1 and p70S6k phosphorylation are not mediated via the classical ␤2-adrenoceptor
signaling process. However, whether activation of this
latter receptor may play a role in the induction of
IGF-II mRNA is not known.
IGF-II and H19 genes show a very similar spatial/
temporal expression pattern during development (11,
13), and their reciprocal imprinting has been proposed
to be controlled by the possession of common sets of
regulatory elements (17). The transient changes in
H19 gene expression after clenbuterol treatment, occurring over a time scale very similar to those for
IGF-II transcripts, support this proposal. Because the
expression of H19 is downregulated in most adult tissues except skeletal muscle (28), this implicates H19 as
harboring a muscle-specific function. The finding of
increased H19 transcript levels is the first to be reported in skeletal muscle hypertrophy in vivo; however, the precise role of H19 in this case is unclear.
Previous studies implicating IGF-II in the regulation
of hypertrophy have also suggested that autocrine or
paracrine production is important (36). The lack of an
effect of clenbuterol on IGF-II expression in nonmuscle
tissues such as liver and gut, together with the myonuclear localization of IGF-II in the present study,
would be consistent with this suggestion. Furthermore,
the association between elevated IGF-II expression
and increased fractional rate of protein synthesis in
muscle is strengthened by the above finding that clenbuterol does not induce IGF-II expression in nonmuscle tissues (such as gut or liver), where the fractional rate of protein synthesis and protein accretion
are not affected (1, 26). The immunolocalization of
IGF-II to the nucleus is unclear; however, it does demonstrate that IGF-II protein is present in muscle and
not in nonmuscle cells. Receptor-bound IGF-II (as, say,
occurring at the membrane or binding with IGF binding proteins) may result in a masking of the epitope for
the anti-IGF-II antibody and hence remain undetected.
The localization of IGF-II to satellite cells during regeneration (14) and studies showing clenbuterol-induced satellite cell activation (21) could reflect localized production of IGF-II in a manner similar to that
proposed in regenerating muscle. Such local production of growth factors would then lead to receptorligand interactions driving downstream consequences
such as increased protein synthesis, accretion, and the
observed hypertrophy. Future studies will no doubt
address the precise signaling pathway(s) involved in
the phosphorylation of 4E-BP1 and activation of p70S6k
and the role that IGF-II plays in the clenbuterol-mediated process of skeletal muscle anabolism.
We thank the staff of the Small Animal Unit for help and care of
the animals.
This work was funded by a program grant from the Scottish
Executive Rural Affairs Department.
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step in the regulation of protein synthesis, since it
liberates eIF4E for direct binding with mRNA and
subsequent complex formation with eIF4G and the
ribosome. Because it is known that IGF-II binds to both
the insulin and IGF-I receptor (as well as the IGF-II
receptor), it is probable that the downstream consequences of IGF-II binding are similar to those documented for insulin and IGF-I. Indeed, in two different
muscle cell lines, IGF-II administration has been
shown to lead to activation of p70S6k (4). Furthermore,
limited data also indicate a potential role for IGF-II in
the regulation of muscle mass. In response to workinduced hypertrophy (7), IGF-II mRNA levels increased after 2 days of treatment and continued to
remain elevated for the time that hypertrophy was
observed. Additionally, a role for IGF-II in the stimulation/maintenance of protein synthesis has been proposed in cardiac myocytes (9), allowing speculation
that it may play a similar role in skeletal muscle.
Hence, it is possible that the elevated transcript levels
for IGF-II (and H19), the increased phosphorylation of
4E-BP1 and p70S6k, and the increased rates of protein
synthesis represent part of a drug-stimulated pathway
from IGF-II expression to protein synthesis. Previous
data showing clenbuterol-stimulated increases in synthesis rates between 1.5 and 3 days (24) and the
present data demonstrating a significant increase in
rates at 2 and 3 days would support that contention.
The finding, however, that the stimulation of protein
synthesis at day 3 occurred when both IGF-II/H19
levels and 4E-BP1 and p70S6k phosphorylation had
returned to control levels would seem inconsistent with
the proposed model. One explanation may be that
p70S6k activation leads to increased translation of a
number of ribosomal proteins and elongation factors,
resulting in increased overall translational capacity of
the muscle. This may provide a delayed effect in terms
of increased protein synthesis subsequent to p70S6k
inactivation. In addition, the activation of additional
IGF-II pathways (to increase eIF2B or eIF-4E䡠eIF4G
complexes independent of 4E-BP1 phosphorylation;
Ref. 37) may also contribute to the observed increase in
protein synthesis before day 3.
A time course of the precise induction of IGF-II mRNA
levels and 4E-BP1 and p70S6k activation before day 1
after clenbuterol treatment, together with IGF-I/insulin
receptor activation studies, would substantiate the validity of the proposed pathway. Moreover, although clenbuterol has been shown not to alter IGF-I levels, at least
in plasma (2), we cannot rule out a potential role for IGF-I
in muscle in addition to IGF-II in this response. These
possibilities must be investigated.
Whether the hypertrophic action of clenbuterol is
mediated through the ␤2-adrenoceptor and the welldocumented increases in intracellular cAMP has been
the source of much debate (5, 25). However, the present
data may provide additional evidence that drug-induced anabolism does not arise from ␤2-adrenoceptormediated increases in intracellular cAMP. In general,
increasing cAMP with agents such as forskolin promotes decreased 4E-BP1 and p70S6k phosphorylation
MRNA
E682
CLENBUTEROL ACTIVATION OF 4E-BP1, P70S6K, AND IGF-II
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MRNA