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Differences in Muscle Protein Synthesis and Anabolic
Signaling in the Postabsorptive State and in Response to
Food in 65–80 Year Old Men and Women
Gordon I. Smith
1.
, Philip Atherton
2.
, Dennis T. Villareal
1
, Tiffany N. Frimel
1
, Debbie Rankin
2
, Michael J.
Rennie
2
, Bettina Mittendorfer
1
*
1 School of Medicine, Washington University, St. Louis, Missouri, United States of America, 2 School of Graduate Entry Medicine and Health, University of Nottingham,
Derby, United Kingdom
Abstract
Women have less muscle than men but lose it more slowly during aging. To discover potential underlying mechanism(s) for
this we evaluated the muscle protein synthesis process in postabsorptive conditions and during feeding in twenty-nine 65–
80 year old men (n = 13) and women (n = 16). We discovered that the basal concentration of phosphorylated eEF2
Thr56
was
,40% less (P,0.05) and the basal rate of MPS was ,30% greater (P = 0.02) in women than in men; the basal concentrations
of muscle phosphorylated Akt
Thr308
, p70s6k
Thr389
, eIF4E
Ser209
, and eIF4E-BP1
Thr37/46
were not different between the sexes.
Feeding increased (P,0.05) Akt
Thr308
and p70s6k
Thr389
phosphorylation to the same extent in men and women but
increased (P,0.05) the phosphorylation of eIF4E
Ser209
and eIF4E-BP1
Thr37/46
in men only. Accordingly, feeding increased
MPS in men (P,0.01) but not in women. The postabsorptive muscle mRNA concentrations for myoD and myostatin were
not different between sexes; feeding doubled myoD mRNA (P,0.05) and halved that of myostatin (P,0.05) in both sexes.
Thus, there is sexual dimorphism in MPS and its control in older adults; a greater basal rate of MPS, operating over most of
the day may partially explain the slower loss of muscle in older women.
Citation: Smith GI, Atherton P, Villare al DT, Frimel TN, Rankin D, et al. (2008) Differences in Muscle Protein Synthesis and Anabolic Signaling in the Postabsorptive
State and in Response to Food in 65–80 Year Old Men and Women. PLoS ONE 3(3): e1875. doi:10.1371/journal.pone.0001875
Editor: Alejandro Lucia, Universidad Europea de Madrid, Spain
Received January 3, 2008; Accepted February 21, 2008; Published March 26, 2008
Copyright: ß 2008 Smith et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by US National Institutes of H ealth grants AR 49869, AG 025501, RR 00036 (General Clinical Research Center), RR 00954
(Biomedical Mass Spectrometry Resource), and DK 56341 (Clinical Nutrition Research Unit), the University of Nottingham, the UK Biotechnology and Biological
Sciences Research Council grants BB/XX510697/1 and BB/C51 6779/1, and a European Union EXEGENESIS program grant. Philip Atherton is a designated Research
Councils UK fellow.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mittendb@wustl.edu
. These authors contributed equally to this work.
Introduction
Adequate maintenance of muscle mass throughout life is
important to preserve locomotor functions and diminish the risk
of falling. Furthermore, muscle is the predominant site of body
glucose uptake [1–3] and contributes ,25% to basal energy
expenditure, and even more during physical activity [4]. Muscle,
therefore, not only serves mechanical functions but also contrib-
utes to metabolic and energy homeostasis.
It is common knowledge that healthy adult women have less
lean body mass (mostly muscle) and more fat than men [5,6].
However, the age-associated decrease in lean body mass and
muscle mass is slower in women than in men [5,7–9].
Unfortunately, little is known about the mechanisms that lead to
sexual dimorphism in body composition. It is thought that most of
it is due to differences in the sex-hormone milieu in men and
women. It has been repeatedly demonstrated in vitro [10,11], in vivo
in animal models [10,12–14], and in human subjects [15,16] that
testosterone stimulates skeletal muscle protein synthesis (MPS) and
increases muscle mass. There is also evidence that ovarian
hormones inhibit MPS [17] and muscle growth [18,19] in rats.
Nevertheless, several investigators who have measured the rates of
MPS in men and women have found no differences [20–22].
The fact that no sex differences in MPS have been reported in
the literature might be because these studies were conducted in
young and middle-age adults with a constant muscle mass during
postabsorptive conditions, when sex differences may be small or
non-existent. We reasoned that sex differences in muscle protein
turnover would become apparent at life stages when muscle mass
is changing (e.g., during growth in adolescence or wasting during
aging) and/or during acute anabolic or catabolic challenges (e.g.,
feeding or injury). Accordingly, we hypothesized that such
differences might be revealed by a comparison of rates of MPS
in older men and women during basal, postabsorptive conditions
and feeding. We, therefore, measured the fractional rate of MPS
during basal, postabsorptive conditions and during feeding by
using stable-isotope labeled amino acid tracer techniques in 65–
80 year old men and women; we also measured the concentrations
of total muscle RNA and protein to gain insight into the protein
synthetic capacity and translational efficiency of the muscle
[23,24]. Furthermore, we measured the activation (as phosphor-
ylation) of elements of intracellular signaling pathways involved in
the regulation of MPS (Akt; ribosomal protein S6 protein kinase
[p70s6k]; eukaryotic initiation factor 4E [eIF-4E]; eIF4E binding
protein 1 [eIF4E-BP1]; and eukaryotic elongation factor 2 [eEF2])
[25,26] and the mRNA expression of proteins involved in the
PLoS ONE | www.plosone.org 1 March 2008 | Volume 3 | Issue 3 | e1875
regulation of muscle mass (i.e., the muscle growth inhibitor
myostatin [27,28] and the muscle growth factor myoD [29]). We
also measured plasma C-reactive protein (CRP) concentration as
an index of inflammation because the concentrations of CRP and
pro-inflammatory cytokines in blood have been found to be
negatively associated with rates of MPS [30] and may contribute
to skeletal muscle atrophy and reduced functional capacity,
especially during aging [31–33].
Methods
Subjects
We studied 13 men and 16 women, aged 65 to 80 y; men and
women were matched for age and body mass index (Table 1). All
subjects were considered to be in good health after completing a
comprehensive medical evaluation. None of the subjects engaged
in regular exercise, reported excessive alcohol intake, smoked, or
received hormone replacement therapy. Ten women and six men
were treated for hypertension, and four women and four men were
treated for hypercholesteremia; the drug regimen had been
initiated several years before subjects entered the study and had
been stable for several months before beginning the study.
Written, informed consent was obtained from all subjects before
their participation in the study, which was approved by the
Human Studies Committee and the General Clinical Research
Center (GCRC) Advisory Committee at Washington University
School of Medicine in St. Louis, MO.
Experimental protoc ol
Approximately two weeks before the protein metabolism study,
subjects’ total body fat-free mass (FFM) and appendicular muscle
mass [31] were measured by using dual-energy X-ray absorpti-
ometry (Delphi-W densitometer, Hologic, Waltham, MA). Mag-
netic resonance imaging (MRI) was used to quantify thigh muscle
volume; images were acquired with a 1.5-T superconducting
magnet (Siemens, Iselin, NJ) and a T1-weighted pulse sequence.
Eight 8-mm-thick axial images, starting 10 cm proximal to the
distal edge of the femur, with a 7-mm intersection gap, were
acquired and muscle volume in each of the images was determined
with the NIH Image Analysis Software (Analyze Direct software
(version 7.0; Mayo Clinic, Rochester, MN), which utilizes pixel
brightness to distinguish muscle from other tissues. Thigh muscle
volume in the region of interest was calculated as the sum of the
individual muscle volumes and the sum of the muscle volumes in
the intersection gap, which were assumed to be the same as in the
preceding image.
Three days before the protein metabolism study, subjects were
instructed to adhere to their usual diet and to refrain from vigorous
exercise until completion of the study. The evening before the
study, subjects were admitted to the GCRC. At 2000 h, they
consumed a standard meal which provided 50.2 kJ per kg body
weight; 15% of the meal energy was provided as protein, 55% as
carbohydrates and 30% as fat. Subjects then rested in bed and
fasted (except for water) until completion of the study the next day.
At ,0600 h on the following morning, a cannula was inserted into
an antecubital vein for the infusion of a stable isotope labeled
leucine tracer; another cannula was inserted into a vein of the
contralateral hand for blood sampling. At ,0800 h, a blood
sample and a muscle biopsy from the quadriceps femoris were
obtained to determine the background leucine enrichment in
plasma, the concentrations of testosterone, progesterone, 17ß-
estradiol, sex hormone binding globulin (SHBG), and CRP in
plasma, the background leucine enrichment in muscle tissue fluid
and muscle protein, and the concentration of total RNA and
protein in muscle. Muscle tissue (,50–100 mg) was obtained
under local anesthesia (lidocaine, 2%) by using Tilley-Henkel
forceps [34]. Immediately afterwards, a primed, constant infusion
of [5,5,5-
2
H
3
] L-leucine (98 Atoms % purchased from Cambridge
Isotope Laboratories Inc, Andover, MA; priming dose:
4.8 mmol kg body wt
21
, infusion rate: 0.08 mmol kg body
wt
21
?min
21
) was started and maintained until completion of the
study ,6 h later. At 210 min after the start of the leucine tracer
infusion, a second muscle biopsy was obtained to determine the
basal rate of MPS (as incorporation of [5,5,5-
2
H
3
]leucine into
muscle protein; see Calculations) and the basal concentrations of
phosphorylated elements of intramuscular signal transduction
proteins (Akt; p70s6k; eIF-4E; eIF4E-BP1; and eEF2) involved in
the regulation of MPS. Immediately after the second biopsy, a
liquid meal (EnsureH, Abbott Laboratories, Abbott Park, IL, USA,
containing 15% of energy as protein, 55% as carbohydrate and
30% as fat) was given intermittently in small boluses every
10 minutes for 150 min so that every subject received a priming
dose of 23 mg protein?kg FFM
21
and 70 mg protein?kg
FFM
21
?h
21
during the 2.5 h feeding period. At the onset of
feeding, the infusion rate of labeled leucine was increased to
0.12 mmol kg body wt
21
?min
21
to adjust for the increased plasma
leucine availability. A third muscle biopsy was obtained at
360 min (i.e., 150 min after the first food aliquot) to determine
both MPS and the intracellular signaling responses to feeding. The
second and third biopsies were obtained from the leg contralateral
to that biopsied initially through the same incision, but with the
forceps directed in proximal and distal direction so that the two
biopsies were collected ,5–10 cm apart. Blood samples were
obtained every 30 min during the entire study period to determine
plasma leucine enrichment and concentration, and concentrations
of glucose, and insulin. The tracer infusions were stopped and
cannulae were removed after the last (third) biopsy and the final
blood draw were completed.
Sample collection and storage
Approximately 4 ml of blood was collected on each occasion.
One milliliter was collected in pre-chilled tubes containing
heparin, plasma separated immediately by centrifugation and
glucose concentration measured immediately. The remaining
blood (,3 ml) was collected in pre-chilled tubes containing
EDTA, plasma was separated by centrifugation within 30 min of
collection and then stored at 280uC until final analysis. Muscle
samples were rinsed in ice-cold saline immediately after collection,
Table 1. Subject characteristics
Men Women P-value
Age (years) 71626961 0.16
Body mass index (kg?m
22
)36613862 0.34
Body mass (kg)
a
108639864 0.09
Fat free mass (kg)
a
67625162 ,0.001
Fat free mass (% body weight) 62615261 ,0.001
Appendicular muscle mass (kg)
a
29612261 ,0.001
Appendicular muscle mass (% FFM) 43614361 0.40
Thigh muscle volume (cm
3
)
b
24776113 1963693 0.002
Values are means6SEM.
a
Measured by DEXA as described in the Experimental protocol section.
b
Measured by MRI as described in the Experimental protocol section.
doi:10.1371/journal.pone.0001875.t001
Muscle Anabolism
PLoS ONE | www.plosone.org 2 March 2008 | Volume 3 | Issue 3 | e1875
cleared of visible fat and connective tissue, frozen in liquid
nitrogen and stored at 280uC until final analysis.
Sample processing and analyses
Plasma glucose concentration was determined on an automated
glucose analyzer (Yellow Spring Instruments, Yellow Springs,
OH). Plasma insulin concentration was determined by radioim-
munoassay (Linco Research, St. Louis, MO). ELISA was used to
determine plasma concentrations of testosterone, progesterone,
17ß-estradiol, SHBG (all Immuno-Biological Laboratories, IBL-
America, Minneapolis, MN), and CRP (ALPCO Diagnostics,
Salem, NH).
To determine plasma leucine concentration and labeling of
plasma leucine and a-ketoisocaproate (KIC), a known amount of
norleucine was added to the plasma, proteins were precipitated,
and the supernatant, containing free amino acids and their keto-
analogues, was collected to prepare the t-butyldimethylsilyl (t-
BDMS) and trimethylsilyl derivative of leucine and KIC,
respectively, to determine their tracer-to-tracee ratios (TTR) by
gas-chromatography/mass-spectrometry (GC-MS; MSD 5973
System, Hewlett-Packard) [35].
To determine leucine labeling of muscle proteins and tissue
fluid, samples (,20 mg) were homogenized in 1 ml trichloroacetic
acid solution (3% w/v), proteins precipitated by centrifugation,
and the supernatant, containing free amino acids, collected. The
pellet containing muscle proteins was washed and then hydrolyzed
in 6 N HCl at 110 uC for 24 h. Amino acids in the protein
hydrolysate and supernatant samples were purified on cation-
exchange columns (Dowex 50W-X8-200, Bio-Rad Laboratories,
Richmond, CA), and the t-BDMS derivative of leucine prepared
to determine its TTR by GC-MS (MSD 5973 System, Hewlett-
Packard) analysis [35]. The extent of leucine labeling in plasma,
muscle tissue fluid, and muscle protein were calculated based on
the simultaneously measured TTR of standards of known isotope
labeling.
Western analysis was used to measure the phosphorylation of
Akt, p70s6k, eIF-4E, eIF4E-BP1, and eEF2. Briefly, frozen muscle
tissue (,20 mg) was rapidly homogenized with scissors in ice-cold
buffer (50 mM Tris-HCL pH 7.5, 1 mM EDTA, 1 mM EGTA,
10 mM glycerophosphate, 50 mM NaF, 0.1% Triton-X, 0.1% 2-
mercaptoethanol, 1 complete protease inhibitor tablet [Roche
Diagnostics Ltd, Burgess Hill, UK]) at 10 ml?mg
21
tissue. Proteins
were extracted by shaking for 15 min at 4uC and samples were
then centrifuged at 130006g for 10 min at 4uC and the
supernatant, containing the proteins was collected. The protein
concentration in the supernatant was determined by the Bradford
method with a commercial reagent (B6916, Sigma-Aldrich, St.
Louis, MO) and adjusted to 3 mg ml
21
in 36Laemmli buffer.
Fifty micrograms of protein from each sample were loaded onto
12% XT-Bis Tris gels, separated by SDS PAGE, and transferred
on ice at 100 V for 45 min to methanol pre-wetted 0.2 mm PVDF
membranes. Blots were then incubated sequentially with 5% (w/v)
non-fat milk for 1 h, primary antibodies overnight at 4uC, and
then secondary antibody (1:2000 anti-rabbit; New England
Biolabs, Ipswich, MA) for 1 h. The following primary antibodies
were used at a concentration of 1:1000: Akt
Thr308
, p70s6k
Thr389
;
4E-BP1
Thr37/46
, eEF2
Thr56
, and GADPH (loading control),
purchased from New England Biolabs, and eIF4E
Ser209
purchased
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Mem-
branes were developed using Immunstar (Bio-Rad Laboratories,
Richmond, CA) and the protein bands were visualized and
quantified by densitometry on a Chemidoc XRS (Bio-Rad
Laboratories, Inc. Hercules, CA) ensuring no pixel saturation.
Data were expressed in relation to GADPH.
The expression of genes involved in the regulation of muscle
mass was evaluated by real-time, reverse transcription polymerase
chain reaction (RT-PCR). Frozen tissue samples (5–10 mg) were
homogenized in TRIZOLH using a Polytron for 15 s on ice. Total
RNA was extracted according to the instructions provided by the
manufacturer (Sigma-Aldrich, St. Louis, MO) and quantified by
spectrophotometry at 260 nm. An aliquot (0.5 mg) was loaded
onto a 1% agarose gel to check RNA quality and loading by
visualization of 28s and 18s rRNA. Reverse transcription was
performed using the iScript synthesis kit (Bio-Rad Laboratories,
Richmond, CA) with 1 mg of total RNA in a reaction volume of
20 ml(4ml iScript reaction mix, 1 ml iScript reverse transcriptase,
1 ml RNA template, 14 ml RNase-free water). The final RT
products were adjusted to 100 ml each using RNase free water.
The following primers were used for myostatin and myoD (all 5 9 to
39). Myostatin forward: CTA CAA CGG AAA CAA TCA TTA
CCA, reverse: GTT TCA GAG ATC GGA TTC CAG TAT;
MyoD forward: CCG CCT GAG CAA AGT AAA TG, reverse:
GCC CTC GAT ATA GCG GAT G. Sybr GreenH PCR analyses
were carried out on the iQ5 Real-Time PCR Detection System
(Bio-Rad Laboratories, Richmond, CA) using the following cycle
conditions: 3 min at 95uC, followed by 40 cycles of 1 min at 60uC,
and 15 s at 95uC. For each gene, real time RT-PCR was
conducted in duplicate in 25 ml reaction volumes containing
12.5 ml qPCR SuperMix (Bio-Rad Laboratories, Richmond, CA),
0.75 ml of each primer (10 pmol ml
21
), 9 ml RNAse-free water and
2 ml of 1:5 diluted cDNA. PCR products were checked for
amplicon specificity by both melting curve and agarose gel
electrophoresis. Results were analyzed using the 2-DDCt method
with 28s as internal control [36]. Due to lack of sufficient muscle
tissue, these analyses were carried out in only 7 of the 13 men and
7 of the 16 women.
To determine the total RNA to protein ratio in muscle, an index of
the capacity for protein synthesis, an aliquot of the total RNA
preparation prepared for RT-PCR was sequentially extracted
(RNA.DNA.protein) according to the manufacturers (Sigma-
Aldrich, St. Louis, MO) protocol. Total RNA was quantified after
complete removal of the upper phase following phase separation;
protein concentration was quantified after removal of the DNA
interphase and precipitation of proteins with acetone from the
bottom layer, which were then washed and resuspended in 1% SDS.
Total RNA was quantified (in mg per g wt weight) spectrophotomet-
rically at 260 nm and protein was quantified (in mg per g wt weight)
at 595 nm using Bradford reagents at a 1:10 dilution to reduce SDS
interference. Due to lack of sufficient muscle tissue, these analyses
were carried out in only 9 of the 13 men and 7 of the 16 women.
Calculations
Leucine rate of appearance (Ra) in plasma was calculated by
dividing the rate of [5,5,5–
2
H
3
]leucine infusion by the steady state
plasma KIC TTR during basal, postabsorptive conditions and
feeding. Leucine Ra during basal conditions is an index of the rate
of whole-body proteolysis; during feeding, leucine Ra represents
the sum of the rate of leucine release into plasma from proteolysis
plus the rate of transfer of absorbed leucine from the meal into the
systemic circulation.
The fractional synthesis rate (FSR) of mixed muscle protein was
calculated from the rate of incorporation of [5,5,5-
2
H
3
]leucine
into muscle protein, using a standard precursor-product model as
follows: FSR = DE
p
/E
ic
61/t6100; where DE
p
is the change
between two consecutive biopsies in extent of labeling (TTR) of
protein-bound leucine. E
ic
is the mean labeling over time of the
precursor for protein synthesis and t is the time between biopsies.
The free leucine labeling in muscle tissue fluid was chosen to
Muscle Anabolism
PLoS ONE | www.plosone.org 3 March 2008 | Volume 3 | Issue 3 | e1875
represent the immediate precursor for MPS (i.e., aminoacyl-t-
RNA) [37]. Values for FSR are expressed as %?h
21
. The absolute
rate of muscle protein synthesis (g protein per hour) was calculated
by multiplying the FSR by the total appendicular muscle protein
mass, which was assumed to be 20% of total appendicular muscle
mass. We have recently found that differences in the rates of
muscle protein synthesis in different muscles are negligible[38];
thus, it is reasonable to extrapolate our data obtained in the vastus
lateralis to all skeletal muscles in the body.
The translation efficiency (mg protein produced per m g RNA
per hour) was calculated by dividing the product of the muscle
protein FSR (in %?h
21
) and the muscle protein concentration (in
mg per g wet tissue) by the muscle total RNA concentration (in mg
per g wet tissue) [23,24].
Statistical analysis
All data sets were tested for normality. Differences between men
and women in subject characteristics and single time-point
measurements (e.g., plasma sex hormone and CRP concentrations)
were evaluated by using Student’s t-test for normally distributed data
and the Mann-Whitney U test for data which were not normally
distributed (i.e., plasma SHBG, testosterone, progesterone, 17ß-
estradiol and CRP concentrations). Analysis of variance (ANOVA)
was used to evaluate possible differences between men and women in
plasma glucose, insulin, and leucine concentrations, muscle protein
FSR, muscle intracellular signaling elements, and muscle mRNA
expression during postabsorptive and fed conditions. If necessary,
data were log transformed to achieve normally distributed data sets
before analysis. A P value of #0.05 was considered statistically
significant. Data in the text are presented as mean6SEM or median
with 25
th
and 75
th
percentiles in brackets for skewed data sets; data in
tables and figures are presented as indicated in the legends.
Results
Subjects’ age and body-composition
Men and women were matched for age and BMI (Table 1).
Total body FFM, total muscle mass and leg muscle volume were
,25% less in women than in men; however, the relative
contribution of muscle mass to total body FFM was not different
in men and women (Table 1).
Plasma sex hormone and CRP concentrations
Plasma SHBG concentration was not different between men
and women (Table 2). Plasma testosterone concentration was 10
times greater ( P,0.001) in men than in women whereas plasma
progesterone and 17ß-estradiol concentrations were not different
between the sexes (Table 2). Plasma CRP concentration was not
different between men and women (Table 2).
Plasma glucose, insulin, and leucine concentrations
Basal plasma glucose and insulin concentrations were not
different between men and women (Table 3). Feeding increased
plasma glucose concentration by ,25% (P,0.001) and plasma
insulin concentration by ,200% (P,0.001) with no differences
between the sexes (Table 3). Basal plasma leucine concentration
was ,15% less in women than in men (Table 3), and feeding
increased plasma leucine concentration by ,15% (P,0.001) in
both sexes (Table 3).
Whole-body leucine Ra
Rates of leucine Ra during basal, postabsorptive conditions (an
index of whole-body protein breakdown) and total leucine Ra
during feeding were not different in men (2.2460.08 and
2.5660.08 mmol kg
21
FFM?min
21
, respectively) and women
(2.2060.09 and 2.6260.09 mmol kg
21
FFM?min
21
, respectively).
Muscle protein synthesis
The capacity for MPS (i.e., total RNA-to-protein ratio in
muscle) tended to be greater (by ,20%; P = 0.18) in women than
in men (5.860.6 vs. 4.760.5 mg RNA?mg protein
21
, respectively).
Mixed muscle protein FSR during basal, postabsorptive
conditions was , 30% greater (P = 0.02) in women than in men
(Figure 1). Feeding had no effect on the FSR in women but
increased (P,0.01) it in men to values similar to those in women
(Figure 1). The absolute rate of muscle protein synthesis, adjusted
for differences in total muscle mass between the sexes, was 116
[104, 143] mg of muscle protein per hour per kg of appendicular
skeletal muscle mass in women and 90 [78, 115] mg of muscle
protein per hour per kg of appendicular skeletal muscle mass in
men (P = 0.02); it increased by 35 [13, 79] mg per hour per kg of
appendicular skeletal muscle mass in response to the meal in men
(P,0.01), but did not change significantly from basal values (by 11
[210, 41] mg of muscle protein per hour per kg of appendicular
skeletal muscle mass) in women.
The rate of MPS in relation to muscle RNA concentration, a
measure of the translational efficiency in muscle, was not different
between men and women during basal, postabsorptive conditions
(0.01260.003 vs. 0.01360.002 mg protein?mg RNA
21
?h
21
) and
increased with feeding in men (to 0.01960.006 mg protein?mg
RNA
21
?h
21
; P = 0.058 vs basal) but not in women (to
0.01560.001 mg protein?mg RNA
21
?h
21
).
Phosphorylation of signaling transduction proteins in
muscle
In the postabsorptive state, the extent of phosphorylation of
Akt
Thr308
, p70s6k
Thr389
, eIF4E
Ser209
and eIF4E-BP1
Thr37/46
in
muscle was not different in men and women (Figure 2). The
Table 2. Plasma sex hormone and CRP concentrations.
Men Women
SHBG (nmol l
21
) 23.1 (20.1, 26.0) 25.9 (19.2, 44.8)
Testosterone (nmol l
21
) 12.2 (8.8, 18.1) 1.1 (0.9, 1.7)*
Progesterone (ng ml
21
) 0.12 (0.05, 0.31) 0.04 (0.02, 0.12)
17ß-Estradiol (pg ml
21
) 11.3 (10.8, 30.9) 10.9 (10.0, 11.9)
CRP (mg l
21
) 3.08 (2.43, 4.23) 2.80 (0.98, 3.60)
Values are median with quartiles in parentheses.
*
Value significantly different from corresponding value in men (P,0.001).
doi:10.1371/journal.pone.0001875.t002
Table 3. Plasma glucose, insulin, and leucine concentrations.
Men Women
Fasted Fed Fasted Fed
Glucose (mmol l
21
)5.260.1 6.560.2
{
5.460.2 6.960.1
{
Insulin (mUml
21
) 15.562.2 47.867.3
{
14.463.1 41.565.6
{
Leucine (mmol l
21
)13364 14464
{
11166* 12666*
{
Values are mean6SEM.
*
Value significantly different from corresponding value in men (P = 0.011);
{
value significantly different from corresponding value during basal,
postabsorptive (fasted) conditions (P,0.001).
doi:10.1371/journal.pone.0001875.t003
Muscle Anabolism
PLoS ONE | www.plosone.org 4 March 2008 | Volume 3 | Issue 3 | e1875
feeding-induced increases in the phosphorylation of Akt
Thr308
,
p70s6k
Thr389
(both P,0.01) were also not different in the two sexes
(Figure 2). In contrast, feeding increased (P,0.01) the phosphor-
ylation of eIF4E
Ser209
and eIF4E-BP1
Thr37/46
in men but had no
effect on their phosphorylation in women (Figure 2). Phosphor-
ylated eEF2
Thr56
in muscle was ,40% less (P,0.05) in women
than in men both during postabsorptive conditions and feeding
(Figure 2); there was a tendency for a decrease in the
phosphorylation of eEF2
Thr56
in both sexes with feeding
(Figure 2) but this difference did not reach statistical significance
(P = 0.103).
mRNA expression of proteins involved in the regulation
of muscle mass
The mRNA concentrations of myostatin and myoD during
postabsorptive conditions were not different in men and women
(Figure 3). Feeding decreased the concentration of myostatin
mRNA (P,0.05) and increased the concentration of myoD
mRNA (P,0.05) to the same extent in the two sexes (Figure 3).
Discussion
In this study we uncovered marked sexual dimorphism between
older men and women in a variety of aspects of muscle protein
metabolism. The differences we observed between older men and
women, namely a ,30% greater basal rate of mixed MPS in
women than in men and resistance of MPS to feeding a liquid
mixed meal (providing a total of ,10 g of protein) in women, are
consistent with a recent study in which comprehensive oligonu-
cleotide microarrays were used to discover potential differences
between men and women in the expression of genes involved in
the regulation of muscle mass [39]; in this study it was found that
women had a two-fold greater expression of two genes that encode
proteins with inhibitory properties on growth factor pathways in
muscle. On the other hand, the results from our study are in clear
contrast to the results from previous workers who searched for
potential sex differences in human muscle protein metabolism and
found none [20–22]. However, those earlier studies were
conducted in young adults (average age: 23–27 y) and we
hypothesized that sex differences in human muscle protein
turnover would only become apparent at life stages when muscle
mass was changing (e.g., during adolescent growth or wasting
during aging) and/or possibly during acute anabolic or catabolic
challenges (e.g., with feeding or injury). To our knowledge the
current work is the first to demonstrate in human beings sex
differences in the rate of MPS and provides some insight
concerning the control of MPS and the different rates of muscle
loss with aging between men and women.
The greater basal rate of mixed MPS in older women was
probably mediated by a combination of a greater capacity for
protein synthesis combined with a relatively more active
translational process at the elongation stage of protein synthesis
because first, there was the trend for a ,20% greater muscle
RNA-to-protein ratio in women than in men, indicating a greater
capacity for MPS [24] in them and secondly, muscle of older
women had a 40% smaller degree of phosphorylation of
eEF2
Thr56
, a molecule regulating elongation of nascent protein
chains which is deactivated by phosphorylation [25]. We found no
differences between sexes in the extent of phosphorylation for
components of the PKB/mTOR/p70s6k signaling pathway, or
the phosphorylation of elements involved in the regulation of
translation initiation (eIF4E and eIF4e-BP1) at baseline (fasted).
Furthermore, the expression in muscle of mRNA for myostatin
and myoD, cell regulatory proteins affecting muscle size [26–29],
was not different between the sexes, which makes it unlikely that
they are involved in regulating the basal rate of MPS, although, we
cannot from our data rule out differences in the muscle myostatin
and myoD protein concentration (and thus in cell function).
Indeed, a greater myostatin protein concentration has been found
in female than male mice [40], which is consistent with the
observed sexual dimorphism in muscle mass but not with our
results of a lower basal rate of MPS in older men, assuming
extrapolation between species is valid. Plasma glucose, insulin, and
CRP concentrations were not different in men and women, so
these results also provide no explanation of baseline sex differences
in MPS.
Figure 1. Muscle protein synthesis rate in men and women.
Panels A and B show the mixed skeletal muscle protein fractional
synthesis rate (FSR) during basal, post-absorptive conditions (fasted)
and liquid mixed meal consumption (fed) in men and women (A) and
the meal-induced change in the FSR in men and women (B). Graphs
show the median (central horizontal line), 25
th
and 75
th
percentiles
(box), and minimum and maximum values (vertical lines). * Value
significantly different from corresponding value in men.
{
Value
significantly different from corresponding value during basal, postab-
sorptive (fasted) conditions.
doi:10.1371/journal.pone.0001875.g001
Muscle Anabolism
PLoS ONE | www.plosone.org 5 March 2008 | Volume 3 | Issue 3 | e1875
The proximal, underlying, biological reasons that the sexua l
dimorphism in muscle protein metabolism and its control is
apparently of late onset (aging, as in the p resent study, vs. young
and middle-age adulthood [20–22]) are not clear but are most
probably related to the changes with advancing age in the sex
hormone milieu. This may at first seem counterintuitive, because
men had a ten-fold excess of testosterone co mpared with women,
with a concomitant relatively s mall or no difference in plasma
progesterone and estradiol concentration between the sexes.
Testosterone is wel l known to be an abolic and increases the basa l
rate of MPS in both healthy and hypo gonadal young men
[15,16,41]. Oddly, however, the effect of testosterone therapy in
older men is unclear; it has been shown by the same group, in
different studies, to either increase [42] or not to affect [43] the
basal rate of MPS. This discrepancy might be a dose- or
treatment duration-related phenomenon, or depend upon the
extent of initial testosterone deficienc y. More importantly,
however, we [44] and others [45] measured the basal rate of
MPS in large cohorts of healthy young (n$22) and old (n$22)
men and found that it was not affected by old age. This suggests
that the normal decline in testosterone with aging, which is small
[45], probably has little effect on the basal rate of MPS. On the
other han d, there is evidenc e from studies in rats that
progesterone and estrogen inhibit MPS. Specifically, it was found
that in ovariectomized rats the rate of MPS was higher than in
sham-operated, intact controls and ovariectomy with either
progesterone or estrogen replacement pr evented the incre ase
[17]. Thus, it appears that the basal rate of MPS probabl y
increases after menopause due to a lack of female se x steroids,
which leads to pronounced differences in the basal rate of MPS
between men and women not apparent in younger adults.
Moreo ver, these and our findings (i.e., greater basal rate of MPS
in women than men despite 10-fold difference in plasma
testosterone concentration) suggest that the anti-anabolic effect
of female sex steroids on MPS may by far outweigh the anabolic
effect of testosterone.
Figure 2. Phosphorylation of anabolic signaling transduction molecules in muscle of men and women. Panels A–E show concentrations
of phosphorylated Akt
Thr308
(A), p70s6k
Thr389
(B), eIF4E-BP1
Thr37/46
(C), eIF4E
Ser209
(D), and eEF2
Thr56
(E) during basal, postabsorptive conditions
(fasted) and liquid mixed meal consumption (fed) in men and women. Values are means6SEM. * Value significantly different from corresponding
value in men;
{
Value significantly different from corresponding value during basal, postabsorptive (fasted) conditions.
doi:10.1371/journal.pone.0001875.g002
Muscle Anabolism
PLoS ONE | www.plosone.org 6 March 2008 | Volume 3 | Issue 3 | e1875
Another somewhat surprising finding was the fact that the
postabsorptive rate of MPS in women was faster despite lower
basal plasma leucine concentrations in women than in men. There
is ample evidence that the rate of MPS is directly related to plasma
amino acid availability [44,46,47], particularly that of plasma
leucine [48–51]. However, this relationship has been established
when plasma amino acid/leucine concentrations were varied
several-fold, whereas the difference in plasma leucine concentra-
tion in our men and women was only ,15%, possibly too small to
exert a noticeable effect on the rate of MPS as MPS only increases
by 50% with a doubling of leucine concentration [46].
The slightly greater postabsorptive plasma leucine concentra-
tion in men, which is in excellent agreement with an earlier report
on sex differences in the plasma amino acid profile [52], probably
reflects greater net negative protein balance in men than in women
but it is difficult to assign components to this, beyond the
decreased MPS in men. We did not measure the rate of muscle
protein breakdown (MPB) (which was unwarranted without more
indicative data of possible sex differences given the already sizeable
investigational burden on our subjects). Therefore we are unable
to estimate muscle protein net balance. However, whole-body
protein breakdown (indicated by leucine Ra) was not different
between our men and women and as MPB normally accounts for
,20–40% of a healthy person’s postabsorptive whole-body
protein breakdown rate [53,54] it is unlikely that MPB was
markedly accelerated in men compared with women.
The differences in the anabolic response to feeding between the
sexes, with men showing a significant increase and women no
significant increase in MPS, was probably partially mediated by a
lack of stimulation by feeding of protein translation initiation in
female muscle, given that the feeding increased the phosphoryla-
tion of muscle eIF4E
Ser209
and eIF4E-BP1
Thr37/46
in men but not
in women. This is particularly interesting given the facts that other
indices of anabolic signaling, like the feeding-induced changes in
the phosphorylation of Akt
Thr308
, p70s6k
Thr389
, and eEF2
Thr56
were similar in men and women. An intriguing new finding was
the marked (and so far as we can tell previously unreported)
changes in the expression of myostatin and myoD with feeding,
which are consistent with an acute nutritional control at the level
of the nucleus of processes regulating muscle mass [55].
Nevertheless, there was no apparent sex difference in these
responses, which makes it unlikely that the feeding-induced
changes in myostatin and myoD expression contributed to the
blunted increase of MPS to feeding in women. The feeding-
induced increases in plasma glucose, insulin, and leucine
concentrations were not different in men and women, which
suggests that these also were not involved in the apparent anabolic
resistance in women. It is possible, however, that there were sex
differences in the insulin-stimulated increase in muscle nutritive
blood flow which would differentially affect amino acid delivery to
the muscle. Although in young adults, the insulin mediated
vasodilation is greater in women than in men [56], there is
evidence for a greater decline with aging in women compared with
men in endothelium dependent dilation induced pharmacologi-
cally or by hypoxia [57–59], which may abolish this difference at a
more advanced age or even result in reduced flow in women.
However all these observations were for measures of bulk blood
flow and no pertinent results exist, to our knowledge, for muscle
microvascular perfusion [60]. The impact of the sex hormone
milieu on the anabolic effect of feeding in muscle is unknown
largely due to a lack of data in the literature. To our knowledge, no
one has determined the effect of female sex steroids on the
anabolic effects of amino acids or food in muscle; however, neither
testosterone [43] nor oxandrolone [61] were found to exert an
acute additive effect to the stimulatory effect of amino acids on
MPS.
Our findings provide a potential mechanism that may help to
explain the slower rate of muscle wasting during aging in women
than in men [5,7–9]. There is no data available for diurmal
variation in human muscle MPS but we suggest that the basal,
postabsorptive rate, which was ,30% greater in our women than
in the men, most likely predominates over most of the day. Our
arguments depend upon a number of observations. First, the data
available from protein intakes in French [62] and North American
[63,64] older adults indicate that individuals between 65–80 y eat
on average approximately 9–14 g protein at breakfast, 38–64 g
protein at lunch, 19–33 g protein at dinner and 1.2–1.7 g protein
in snacks [62]. Secondly, meal feeding causes an increase of
plasma total amino acid concentrations that is proportional to the
amount of protein consumed for approximately 3–5 h, after which
the change returns to within 25% of baseline [65–67]. Thirdly, the
results of a study of the circadian variations of amino acids during
a 24-h long study of the effects of 3 meals and two snacks suggested
Figure 3. Muscle mRNA expression of proteins involved in the
regulation of muscle mass in men and women. Panels A and B
show myostatin (A) and myoD (B) gene expression during basal,
postabsorptive conditions (fasted) and liquid mixed meal consumption
(fed) in men (n = 7) and women (n = 7). Values are means6SEM.
{
Value
significantly different from corresponding value during basal, postab-
sorptive (fasted) conditions; P,0.05.
doi:10.1371/journal.pone.0001875.g003
Muscle Anabolism
PLoS ONE | www.plosone.org 7 March 2008 | Volume 3 | Issue 3 | e1875
that the period of plasma essential amino acids being raised above
the postabsorptive morning value was approximately 7 h [68].
Fourth, there is evidence that a sustained hyperaminoacidemia
does not lead to a sustained increase in MPS, which returns to
baseline values within approximately 2–2.5 h of the initial increase
of amino acid availability [69]. Lastly, the relationship between
amino acid availability and MPS is saturable both for orally
delivered free amino acids [44] and orally delivered protein [70],
with excess essential amino acids being oxidized above delivery of
approximately 15 g of protein per meal in young trained
individuals [70] and probably even less in older adults [44]. Thus,
we suggest that MPS proceeds at a rate corresponding closely to
fed values in our study for approximately 6–8 h; for the remainder
of the day, MPS would presumably operate at or near
postabsorptive values. This then would result in a lower than
average rate of MPS over the course of the day than in women.
We recognize that a complete description of the mechanisms
involved in nutritional regulation of muscle mass in the elderly will
depend on information about the meal induced changes in muscle
protein breakdown. To date, there is no evidence that the
sensitivity of MPS in older men and women to insulin released by
feeding is different. Furthermore, in a study of 6 older men and 6
older women (68–70 y), who received two doses of insulin during
hyperinsulinaemic, isoaminoacidemic clamps, leucine whole body
Ra (an index of whole-body proteolysis) was not different between
the sexes [71]. Additional support comes from a study of 30 y old
men and women, in which it was observed that the inhibitory
effect of insulin on whole-body proteolysis (assessed during a
hyperinsulinemic, euglycemic, isoaminoacidemic clamp) was not
different between the sexes but, consonant with our results, in
women there appeared to be an anabolic resistance to the
stimulatory effect of insulin on whole-body protein synthesis [72].
In summary, we have demonstrated that there is significant
sexual dimorphism in MPS and its control in older adults; a
greater basal rate of MPS, operating over a large portion of the
diurnal cycle, may be, at least in part, responsible for the slower
loss of muscle in women than in men.
Acknowledgments
The authors wish to thank Nicole Wright for subject recruitment and
technical assistance, and the study subjects for their participation.
Author Contributions
Conceived and designed the experiments: BM DV MR. Performed the
experiments: GS DV. Analyzed the data: BM GS PA DV TF DR MR.
Contributed reagents/materials/analysis tools: BM GS PA DV MR. Wrote
the paper: BM GS PA DV MR.
References
1. Horowitz JF, Coppack SW, Klein S (2001) Whole-body and adipose tissue
glucose metabolism in response to short-term fasting in lean and obese women.
Am J Clin Nutr 73: 517–522.
2. Zierler K (1999) Whole body glucose metabolism. Am J Physiol 276: E409–426.
3. Kraegen EW, Jenkins AB, Storlien LH, Chisholm DJ (1990) Tracer studies of in
vivo insulin action and glucose metabolism in individual peripheral tissues.
Horm Metab Res Suppl 24: 41–48.
4. Radegran G, Blomstrand E, Saltin B (1999) Peak muscle perfusion and oxygen
uptake in humans: importance of precise estimates of muscle mass. J Appl
Physiol 87: 2375–2380.
5. Forbes GB, Reina JC (1970) Adult lean body mass declines with age: some
longitudinal observations. Metabolism 19: 653–663.
6. Mingrone G, Marino S, DeGaetano A, Capristo E, Heymsfield SB, et al. (2001)
Different limit to the body’s ability of increasing fat-free mass. Metabolism 50:
1004–1007.
7. Gallagher D, Ruts E, Visser M, Heshka S, Baumgartner RN, et al. (2000)
Weight stability masks sarcopenia in elderly men and women. Am J Physiol
Endocrinol Metab 279: E366–375.
8. Hughes VA, Frontera WR, Roubenoff R, Evans WJ, Singh MA (2002)
Longitudinal changes in body composition in older men and women: role of
body weight change and physical activity. Am J Clin Nutr 76: 473–481.
9. Zamboni M, Zoico E, Scartezzini T, Mazzali G, Tosoni P, et al. (2003) Body
composition changes in stable-weight elderly subjects: the effect of sex. Aging
Clin Exp Res 15: 321–327.
10. Bhasin S, Woodhouse L, Storer TW (2001) Proof of the effect of testosterone on
skeletal muscle. J Endocrinol 170: 27–38.
11. Buresova M, Gutmann E (1971) Effect of testosterone on protein synthesis and
contractility of the levator ani muscle of the rat. J Endocrinol 50: 643–651.
12. Grigsby JS, Bergen WG, Merkel RA (1976) The effect of testosterone on skeletal
muscle development and protein synthesis in rabbits. Growth 40: 303–316.
13. Rooyackers OE, Nair KS (1997) Hormonal regulation of human muscle protein
metabolism. Annu Rev Nutr 17: 457–485.
14. Umpleby AM, Russell-Jones DL (1996) The hormonal control of protein
metabolism. Baillieres Clin Endocrinol Metab 10: 551–570.
15. Ferrando AA, Tipton KD, Doyle D, Phillips SM, Cortiella J, et al. (1998)
Testosterone injection stimulates net protein synthesis but not tissue amino acid
transport. Am J Physiol 275: E864–871.
16. Griggs RC, Kingston W, Jozefowicz RF, Herr BE, Forbes G, et al. (1989) Effect
of testosterone on muscle mass and muscle protein synthesis. J Appl Physiol 66:
498–503.
17. Toth MJ, Poehlman ET, Matthews DE, Tchernof A, MacCoss MJ (2001) Effects
of estradiol and progesterone on body composition, protein synthesis, and
lipoprotein lipase in rats. Am J Physiol Endocri nol Metab 280: E496–501.
18. McCormick KM, Burns KL, Piccone CM, Gosselin LE, Brazeau GA (2004)
Effects of ovariectomy and estrogen on skeletal muscle function in growing rats.
J Muscle Res Cell Motil 25: 21–27.
19. Piccone CM, Brazeau GA, McCormick KM (2005) Effect of oestrogen on
myofibre size and myosin expression in growing rats. Exp Physiol 90: 87–93.
20. Fujita S, Rasmussen BB, Bell JA, Cadenas JG, Volpi E (2007) Basal muscle
intracellular amino acid kinetics in women and men. Am J Physiol Endocrinol
Metab 292: E77–83.
21. Parise G, Mihic S, MacLennan D, Yarasheski KE, Tarnopolsky MA (2001)
Effects of acute creatine monohydrate supplementation on leucine kinetics and
mixed-muscle protein synthesis. J Appl Physiol 91: 1041–1047.
22. Jahn LA, Barrett EJ, Genco ML, Wei L, Spraggins TA, et al. (1999) Tissue
composition affects measures of postabsorptive human skeletal muscle
metabolism: comparison across genders. J Clin Endocrinol Metab 84:
1007–1010.
23. Vary TC, Jefferson LS, Kimball SR (1999) Amino acid-induced stimulation of
translation initiation in rat skeletal muscle. Am J Physiol 277: E1077–1086.
24. Millward DJ, Garlick PJ, James WP, Nnanyelugo DO, Ryatt JS (1973)
Relationship between protein synthesis and RNA content in skeletal muscle.
Nature 241: 204–205.
25. Proud CG (200 7) Signalling to translation: how signal transduction pathways
control the protein synthetic machinery. Biochem J 403: 217–234.
26. Frost RA, Lang CH (2007) Protein kinase B/Akt: a nexus of growth factor and
cytokine signaling in determining muscle mass. J Appl Physiol 103: 378–387.
27. Lee SJ, McPherron AC (2001) Regulation of myostatin activity and muscle
growth. Proc Natl Acad Sci U S A 98: 9306–9311.
28. McPherron AC, Lee SJ (2002) Suppression of body fat accumulation in
myostatin-deficient mice. J Clin Invest 109: 595–601.
29. Guttridge DC (2004) Signaling pathways weigh in on decisions to make or break
skeletal muscle. Curr Opin Clin Nutr Metab Care 7: 443–450.
30. Toth MJ, Matthews DE, Tracy RP, Previs MJ (2005) Age-related differences in
skeletal muscle protein synthesis: relation to markers of immune activation.
Am J Physiol Endocrinol Metab 288: E883–891.
31. Schaap LA, Pluijm SM, Deeg DJ, Visser M (2006) Inflammatory markers and
loss of muscle mass (sarcopenia) and strength. Am J Med 119: 526 e529–517.
32. Visser M, Pahor M, Taaffe DR, Goodpaster BH, Simonsick EM, et al. (2002)
Relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass
and muscle strength in elderly men and women: the Health ABC Study.
J Gerontol A Biol Sci Med Sci 57: M326–332.
33. Roubenoff R (2003) Catabolism of aging: is it an inflammatory process? Curr
Opin Clin Nutr Metab Care 6: 295–299.
34. Dietrichson P, Coakley J, Smith PE, Griffiths RD, Helliwell TR, et al. (1987)
Conchotome and needle percutaneous biopsy of skeletal muscle. J Neurol
Neurosurg Psychiatry 50: 1461–1467.
35. Patterson BW, Zhang XJ, Chen Y, Klein S, Wolfe RR (1997) Measurement of
very low stable isotope enrichments by gas chromatography/mass spectrometry:
application to measurement of muscle protein synthesis. Metabolism 46:
943–948.
36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:
402–408.
37. Watt PW, Lindsay Y, Scrimgeour CM, Chien PA, Gibson JN, et al. (1991)
Isolation of aminoacyl-tRNA and its labeling with stable-isotope tracers: Use in
Muscle Anabolism
PLoS ONE | www.plosone.org 8 March 2008 | Volume 3 | Issue 3 | e1875
studies of human tissue protein synthesis. Proc Natl Acad Sci U S A 88:
5892–5896.
38. Mittendorfer B, Andersen JL, Plomgaard P, Saltin B, Babraj JA, et al. (2005)
Protein synthesis rates in human muscles: neither anatomical lo cation nor fibre-
type composition are major determinants. J Physiol 563: 203–211.
39. Welle S, Tawil R, Thornton CA (2008) Sex-related differences in gene
expression in human skeletal muscle. PLoS ONE 3: e1385.
40. McMahon CD, Popovic L, Jeanplong F, Oldham JM, Kirk SP, et al. (2003)
Sexual dimorphism i s ass ociated with decreased expression of processed
myostatin in males. Am J Physiol Endocri nol Metab 284: E377–381.
41. Brodsky IG, Balagopal P, Nair KS (1996) Effects of testosterone replacement on
muscle mass and muscle protein synthesis in hypogonadal men–a clinical
research center study. J Clin Endocrinol Metab 81: 3469–3475.
42. Urban RJ, Bodenburg YH, Gilkison C, Foxworth J, Coggan AR, et al. (1995)
Testosterone administration to elderly men increases skeletal muscle strength
and protein synthesis. Am J Physiol 269: E820–826.
43. Ferrando AA, Sheffield-Moore M, Paddon-Jones D, Wolfe RR, Urban RJ
(2003) Differential anabolic effects of testosterone and amino acid feeding in
older men. J Clin Endocrinol Metab 88: 358–362.
44. Cuthbertson D, Smith K, Babraj J, Leese G, Waddell T, et al. (2005) Anabolic
signaling deficits underlie amino acid resistance of wasting, aging muscle. Faseb J
19: 422–424.
45. Volpi E, Sheffield-Moore M, Rasmussen BB, Wolfe RR (2001) Basal muscle
amino acid kineti cs and protein synthesis in healthy young and older men.
JAMA 286: 1206–1212.
46. Bohe J, Low A, Wolfe RR, Rennie MJ (2003) Human muscle protein synthesis is
modulated by extracellular, not intramuscular amino acid availability: a dose-
response study. J Physiol 552: 315–324.
47. Kobayashi H, Borsheim E, Anthony TG, Traber DL, Badalamenti J, et al.
(2003) Reduce d amino acid availability inhibits muscle protein synthesis and
decreases activity of initiation factor eIF2B. Am J Physiol Endocrinol Metab 284:
E488–498.
48. Rennie MJ, Bohe J, Smith K, Wackerhage H, Greenhaff P (2006) Branc hed-
chain amino acids as fuels and anabolic signals in human muscle. J Nutr 136:
264S–268S.
49. Crozier SJ, Kimball SR, Emmert SW, Anthony JC, Jefferson LS (2005) Oral
leucine administration stimulates protein synthesis in rat skeletal muscle. J Nutr
135: 376–382.
50. Garlick PJ (2005) The role of leucine in the regulation of protein metabolism.
J Nutr 135: 1553S–1556S.
51. Rieu I, Balage M, Sornet C, Giraudet C, Pujos E, et al. (2006) Leucine
supplementation improves muscle protein synthesis in elderly men indepen-
dently of hyperaminoacidaemia. J Physiol 575: 305–315.
52. Pitkanen HT, Oja SS, Kemppainen K, Seppa JM, Mero AA (2003) Serum
amino acid concentrations in aging men and women. Amino Acids 24: 413–421.
53. Long CL, Birkhahn RH, Geiger JW, Blakemore WS (1981) Contribution of
skeletal muscle protein in elevated rates of whole body protein catabolism in
trauma patients. Am J Clin Nutr 34: 1087–1093.
54. Morais JA, Gougeon R, Pencharz PB, Jones PJ, Ross R, et al. (1997) Whole-
body protein turnover in the healthy elderly. Am J Clin Nutr 66: 880–889.
55. Partridge T (2000) The current status of myoblast transfer. Neurol Sci 21:
S939–942.
56. Lind L, Fugmann A, Mill gard J, Berne C, Lithell H (2002) Insulin-mediated
vasodilatation, but not glucose uptake or endothelium-mediated vasodilatation,
is enhanced in young females compared with males. Clin Sci (Lond) 102:
241–246.
57. Celermajer DS, Sorensen KE, Spiegelhalter DJ, Georgakopoulos D, Robinson,
et al. (1994) Aging is associated with endothelial dysfunction in healthy
men years before the age-related decline in women. J Am Coll Cardiol 24:
471–476.
58. Sarabi M, Millgard J, Lind L (1999) Effects of age, gender and metabolic factors
on endothelium-dependent vasodilation: a population-based study. J Intern Med
246: 265–274.
59. Jensen-Urstad K, Johansson J (2001) Gender difference in age-related changes in
vascular function. J Intern Med 250: 29–36.
60. Clark MG, Rattigan S, Barrett EJ (2006) Nutritive blood flow as an essential
element supporting muscle anabolism. Curr Opin Clin Nutr Metab Care 9:
185–189.
61. Sheffield-Moore M, Urban RJ, Wolf SE, Jiang J, Catlin DH, et al. (1999) Short-
term oxandrolone administration stimulates net muscle protein synthesis in
young men. J Clin Endocrinol Metab 84: 2705–2711.
62. Rousset S, Patureau Mirand P, Brandolini M, Martin JF, Boirie Y (2003) Daily
protein intakes and eating patterns in young and elderly French. Br J Nutr 90:
1107–1115.
63. Cid-Ruzafa J, Caulfield LE, Barron Y, West SK (1999) Nutrient intakes and
adequacy among an older population on the eastern shore of Maryland: the
Salisbury Eye Evaluation. J Am Diet Assoc 99: 564–571.
64. Ledikwe JH, Smiciklas-Wright H, Mitchell DC, Miller CK, Jensen GL (2004)
Dietary patterns of rural older adults are associated with weight and nutritional
status. J Am Geriatr Soc 52: 589–595.
65. Adibi SA, Mercer DW (1973) Protein digestion in human intestine as reflected in
luminal, mucosal, and plasma amino acid concentrations after meals. J Clin
Invest 52: 1586–1594.
66. Bergstrom J, Furst P, Vinnars E (1990) Effect of a test meal, without and with
protein, on muscle and plasma free amino acids. Clin Sci (Lond) 79: 331–337.
67. Remond D, Machebeuf M, Yven C, Buffiere C, Mioche L, et al. (2007)
Postprandial whole-body protein metabolism after a meat meal is influenced by
chewing efficiency in elderly subjects. Am J Clin Nutr 85: 1286–1292.
68. Tsai PJ, Wu WH, Huang PC (2000) Circadian variations in plasma neutral and
basic amino acid concentrations in young men o n an ordinary Taiwanese diet.
J Formos Med Assoc 99: 151–157.
69. Bohe J, Low JF, Wolfe RR, Rennie MJ (2001) Latency and duration of
stimulation of human muscle protein synthesis during continuous infusion of
amino acids. J Physiol 532: 575–579.
70. Moore D, Phillips S (2008) Dose response relationship between protein ingestion
and muscle protein synthesis post exercise in young men. In Experimental
Biology 2008. San Diego, CA.
71. Boirie Y, Gachon P, Cordat N, Ritz P, Beaufrere B (2001) Differential insulin
sensitivities of glucose, amino acid, and albumin metabolism in elderly men and
women. J Clin Endocrinol Metab 86: 638–644.
72. Chevalier S, Marliss EB, Morais JA, Lamarche M, Gougeon R (2005) The
influence of sex on the protein anabolic response to insulin. Metabolism 54:
1529–1535.
Muscle Anabolism
PLoS ONE | www.plosone.org 9 March 2008 | Volume 3 | Issue 3 | e1875
