Am J Physiol Endocrinol Metab 308: E1056–E1065, 2015.

First published March 31, 2015; doi:10.1152/ajpendo.00481.2014.

CALL FOR PAPERS Endocrine and Metabolic Dysfunction During Aging and
Senescence

Intake of low-dose leucine-rich essential amino acids stimulates muscle anabolism

equivalently to bolus whey protein in older women at rest and after exercise
Syed S. I. Bukhari,1* Bethan E. Phillips,1* Daniel J. Wilkinson,1 Marie C. Limb,1 Debbie Rankin,1
William K. Mitchell,1 Hisamine Kobayashi,2 Paul L. Greenhaff,1 Kenneth Smith,1 and Philip J. Atherton1
1
Medical Research Council/Arthritis Research United Kingdom Centre of Excellence for Musculoskeletal Ageing Research,
University of Nottingham, Derby, United Kingdom; and 2Ajinomoto Company Incorporated, Tokyo, Japan
Submitted 20 October 2014; accepted in final form 26 March 2015

Bukhari SS, Phillips BE, Wilkinson DJ, Limb MC, Rankin D, Mitch-
ell WK, Kobayashi H, Greenhaff PL, Smith K, Atherton PJ. Intake of
low-dose leucine-rich essential amino acids stimulates muscle anabolism
equivalently to bolus whey protein in older women at rest and after exercise.
Am J Physiol Endocrinol Metab 308: E1056–E1065, 2015. First published
March 31, 2015; doi:10.1152/ajpendo.00481.2014.—Dysregulated ana-
bolic responses to nutrition/exercise may contribute to sarcopenia;
however, these characteristics are poorly defined in female popula-
tions. We determined the effects of two feeding regimes in older
women (66 ⫾ 2.5 yr; n ⫽ 8/group): bolus whey protein (WP-20 g) or
novel low-dose leucine-enriched essential amino acids (EAA)
[LEAA; 3 g (40% leucine)]. Using [13C6]phenylalanine infusions, we
quantified muscle (MPS) and albumin (APS) protein synthesis at
baseline and in response to both feeding (FED) and feeding plus
exercise (FED-EX; 6 ⫻ 8 knee extensions at 75% 1-repetition max-
imum). We also quantified plasma insulin/AA concentrations, whole
leg (LBF)/muscle microvascular blood flow (MBF), and muscle
anabolic signaling by phosphoimmunoblotting. Plasma insulinemia
and EAA/aemia were markedly greater after WP than LEAA (P ⬍
0.001). Neither LEAA nor WP modified LBF in response to FED or
FED-EX, whereas MBF increased to a similar extent in both groups
only after FED-EX (P ⬍ 0.05). In response to FED, both WP and
LEAA equally stimulated MPS 0 –2 h (P ⬍ 0.05), abating thereafter
(0 – 4 h, P ⬎ 0.05). In contrast, after FED-EX, MPS increased at 0 –2
h and remained elevated at 0 – 4 h (P ⬍ 0.05) with both WP and
LEAA. No anabolic signals quantifiably increased after FED, but p70
S6K1 Thr389 increased after FED-EX (2 h, P ⬍ 0.05). APS increased
similarly after WP and LEAA. Older women remain subtly responsive
to nutrition ⫾ exercise. Intriguingly though, bolus WP offers no
trophic advantage over LEAA.
skeletal muscle; blood flow; protein synthesis; aging; amino acids;
exercise
ILL HEALTH ASSOCIATED WITH AGING represents a major socioeco-
nomic burden, especially given shifting demographics toward a
more aged, populous world. In particular, the loss of skeletal
muscle mass associated with aging, or sarcopenia, is a major
clinical issue. For instance, not only are there established links
between low muscle mass and all-cause mortality per se (1),
but also, lower skeletal muscle mass associated with sar-
* S. S. I. Bukhari and B. E. Phillips contributed equally to this article.
Address for reprint requests and other correspondence: P. J. Atherton,
MRC-ARUK Centre of Excellence for Musculoskeletal Ageing Research,
School of Medicine, Derby, DE22 3DT, UK (e-mail: philip.atherton@nottingham.
ac.uk).
E1056 0193-1849/15 Copyright © 2015 the American Physiological Society
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copenia leads to increased frailty, risk of falls, sedentarism,
poor quality of life, and prevalence of metabolic comorbidi-
ties (17, 53).
The two major extrinsic influences over muscle mass are
nutrition and physical activity. For example, oral intake of
protein-based foods containing essential amino acids (EAA)
leads to a transient (2–3 h; see Ref. 2) stimulation of muscle
protein synthesis (MPS) in younger men. This brief increase in
MPS above postabsorptive rates serves the purpose of replen-
ishing protein stores lost during fasting, ensuring preservation
of muscle protein mass. Similarly, physical activity is a pre-
requisite for maintenance of a healthy muscle mass. For ex-
ample, inactivity (6, 20) causes muscle atrophy by inducing
“anabolic resistance” to nutrition. Conversely, physical activ-
ity/exercise potentiates the trophic effects of nutrition; i.e.,
anabolic responses are greater when exercise and nutrient
intake are combined to facilitate adaptation and repair. Thus,
investigations into the effects of nutrition and the combination
of nutrition and exercise are key to determining the regulation
and dysregulation of muscle protein metabolism in older age.
The anabolic effects of protein and EAA have long been
defined. However, a significant body of recent work has shown
that the EAA leucine (LEU) serves not only as substrate for
MPS but also as an anabolic signal to the MPS machinery (27,
47); this is highlighted by the fact that the provision of small
quantities of LEU robustly stimulates MPS even in the absence
of other exogenous EAA (61). On this basis, the “dose re-
sponse” of MPS to EAA/protein is not apparently driven by
AA quantity per se but LEU content instead. This leaves open
the possibility that lower doses of EAA enriched with LEU
may provide a robust and less satiating (or caloric) anabolic
alternative, e.g., to large boluses of high-quality protein in
older age.
The rationale for the present study was threefold. First, older
women are grossly underrepresented in terms of studies on the
regulation of muscle protein metabolism with aging. Second,
since there exists sexual dimorphism in muscle protein turn-
over in older age (50, 51), with markedly different feeding
responses observed between older men and women (50), one
cannot assume that prior research findings in older men are
germane to older women. Therefore, an improved understand-
ing of the regulation of muscle protein metabolism in older
women, i.e., by nutrition and exercise combinations, is needed.
Third, in the context of older women, who show an apparent
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 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN
Table 1. Subject characteristics
LEAA (3 g) WP (20 g)
Age, yr 66 ⫾ 1 66 ⫾ 1
BMI, kg/m2 29 ⫾ 1 29 ⫾ 2
LBM, kg 41 ⫾ 2 40 ⫾ 2
EAA dose, g/kg LBM 0.075 ⫾ 0.003 0.25 ⫾ 0.01*
Appendicular lean mass, kg 17 ⫾ 1 17 ⫾ 1
SMI 25 ⫾ 1 26 ⫾ 2
Values are means ⫾ SE. LEAA, leucine-enriched amino acids; WP, whey
protein; BMI, body mass index; LBM, lean body mass; EAA, essential amino
acids; SMI, skeletal muscle index. *P ⬍ 0.001 vs. LEAA.
increased level of anabolic resistance (vs. men; see Ref. 51), an
investigation into novel nutritional interventions, which max-
imize anabolic responses to nutrition, is sorely needed. Al-
though increasing protein content of meals and providing large
boluses of protein are known to maximally stimulate MPS
(62), few older individuals may be able or willing to consume
such a satiating dose of protein without impacting habitual
nutrient intake, hence the need for lower caloric supplements.
Finally, albumin is an abundant protein used as a marker of
general nutritional status, and low levels are associated with
low protein intake (36), functional decline (49), reduced mus-
cle mass (4), reduced strength (48), and numerous age-related
diseases. Moreover, albumin synthesis is also affected by sex
difference (females have lower synthesis rates than males)
(56). Therefore, the albumin-synthetic response to anabolic
stimuli, such as nutrition, provides another window of insight
into sex differences in protein metabolism and is linked to
skeletal muscle outcomes in ageing. Therefore, in this study we
quantified the effects of two distinct nutritional interventions:
1) low-dose leucine-enriched amino acids [LEAA; 3 g of EAA
(40% LEU)] nutrition or 2) a large whey protein (WP) bolus
(20 g) under rested conditions and in tandem with an acute bout
of resistance exercise (RE; which is well known to potentiate
the anabolic response; see Ref. 16) on MPS and plasma
albumin synthesis in older women. It was hypothesized that
LEAA would provide equivalent stimulation of MPS and
albumin protein synthesis (APS) due principally to its leucine
content and to WP in older women, and the addition of RE
would potentiate this response.
MATERIALS AND METHODS
Subject characteristics and ethics. Ethical approval was obtained
from the University of Nottingham Medical School Ethics Committee
(United Kingdom), with all studies conducted in accordance with the
Declaration of Helsinki and preregistered at www.clinicaltrials.gov
(registration no. NCT02053441). Sixteen older postmenopausal
women (n ⫽ 8 in each group) matched for age and BMI [age 66 ⫾ 3
yr, BMI 29 ⫾ 1 (means ⫾ SE); see Table 1 for subject characteristics]
were recruited locally via advertisement through the mail. Exclusion
criteria included impaired mobility, history of diabetes, cardiovascu-
lar, pulmonary, liver, or kidney disorders, those on contraindicated
medications (nonsteroidal anti-inflammatory drugs, acetaminophen,
hormone replacement therapy) and those currently undergoing active
cancer therapies. All volunteers were then screened by a physician
(ⱖ1 wk prior to the study day) by means of a medical questionnaire,
physical examination, and resting electrocardiogram, with exclusions
for metabolic, respiratory, cardiovascular/vascular, or claudication-
related (either symptomatic or on treatment) disorders or other symp-
toms of ill health. Subjects had normal blood chemistry and were
normotensive (blood pressure ⬍ 140/90), and all subjects performed
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E1057
activities of daily living and recreation but did not routinely partici-
pate in any formal strenuous exercise regimes and were not on a
weight loss diet. During the screening visit, knee extension one-
repetition maximum (1-RM) was assessed, using the participants’
dominant/preferred leg, on a standard weighted gym knee extension
machine (Technogym, Gambettola, Italy). In addition, lean body mass
was assessed via dual X-ray absorptiometry and used to measure
appendicular muscle mass and calculate skeletal muscle index (SMI)
according to the following equation (38): SMI ⫽ (total body skeletal
muscle mass/total body mass) ⫻ 100. All subjects gave their written,
informed consent to participate after all procedures and risks were
explained. Following screening subjects were randomly assigned, on
the day of the acute study, to one of two groups receiving either 1) WP
(n ⫽ 8) or 2) LEAA (n ⫽ 8) (Table 2). Subjects were requested not
to start any new diet or exercise program between screening and the
study day and to refrain from vigorous exercise for 48 h prior to the
acute study visit and arrive at 0800 on the morning of the study fasted
overnight from 2000 the night before.
Study procedures. On the morning of the study (0800), subjects had
an 18-g cannula inserted into the antecubital vein of one arm for a
primed (0.4 mg/kg), constant infusion (0.6 mg·kg⫺1·h⫺1) of L-[ring-
13
C6]phenylalanine (Isotec; Sigma Aldrich) tracer and a retrograde
14-g cannula inserted to sample arterialized blood from the dorsal
capillary bed of the hand (using the “hot hand” method). Biopsies
were taken 1 and 3 h after the commencement of tracer infusion to
permit assessment of basal (postabsorptive) MPS. Subjects then per-
formed a bout of unilateral knee extension RE previously shown (34,
35) to maximally stimulate MPS (6 ⫻ 8 repetitions at 75% of their
predetermined 1-RM using the subjects’ dominant/preferred leg with
2 min interset rest) and also elevate leg blood flow (LBF) in elderly
men (44) before their supplement was consumed. Each bout lasted
40 – 60 s. If the subject failed to complete eight repetitions, then an
interset break was allowed before moving on to the next set. This
happened regularly with sets 5 and 6. Immediately after the exercise,
subjects consumed either 20 g of WP or 3 g of LEAA (“Amino L40”;
Ajinomoto) prepared in water (250 ml). The AA composition of each
supplement is given in Table 2. This unilateral study design meant that
the nonexercised leg was exposed to the effect of feeding alone
(“FED”), whereas the exercised leg was exposed to the combination
of feeding and exercise (“FED-EX”). Subsequent biopsies were then
taken 2 and 4 h after feeding to permit assessment of MPS over and
within the intervening periods. Blood samples and blood flow/vascu-
lar measurements were collected as outlined in Fig. 1. Muscle biopsies
were collected from m. vastus lateralis using the conchotome tech-
nique (14) after induction of local anesthesia via infiltration of 5 ml of
1% lignocaine. Muscle was washed in ice-cold phosphate-buffered
saline, and visible fat and connective tissue removed were before
being snap-frozen in liquid N2 and stored at ⫺80°C until analysis.
After completion of the study, cannulae were removed and subjects
fed and monitored for a further 30 min before being provided
transportation home.
Measurement of plasma insulin and AA concentrations. Arterial-
ized venous plasma insulin concentration was measured using a
Table 2. Essential amino acid composition of both types
of feed
LEAA (3 g), g WP (20 g), g
L-Leucine 1.2 2
L-Isoleucine 0.32 1.4
L-Valine 0.33 1.2
L-Threonine 0.28 1.4
L-Lysine 0.5 1.8
L-Methionine 0.1 0.4
L-Histidine 0.05 0.4
L-Phenylalanine 0.2 0.6
L-Tryptophan 0.02 0.4
E1058 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN
-1
Fig. 1. Study protocol: effects of leucine-en-
riched amino acids (LEAA) and whey protein
(WP) at rest and after resistance exercise (RE)
in older women. 1-RM, 1-repetition maximum;
CEUS, contrast-enhanced ultrasound.
Arterialized Bloods
Leg Blood Flow (Doppler)
Muscle Nutritive Flow (CEUS)
Muscle Biopsies - Rest Leg
Exercise Leg
high-sensitivity human insulin enzyme-linked immunosorbent assay
(ELISA; DRG Instruments, Marburg, Germany). For AA analyses, equal
volumes of arterialized plasma and 10% sulfosalicyclic acid were mixed
and cooled to 4°C for 30 min. Samples were centrifuged at 8,000 g to
pellet the precipitated protein, and the supernatant fluid was passed
through a 0.22-␮m filter before analysis with a dedicated AA analyzer
(Biochrom 30; Biochrom, Cambridge, UK) using lithium buffers. All
20 AA concentrations were measured by comparison to a standard AA
mix with norleucine as an internal standard.
Measurement of LBF and muscle microvascular blood flow. LBF
was measured using Doppler ultrasound, with a 9-3 mHz probe
positioned over the origin of the common femoral artery. Flow was
estimated as the product of vessel cross-sectional area and mean
velocity over six cardiac cycles. Contrast-enhanced ultrasound
(CEUS) was used to measure MBF, as described previously (45).
Briefly, Sonovue microbubbles were infused via an antecubital fossa
vein (Bracco, Milan, Italy). An iU22 ultrasound scanner (Phillips
Healthcare, Reigate, UK) with 40-mm linear 9-3 mHz probes (Phillips
L9-3) firmly secured on both anterior thighs (and measured individ-
ually for the FED and FED-EX legs) was used to register microbubble
appearance within the quadriceps muscle at rest and in response to
FED and FED-EX. Intermittent high mechanical index (MI) “flashes”
disrupted microbubbles while continuous low MI recording measured
the rate of microbubble replenishment. Infusion was at 2 ml/min for
1 min and 1 ml/min for 3 subsequent min. During the last 90 s of this
measurement protocol, 3 ⫻ 30 s flash/replenishment recordings were
made. Offline region-of-interest analysis using Q-Lab software (Phil-
lips Healthcare, Surrey, UK) was used to measure the plateau (“A”
value) linear acoustic intensity and also the rate constant (“␤” value),
the product of which is proportional to volume blood ⫻ volume
tissue⫺1 ⫻ s⫺1 and thus skeletal muscle tissue MBF (45). In total, two
measurements per participant were performed, the first one 50 – 60
min before the exercise and second one 50 – 60 min after the exercise
(Fig. 1).
Measurement of APS. Plasma (200 ␮l) was precipitated with
ice-cold 10% trichloroacetic acid. Following centrifugation (13,000
rpm, 4°C) to pellet plasma protein, the albumin from the pellet was
then extracted into ethanol to separate it from the nonalcohol soluble
proteins. The ethanol extract was dried under nitrogen and solubilized
in 0.3 M NaOH at 37°C for 60 min and then precipitated with 1 M
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Time (hours)
0 2 4 6
Primed (0.3 mg·kg-1), constant (0.6 mg·kg-1) infusion of 13C6 Phenylalanine
Postabsorptive
Unilateral Knee-extension:
6 x 8 at 75% of 1RM
Whey or LEAA (+ 6% 13C6 Phe)
perchloric acid. Isolated albumin-bound AA were released by acid
hydrolysis with 0.1 M hydrochloric acid and Dowex H⫹ ion exchange
resin (50W-X8-200; Sigma Aldrich, Poole, UK) heated overnight at
110°C before purification by ion exchange chromatography; AA were
derivatized as their n-acetyl-N-propyl esters, and incorporation of
13
L-[ring- C6]phenylalanine into albumin was assayed by gas chroma-
tography-combustion-isotope ratio mass spectrometry (Delta Plus XP;
Thermofisher Scientific, Hemel Hempstead, UK). Fractional synthesis
rates of albumin were determined using a standard precursor-product
model:
APS共% ⁄ h兲 ⫽ 冋 册
⌬Ea
Ep . t
⫻ 100
where ⌬Ea is the change in labeling of albumin phenylalanine be-
tween two blood samples, EP is the mean enrichment over time of the
precursor (taken as arterialized plasma L-[ring-13C6]phenylalanine
labeling), and t is the time in hours between samples.
Measurement of MPS. Myofibrillar proteins were isolated, hydro-
lyzed, and derivatized using our standard techniques (61). Briefly,
⬃25 mg of muscle biopsy tissue was homogenized in ice-cold
homogenization buffer [50 mm Tris·HCl (pH 7.4), 50 mm NaF, 10
mm ␤-glycerophosphate disodium salt, 1 mm EDTA, 1 mm EGTA,
and 1 mm activated Na3VO4 (all Sigma-Aldrich, Poole, UK)], and a
complete protease inhibitor cocktail tablet (Roche, West Sussex, UK)
at 10 ␮l/␮g of tissue. Homogenates were rotated for 10 min, and the
supernatant was collected by centrifugation at 13,000 g for 5 min at
4°C. The resulting pellet was washed three times with homogeniza-
tion buffer, and 0.3 M NaOH was added to facilitate the separation of
the soluble myofibrillar fraction from the insoluble collagen fraction
by subsequent centrifugation. The myofibrillar fraction was then
removed and precipitated using 1 M perchloric acid and pelleted by
centrifugation. The myofibrillar pellet was then washed twice with
70% ethanol, and the protein-bound AA were released by acid
hydrolysis using 0.1 M HCl and 1 ml of Dowex ion exchange resin
(50W-X8-200) heated overnight at 110°C. The free AA were purified,
derivatized, and analyzed as for APS, and the fractional synthesis rate
(FSR) of the myofibrillar proteins was calculated using the same
precursor-product approach:
 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN E1059

FSR共% ⁄ h兲 ⫽
where ⌬Em is the change in enrichment of bound L-[ring-13C6]phenylalanine
in two sequential biopsies, t is the time interval between two biopsies
in hours, and Ep is the mean free L-[ring-13C6]phenylalanine enrich-
ment in the intramuscular pool.
Immunoblotting for Akt/mTORC1 signaling pathway activity (i.e.,
phosphorylation). Immunoblotting was performed as described pre-
viously (3) using the sarcoplasmic fraction collected during MPS
preparation described above. Sarcoplasmic protein concentrations
were determined using a NanoDrop ND1000 spectrophotometer
(NanoDrop Technologies, Wilmington, DE) and adjusted to 2 ␮l/␮g
in 3⫻ Laemmli buffer. Each sample was loaded onto precast 12%
Bis-Tris Criterion XT gels (Bio-Rad, Hemel Hempstead, UK) at 20
␮g/lane and separated electrophoretically at 200 V for 1 h. Proteins
were wet transferred at 100 V for 45 min and subsequently blocked in
2.5% nonfat milk in 1⫻ Tris buffered saline-Tween 20 (TBST).
Membranes were incubated in primary antibodies (1:2,000 dilution in
2.5% BSA in TBST) rocking overnight at 4°C, Akt Ser473, p70 S6K1
Thr389, 4E-binding protein 1 (4E-BP1) Ser65/70, and eukaryotic initi-
ation factor 2 (eEF2) Thr56 (New England Biolabs, Hertfordshire,
UK). Membranes were subsequently washed for 3 ⫻ 5 min in TBST,
incubated in horseradish peroxidase (HRP)-conjugated secondary an-
tibody (1:2,000 in 2.5% BSA in TBST; New England Biolabs,
Hertfordshire, UK) at ambient temperature for 1 h, followed by 3 ⫻
5 min washes in TBST. Membranes were exposed to Chemilumines-
冋 册
⌬Em
Ep . t
⫻ 100
cent HRP Substrate (Millipore, Billerica, MA) for 5 min and bands
quantified by Chemidoc XRS (Bio-Rad, Hertfordshire, UK). All
signals were within the linear range of detection, and loading anom-
alies were corrected to coomassie (59). Blotting data were captured
using peak density analysis.
Statistical analyses. Data are presented as means ⫾ SE, except
where indicated. Demographic, anthropometric, and CEUS compari-
sons were analyzed within or between groups by unpaired or paired
t-tests (all 2-tailed) as appropriate. All other data sets were analyzed
using two-way repeated measures ANOVA (feed type ⫻ time) with
differences located via Holm Sidak posttests using GraphPad Prism
version 5, with P values calculated using GraphPad Quickcalcs
(GraphPad Software, San Diego, CA). P values of ⬍0.05 were
considered significant.
RESULTS
Plasma AA and insulin concentrations. Groups were
matched for appendicular lean mass (LEAA 17 ⫾ 1 vs. WP
17 ⫾ 1 kg) and skeletal muscle index (LEAA 25 ⫾ 1 vs. WP
26 ⫾ 2) but received markedly different (P ⬍ 0.01) quantities
of EAA (i.e., LEAA 0.075 ⫾ 0.003 vs. WP 0.25 ⫾ 0.01 g/kg
lean body mass). Plasma AA concentrations increased rapidly
in response to LEAA and WP and peaked at ⬃40 – 60 min
(P ⬍ 0.05; Fig. 2, A–E). Plasma availability of AA, EAA,
branched-chain AA, and LEU was significantly greater and

A B
5000 a,b a,b LEAA (3 g) 2500
a,b a,b
Total AA (μM)

a,b a,b
4000 a,b WP (20 g) 2000
EAA (μM)

a a,b
a
3000 1500 a,b
a a
2000 1000
1000 500
0 0
20

0
20
40
60
80

5
0
0
0
20
40
60
80

5
0
0
20

11
16
22
11
16
22

-1
-1

Time (min) Time (min)

C D
3000 1500 Fig. 2. Time course effects of 3 g of LEAA or 20
a,b a,b
g of WP on plasma amino acids (AA) and insulin
NEAA (μM)

BCAA (μM)

a,b a,b a,b concentrations: total AA (A), essential AA (EAA;

a,b
2000 1000 a,b B), nonessential AA (NEAA; C), branched-chain
a a,b
a AA (BCAA; D), leucine (E), and insulin (F).
1000 500 a a
a
Greater than basal (P ⬍ 0.05); bgreater than other
group at the specific time point indicated (P ⬍
0 0
0.05). Main effects of feeding, P ⬍ 0.001; effects
of feed-type, P ⬍ 0.001; interaction, P ⬍ 0.001.
20

0
20
40
60
80

5
0
0

20

0
20
40
60
80

5
0
0
11
16
22

11
16
22
-1

-1

Time (min) Time (min)

E F
500 30 a,b
Insulin (mU·l-1)
Leucine (μM)

a a,b a
400 a a,b a,b
a,b 20
300 a
a,b
a
200 a a
10 a
100
0
0
20

0
20
40
60
80

5
0
0

20

40

60

5
11
16
22

11
-1

Time (min) Time (min)

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E1060 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN

A B
0.5 0.5
Rest Leg
*

LBF (l·min-1)

MBF (A x β)
0.4 Exercise Leg 0.4

0.3 0.3

0.2 0.2

0.1 0.1
Fig. 3. The effect of 3 g of LEAA or 20 g of WP
0.0 0.0
in skeletal muscle of older women on leg blood

l
g)

g)
sa

sa
flow (LBF) and muscle blood flow (MBF) re-

(3

(3
Ba

Ba
AA

AA
sponses to the feeding (FED; A and B) or feeding

LE

LE
plus exercise (FED-EX; C and D) groups. *P ⬍
0.05 vs. basal. A: main effects of feeding, P ⬍
0.01; effects of feed-type, P ⬎ 0.05; interaction, C D
P ⬎ 0.05. B: main effects of feeding, P ⬍ 0.05; 0.5
0.5
effects of feed-type, P ⬎ 0.05; interaction, P ⬎
LBF (l·min-1)

MBF (A x β)
0.05. 0.4 0.4

0.3 0.3 *
0.2 0.2

0.1 0.1

0.0 0.0
l

g)

g)
l
sa

sa
0
Ba

0
Ba
(2

(2
P

P
W

more sustained in response to WP than LEAA (i.e., returning to
baseline concentrations 60 – 80 min after LEAA vs. 115–160
min after WP, P ⬍ 0.001). Plasma insulin concentration
increased over postabsorptive values (⬃5 mU/l, both groups)
by 20 min in response to both LEAA and WP (to 14 ⫾ 3 vs.
20 ⫾ 3 mU/l, respectively, P ⬍ 0.05). However, whereas
plasma insulin returned to basal concentration 40 min after
LEAA intake, (Fig. 2F), WP led to insulin concentrations that
remained elevated up to (and beyond) 60 min (18 ⫾ 3 mU/l).
LBF and MBF. LBF was not different between groups at
baseline (LEAA 0.27 ⫾ 0.03 vs. WP 0.34 ⫾ 0.04 l/min) and
was unaltered in response to FED and FED-EX 60 – 80 min
postfeeding, when AA concentrations peaked (P ⬎ 0.05; Fig.
3, A and C). Similarly MBF, measured 60 – 80 min postinges-
tion of supplement, was not increased in the rested leg, i.e.,
FED only, in either group, and although MBF was numerically
greater in the LEAA group, this was not consistent in all
individuals and did not reach significance. However, MBF
increased following FED-EX in both the LEAA and WP
groups (LEAA ⫹91 ⫾ 22%, WP ⫹229 ⫾ 23%, P ⬍ 0.05; Fig.
W
0.013%/h, both P ⬍ 0.05), but unlike with FED, MPS re-
mained elevated over the entire 4-h study period (LEAA
BASAL to FED-EX 0.089 ⫾ 0.005%/h, WP BASAL to
FED-EX 0.093 ⫾ 0.007%/h, both P ⬍ 0.05; Fig. 5B). Increases
in MPS in response to both FED and FED-EX were indistin-
guishable between the WP and LEAA groups (P ⬎ 0.05).
Effects on muscle anabolic signaling. Muscle anabolic sig-
nals as determined by measuring the phosphorylation of pro-
tein kinase B (Akt), the mechanistic target of rapamycin
complex 1 (mTORc1) translational initiation substrates ribo-
somal protein S6 kinase (p70 S6K1) and 4E-BP1, and the
elongation factor eEF2 (Fig. 6, A–F) were not increased de-
tectably in FED. Only in response to FED-EX could we detect
significant increases (⫹133 ⫾ 22%, P ⬍ 0.01; Fig. 6D) in
0.35
LEAA (3 g)
0.30 * *
#
Albumin FSR (%·h )

WP (20 g) *
-1

3, B and D). 0.25

Effects of WP and LEAA on APS and MPS. Increases in
0.20
plasma APS were observed in response to WP, whereas a trend
existed for LEAA only at 0 –2 h (LEAA basal to FED 0.19 ⫾ 0.15
0.02 to 0.23 ⫾ 0.03%/h, WP basal to FED 0.17 ⫾ 0.01 to
0.26 ⫾ 0.04%/h, P ⬍ 0.1 and P ⬍ 0.05, respectively; Fig. 4). 0.10
APS remained elevated in both groups at 0 – 4 h (LEAA, FED
increased to 0.25 ⫾ 0.02%/h, WP, FED increased to 0.29 ⫾ 0.05

0.02%/h, P ⬍ 0.05). In response to WP and LEAA, MPS was 0.00

increased at 0 –2 h (LEAA BASAL to FED 0.071 ⫾ 0.004 to
l

h)

h)

0.089 ⫾ 0.008%/h, WP BASAL to FED 0.066 ⫾ 0.006 to

sa

-2

-4
Ba

(0

(0

0.082 ⫾ 0.009%/h, both P ⬍ 0.05); this anabolic effect abated

d

d
Fe

Fe

rapidly, returning to postabsorptive rates (0 – 4 h, P ⬎ 0.05;

Fig. 4. The effect of 3 g of LEAA or 20 g of WP on albumin protein synthesis
Fig. 5A). In the exercised leg, MPS also increased at 0 –2 h (APS) in older women. *P ⬍ 0.05 and #P ⬍ 0.1 vs. basal. Main effects of
(LEAA BASAL to FED-EX 0.071 ⫾ 0.004 to 0.085 ⫾ feeding, P ⬍ 0.001; effects of feed type, P ⬎ 0.05; interaction, P ⬍ 0.05. FSR,
0.014%/h, WP BASAL to FED-EX 0.066 ⫾ 0.006 to 0.095 ⫾ fractional synthesis rate.

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 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN
Fig. 5. The effect of 3 g of LEAA or 20 g of WP in skeletal muscle of older
women on muscle protein synthesis (MPS) responses to the FED (A) and
FED-EX (B) groups. *P ⬍ 0.05 vs. basal. A: main effects of feeding, P ⬍ 0.01;
effects of feed type, P ⬎ 0.05; interaction, P ⬎ 0.05. B: main effects of
feeding, P ⬍ 0.05; effects of feed type, P ⬎ 0.05, interaction, P ⬎ 0.05.
phospho-p70 S6K1, but only after WP and not LEAA and at
only 2 h postfeeding, returning to baseline by 4 h (Fig. 7).
DISCUSSION
In the present study, we investigated in skeletal muscle of
older women the effects of and interaction between the two
major extrinsic drivers of muscle maintenance: nutrition and
exercise. Furthermore, we have investigated the use of a novel
low-dose, low-calorie, and leucine-enriched EAA (LEAA
“amino L-40”) supplement as a nutritional intervention in this
group. We found in older women that 1) both WP and LEAA
transiently (ⱕ2 h) stimulate MPS, 2) RE extends the “anabolic
window” by ⱖ4 h after consumption of WP or LEAA, 3) APS
is stimulated similarly after being fed WP and LEAA, 4) over
⬃4 h WP provides no additional trophic effects to LEAA in
response to either FED or FED-EX.
Whether anabolic resistance to nutrition is an intrinsic fea-
ture of aging remains contentious (24, 41, 54, 55). In our view,
any doubt surrounding the existence of anabolic resistance may
be due primarily to interstudy differences, e.g., not accounting
for differing habitual physical activity, physical status, or
pattern of feeding, e.g., bolus vs. pulse/spread. Many studies
measure MPS over periods longer (i.e., 4 – 6 h) than the
previously demonstrated anabolic window of 90 –120 min (2,
5), thereby missing any detectable effect, when the rate is
averaged. Nonetheless, whatever the standpoint, few would
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E1061
argue against optimizing muscle anabolic responses to nutri-
tion as being paramount in older age. We show here that
increasing the quantity of EAA consumed does not necessarily
augment MPS over and above the ingestion of ⬃1.2 g LEU
(plus a small 1.8-g mixed EAA) in older women. Therefore,
providing greater quantities of AA as meals or supplements to
older women may not bolster MPS beyond, e.g., LEAA.
Nonetheless, since anabolic resistance may be overcome by
providing larger doses of WP to older men (⬃40 g; see Ref.
43), it does remain entirely plausible that neither 20 g of WP
nor 3 g of LEAA maximally stimulates MPS in skeletal
muscles of older women; further studies are needed to define
this. Yet despite this, these findings highlight that a small dose
of LEAA (of low caloric value) provides adequate EAA
substrate to promote robust enhancement of MPS and thereby
could perhaps promote better maintenance of muscle mass in
older women.
Although the stimulation of MPS by EAA has long been
defined, it is becoming increasingly clear that protein or AA
dose response studies belie the importance of certain highly
anabolic EAA. For example, LEU is a major driver of MPS
(61) to the extent that enrichment of “suboptimal” doses of
protein or EAA with LEU robustly stimulates MPS (10), as
does the provision of just ⬃3 g of LEU to ⬃70 kg in younger
men entirely in the absence of other exogenous EAA (61).
Herein, we extend these findings by demonstrating that LEAA
stimulates MPS in older women with equal efficacy to a large
(20 g) bolus of WP despite the latter providing more than three
times more EAA per kilogram of LBM; we anticipate that LEU
was instrumental in this response given its unique potent
anabolic properties (7, 11, 61). This is supported somewhat by
the fact that despite WP having greater EAA and total AA
content, both WP and LEAA showed the same peak in LEU
concentration (Fig. 2E), and despite the WP maintaining sig-
nificantly raised LEU concentrations for longer than the
LEAA, there was no additional stimulation of MPS with WP.
On this basis, research is needed to define the effects of LEU
in the context of AA requirements for muscle health in old age
(7, 8, 15, 19, 46, 55, 58).
Physical activity in the form of RE (9, 26, 29, 35, 42) or even
maintaining habitual movement (57) remains the best-known
countermeasure against sarcopenia. Nonetheless, aging is as-
sociated with impaired anabolic responses to exercise, meaning
that it is essential to study how exercise and nutrition can be
best combined to yield maximal MPS. For example, increases
in MPS in response to exercise (18, 34, 35) and the combina-
tion of exercise and nutrition (18) are blunted in older men, as
is muscle hypertrophy in both men (32) and women (23). In the
present study, we report that older women exhibit prolonged
MPS responses to FED-EX (vs. FED) and that responses are
equal between LEAA and WP. Therefore, intake of low doses
of EAA enriched with LEU robustly stimulates MPS following
exercise in older women, similar to what has been shown in
younger men at rest (10) and after exercise (11). It is notewor-
thy in the latter study that WP bolus, but not LEAA, led to
sustainment of the anabolic effects of the nutrient/exercise
combination. In contrast, we found similarly prolonged ana-
bolic responses to FED-EX after both LEAA and WP; this is in
agreement with what has been observed in older men with
analogous LEAA supplementations post-RE (13). We specu-
late that the lack of difference in MPS between WP and LEAA
E1062 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN

A B

Coomassie (AU)

Coomassie (AU)
3 LEAA (3g) 3 LEAA (3g) + RE

p-Akt Thr473/
Whey (20g)

p-Akt Thr473/
Whey (20g) + RE
2 2

1 1

0 0

2h

4h
l

2h

4h

sa
sa

Ba
Ba
C Time (h)
D
Time (h)

p-p70S6K1Thr389 /

p-p70S6K1Thr389 /
Coomassie (AU)

Coomassie (AU)
4 4

3 3
*
2 2

1 1

Fig. 6. The effect of 3 g of LEAA or 20 g of 0 0

WP in skeletal muscle of older women on
muscle signaling responses to the FED (A)

l
l

2h

4h
2h

4h

sa
sa

Ba
Ba

and FED-EX (B) groups. *P ⬍ 0.05 vs.

basal. AU, arbitrary units; p70 S6K1, ribo-
somal protein S6 kinase; 4E-BP1, 4E-bind-
E Time (h)
F
Time (h)
p-4EBP1 Thr37/46 /
Coomassie (AU)

p-4EBP1 Thr37/46/
Coomassie (AU)
ing protein 1; eEF2, eukaryotic initiation 4 4
factor 2. 3 3

2 2

1 1

0 0
l

2h

4h
sa

2h

4h
sa
Ba

Ba
Time (h) Time (h)
G H
Coomassie (AU)

Coomassie (AU)

2 2
p-eEF2 Thr56 /

p-eEF2 Thr56 /

1 1

0 0
l

2h

4h
2h

4h
sa

sa
Ba
Ba

Time (h) Time (h)

might reflect blunted hypertrophy in older vs. young individ-
uals (23, 37, 60), i.e., being manifested as diminished uptake of
excess AA substrate into muscle proteins. This is also substan-
tiated by the lack of robust increases in anabolic signaling [as
reported in older men (34) and women (50)] and leg/muscle
blood flow, facets that may underlie the moderate anabolic
response to raised AA availability after FED and FED-EX in
older women.
When studying leg and muscle blood flow, we hypothesized
that WP may elicit greater blood flow responses compared with
LEAA due to the vasodilatory properties of arginine and
greater insulin responses associated with higher AA concen-
trations, i.e., through promoting secretory effects of AA upon
pancreatic ␤-cells. Nonetheless, whereas plasma insulin (and
arginine) concentrations were expectedly higher in the WP
group (Fig. 2F), there were no differences in LBF in line with
work we completed recently in older men (39, 44); that is,
older people exhibit “vascular resistance” to nutritional intake.
In contrast, there were significant increases to both WP and
LEAA in MBF, but only in the FED-EX leg (Fig. 3), suggest-
ing that this may have some role in the sustainment of MPS
beyond 2 h in the FED-EX leg. Indeed, despite the fact that we
have shown previously that pharmacological enhancement of
leg and muscle blood flow above the response to FED does not
enhance anabolism (45), when superimposed onto a metabolic
background of a prior bout of RE, our observed sustainment in
MBF with FED-EX may facilitate sustaining anabolic re-
sponses beyond ⬎2 h.
An important discussion point is to what extent, and under
what conditions, such WP or LEAA supplements yield effi-
cacy, e.g., on muscle mass/function. Since protein supplements
have significant effects to positively influence muscle hyper-
trophy in response to resistance exercise training (28, 63), we
speculate based on the information provided by the present
study that low-dose LEAA would be a similarly effective
supplement (at least for older women) without the need for
more satiating higher protein doses that may act as meal
replacements, perhaps negatively impacting overall energy/
protein intake. Moreover, most studies have focused on the
effects of supplements on muscle gains; however, given the

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 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN E1063
FED FED FED+EX particularly with age due to the known associations between
Basal 2h 4h 2h 4h low-serum albumin, health, morbidity, and mortality (12).
Indeed, albumin concentrations decrease with age (31), and
LEAA females have slower rates of APS (56). In this context, we have
demonstrated that LEAA led to significant stimulation of APS
Coomassie
p-AktThr473 equivalent to a large dose of WP, which is sustained for ⱕ4 h
Whey postfeeding. This suggests that the LEAA supplement (and
also the larger WP bolus) may also have implications for
Coomassie sustaining APS in older women. Given the known links to
muscle and general health, this could transpire to be an impor-
tant observation.
Potential study limitations warrant comment. First, we were
FED FED FED+EX unable to define the specific importance of LEU in mediating
Basal 2h 4h 2h 4h the efficacy of LEAA. Previous work by one of the current
authors using intravenous flooding dose techniques revealed
LEAA anabolic effects of other EAA in our LEAA feed (i.e., valine,
Coomassie phenylalanine) in man (52). Nonetheless, given potent
p-p70S6K1Thr389
mTORc1 signaling actions attributed to LEU (3, 21, 25), it is
Whey
speculated LEU was central. Second, we measured neither
Coomassie muscle protein breakdown nor whole body protein turnover;
i.e., age-related differences in splanchnic metabolism (40) and
insulin handling may also impact muscle and whole body
responses to feeding/exercise, particularly because of the anti-
proteolytic effect of insulin on muscle protein breakdown (33).
FED FED FED+EX Also, although 20 g of WP led to a significant stimulation of
Basal 2h 4h 2h 4h MPS, larger amounts of protein (⬎40 g) have been shown to
assist in potentiating MPS responses in older men (63), and
LEAA therefore, the MPS response to WP in the current group of
older women may be suboptimal. That said, our aim was to
Coomassie assess the efficacy of a low-dose, lower-satiating EAA nutri-
p-4EBP1Thr37/46
Whey
tional supplement rather than test the absolute dose of WP for
maximal MPS stimulation. Finally, with regard to anabolic
Coomassie signaling (in which responses were negligible), we may have
missed the “peak” best reflecting MPS responses; nonetheless,
we designed the studies to acquire biopsies at times most
appropriate to our primary outcome measurement of MPS.
FED FED FED+EX Moreover, given “peak signaling,” event(s) may differ between
Basal 2h 4h 2h 4h distinct feeding regimens such as those applied herein (20 g of
WP or 3 g of EAA) in addition to dissociations between
LEAA anabolic signaling and MPS (2, 22); this is relatively uncon-
Coomassie trollable. To conclude, our findings show that low-dose LEAA
p-eEF2Thr56 supplements have potential alone or combined with exercise as
Whey strategies for older women to enhance muscle maintenance.
Coomassie
ACKNOWLEDGMENTS

Fig. 7. Representative blots for signaling proteins. Note that solid lines indicate
noncontinuous lanes where samples were excluded, as they were not included
in the analysis (i.e., 3-h FED) due to incomplete data sets/lack of a comparison
with FED-EX (biopsy dropped midstudy).
incipient nature of sarcopenia, longer-term studies are needed
to determine the efficacy of supplements in offsetting losses in
muscle mass/function. Nonetheless, those studies, which have
been conducted, have indeed shown promise (30). Addition-
ally, although caution should be applied in terms of the extrap-
olation of one-off metabolic studies to the chronic scenario, we
contend that these acute metabolic readouts of protein anabo-
lism form a solid platform for identifying candidate interven-
tions for testing in longer-term supplement studies.
In addition to the muscle-specific effects observed in the
present study, the influence of nutrition on APS is important
We thank Margaret Baker and Amanda Gates for expert technical assis-
tance.
GRANTS
This work was supported by funding from Ajinomoto Co., Inc.
DISCLOSURES
Hisamine Kobayashi is an employee of Ajinomoto Co., Inc. The authors
declare no other conflicts of interest.
AUTHOR CONTRIBUTIONS
S.S.B., B.E.P., D.J.W., M.C.L., D.R., W.K.M., K.S., and P.J.A. performed
experiments; S.S.B., B.E.P., D.J.W., M.C.L., D.R., W.K.M., K.S., and P.J.A.
analyzed data; S.S.B., B.E.P., D.J.W., M.C.L., D.R., W.K.M., H.K., P.L.G.,
K.S., and P.J.A. interpreted results of experiments; S.S.B., D.J.W., and P.J.A.
drafted manuscript; S.S.B., B.E.P., D.J.W., H.K., P.L.G., K.S., and P.J.A.
edited and revised manuscript; S.S.B., B.E.P., D.J.W., M.C.L., D.R., H.K.,

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E1064 NUTRITION, EXERCISE, AND ANABOLISM IN OLDER WOMEN
P.L.G., K.S., and P.J.A. approved final version of manuscript; B.E.P., H.K.,
P.L.G., K.S., and P.J.A. conception and design of research; B.E.P., K.S., and
P.J.A. prepared figures.
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