Content uploaded by Christopher S Fry
Author content
All content in this area was uploaded by Christopher S Fry on Nov 18, 2015
Content may be subject to copyright.
Skeletal muscle amino acid transporter expression is increased in young and
older adults following resistance exercise
Micah J. Drummond,
1,3,5
Christopher S. Fry,
3
Erin L. Glynn,
3
Kyle L. Timmerman,
3,5
Jared M. Dickinson,
3
Dillon K. Walker,
3
David M. Gundermann,
3
Elena Volpi,
2,4,5
and Blake B. Rasmussen
1,3,5
Departments of
1
Nutrition and Metabolism and
2
Internal Medicine, Divisions of
3
Rehabilitation Science and
4
Geriatrics, and
5
Sealy Center on Aging, University of Texas Medical Branch, Galveston, Texas
Submitted 2 December 2010; accepted in final form 25 April 2011
Drummond MJ, Fry CS, Glynn EL, Timmerman KL, Dick-
inson JM, Walker DK, Gundermann DM, Volpi E, Rasmussen
BB. Skeletal muscle amino acid transporter expression is increased
in young and older adults following resistance exercise. J Appl
Physiol 111: 135–142, 2011. First published April 28, 2011;
doi:10.1152/japplphysiol.01408.2010.—Amino acid transporters
and mammalian target of rapamycin complex 1 (mTORC1) signaling
are important contributors to muscle protein anabolism. Aging is
associated with reduced mTORC1 signaling following resistance
exercise, but the role of amino acid transporters is unknown. Young
(n⫽13; 28 ⫾2 yr) and older (n⫽13; 68 ⫾2 yr) subjects performed
a bout of resistance exercise. Skeletal muscle biopsies (vastus latera-
lis) were obtained at basal and 3, 6, and 24 h postexercise and were
analyzed for amino acid transporter mRNA and protein expression
and regulators of amino acid transporter transcription utilizing real-
time PCR and Western blotting. We found that basal amino acid
transporter expression was similar in young and older adults (P⬎
0.05). Exercise increased L-type amino acid transporter 1/solute-
linked carrier (SLC) 7A5, CD98/SLC3A2, sodium-coupled neutral
amino acid transporter 2/SLC38A2, proton-assisted amino acid trans-
porter 1/SLC36A1, and cationic amino acid transporter 1/SLC7A1
mRNA expression in both young and older adults (P⬍0.05). L-type
amino acid transporter 1 and CD98 protein increased only in younger
adults (P⬍0.05). eukaryotic initiation factor 2 ␣-subunit (S52)
increased similarly in young and older adults postexercise (P⬍0.05).
Ribosomal protein S6 (S240/244) and activating transcription factor 4
nuclear protein expression tended to be higher in the young, while
nuclear signal transducer and activator of transcription 3 (STAT3)
(Y705) was higher in the older subjects postexercise (P⬍0.05).
These results suggest that the rapid upregulation of amino acid
transporter expression following resistance exercise may be regulated
differently between the age groups, but involves a combination of
mTORC1, activating transcription factor 4, eukaryotic initiation factor
2␣-subunit, and STAT3. We propose an increase in amino acid
transporter expression may contribute to enhanced amino acid sensi-
tivity following exercise in young and older adults. In older adults, the
increased nuclear STAT3 phosphorylation may be indicative of an
exercise-induced stress response, perhaps to export amino acids from
muscle cells.
mammalian target of rapamycin complex 1; L-type amino acid trans-
porter 1; activating transcription factor 4; proton-assisted amino acid
transporter 1; sarcopenia
AGE-RELATED SARCOPENIA IS characterized by a gradual, but
progressive, loss in skeletal muscle mass and strength (7, 14).
The molecular and cellular mechanisms of sarcopenia are
complex and result from many interacting physiological events
(40). A factor that contributes to the loss of lean mass in older
adults may be the inability to fully activate components of the
mammalian target of rapamycin complex 1 (mTORC1) signal-
ing pathway following resistance exercise (15, 34, 38). The
mTORC1 pathway is sensitive to changes in nutritional, hor-
monal, contractile, and energy status, and its activity notably
leads to downstream phosphorylation of ribosomal S6 kinase 1
and 4E binding protein 1 (10). mTORC1 can also indirectly
upregulate the transcription of several genes associated with
amino acid transport (1, 37, 44, 45). This function appears to be
partly mediated by activating transcription factor 4 (ATF4/
cAMP response element binding protein 2) (1, 16, 31, 42), but
the mechanisms of mTORC1-regulated amino acid transporter
expression in young and older adult skeletal muscle are not yet
understood.
Changes in amino acid availability can profoundly alter
protein metabolism (50). Amino acid transporters have a key
role in the regulation of muscle protein metabolism because of
their ability to activate the mTORC1 signaling pathway.
Amino acid transporters can influence mTORC1 activity, ei-
ther by increasing the delivery of substrates (i.e., leucine) to an
unknown upstream nutrient sensor, or by relaying signals to
downstream targets, independent of amino acid transport (e.g.,
transceptor) (17, 24). Specifically, system L [L-type amino
acid transporter 1 (LAT1)/solute-linked carrier (SLC) 7A5 and
CD98/SLC3A2 heterodimeric complex] and system A [i.e.,
sodium-coupled neutral amino acid transporter 2 (SNAT2)/
SLC38A2] amino acid transporters have received special at-
tention because of their role in sensing changes in amino acid
availability (3, 13, 26, 39, 41), transporting large neutral amino
acids (i.e., branch-chain amino acids) into the cell to activate
mTORC1 (2, 6, 27), and are transcriptionally regulated by a
mTORC1-dependent mechanism (1, 37, 43– 45). More re-
cently, the proton-assisted amino acid transporters (i.e., PAT1)
have been credited with regulating cell size and may also be
important players in transmitting nutrient signals to mTORC1
(17–19, 21). The role of cationic amino acid transporter 1
(CAT1)/SLC7A1 in skeletal muscle is less clear, although it is
a modulator of arginine transport (28, 48). To add further
complexity, amino acid availability influences CAT1 expres-
sion (25, 36), while downregulation of CAT1 transporters by
RNA interference in neuronal cells caused a marked reduction
of mTORC1 activity and nerve growth (23). In support of the
link between mTORC1 signaling and amino acid transporter
expression, we have recently reported that an increase in amino
acid availability increases mTORC1 signaling and protein
Address for reprint requests and other correspondence: M. J. Drummond,
Univ. of Texas Medical Branch, Dept. of Nutrition and Metabolism, Division
of Rehabilitation Sciences, Sealy Center on Aging, 301 Univ. Blvd., Galves-
ton, TX 77555-1144 (e-mail: mjdrummo@utmb.edu).
J Appl Physiol 111: 135–142, 2011.
First published April 28, 2011; doi:10.1152/japplphysiol.01408.2010.
8750-7587/11 Copyright ©2011 the American Physiological Societyhttp://www.jap.org 135
synthesis as well as system L, system A, and PAT1 expression
in human skeletal muscle (12).
Resistance exercise in fasted adults also increases amino
acid transport and, subsequently, muscle protein synthesis, as
assessed with the stable isotope tracer technique (4). To date,
some information is available describing the role of specific
amino acid transporters in rodent (20, 32), but not human,
skeletal muscle following exercise. Since an unaccustomed
bout of resistance exercise stimulates mTORC1 signaling and
muscle protein synthesis in young but not older adults (15, 34,
38), we postulated that signaling and transcriptional events
associated with amino acid transporter expression, downstream
of mTORC1, may be reduced in older adults.
Therefore, the goal of this study was twofold: 1) to deter-
mine whether a single bout of resistance exercise alters amino
acid transporter mRNA and protein expression in human
skeletal muscle; and 2) to determine whether aging is
associated with a differential response following exercise.
We hypothesized that an acute bout of high-intensity resis-
tance exercise would increase amino acid transporter mRNA
and protein expression (LAT1/SLC7A5, CD98/SLC3A2,
SNAT2/SLC38A2, PAT1/SLC36A1, and CAT1/SLC7A1)
and ATF4 nuclear protein expression to a greater extent in
younger than in older adults.
MATERIALS AND METHODS
Subjects. We studied young (n⫽13; 8 male, 5 female) and older
(n⫽13; 8 male, 5 female) subjects, who were a subset from a larger
trial (15). Subjects only differed in age (young 28 ⫾2 yr; old 68 ⫾
2 yr) and leg strength (one-repetition maximum: young 102 ⫾9 kg;
old 69 ⫾5 kg) (P⬍0.05). The subjects were considered physically
active, but did not engage in any regular exercise training program at
the time of enrollment. Screening of subjects was performed with
clinical history, physical exam, and laboratory tests, including com-
plete blood count with differential, liver and kidney function tests,
coagulation profile, fasting blood glucose and oral glucose tolerance
test, hepatitis B and C screening, human immuodeficiency virus test,
TSH, lipid profile, urinalysis, and drug screening. Older subjects
underwent additional screening that included an electrocardiogram
and stress test on a treadmill. On a separate day, a dual-energy X-ray
absorptiometry scan (Hologic QDR 4500W, Bedford, MA) was per-
formed to measure lean and fat mass. Subjects were also tested for
maximal strength by performing a one-repetition maximum on a leg
extension machine (Cybex-VR2, Medway, MA) during the initial
screening. A second one-repetition maximum testing was performed
at least 1 wk before study participation. The higher of the two
maximum tests was recorded as the subjects’ one-repetition maxi-
mum. All subjects gave written, informed consents before participat-
ing in the study, which was approved by the Institutional Review
Board of the University of Texas Medical Branch (which is in
compliance with the Declaration of Helsinki).
Experimental design. Subjects were admitted to the Institutional
Translational Sciences-Clinical Research Center of the University of
Texas Medical Branch the day before the study and refrained from
exercise for 48 h before study participation. Subjects were fed a
standard dinner and a snack at 2200 and were studied following an
overnight fast. Experiments were conducted at the same approximate
time of day to minimize subject-to-subject variation due to circadian
rhythms and lengths of fasting. Further details involving tracer meth-
odologies within the current experiment can be found elsewhere (15).
The morning of the study (day 1), a muscle biopsy was obtained from
the vastus lateralis using a 5-mm Bergström biopsy needle with
suction under local anesthesia (1% lidocaine). Following the basal
biopsy, subjects were transported to the exercise laboratory in which
each subject performed 8 sets of 10 repetitions of bilateral leg
extension exercise (Cybex International) at 70% of their one-repeti-
tion maximum. The rest period between sets was 3 min. On comple-
tion of exercise (⬃40 min), subjects returned to their hospital bed for
the remainder of the study. Muscle biopsies were sampled at 3 and 6
h following the completion of resistance exercise. Following the 6-h
postexercise muscle biopsy, the subjects were fed a standardized
lunch and dinner and a snack at 2200 as on the prior night. The next
day after an overnight fast (day 2), a final biopsy was performed at 24
h postexercise.
On day 1, the basal biopsy was sampled out of a single incision,
while the 3- and 6-h postexercise muscle biopsies were sampled from
another incision site on the same leg ⬃7 cm proximal to the previous
incision site. To minimize trauma to muscle due to the previous
biopsy sampling, muscle biopsies were angled in such a way that ⬃5
cm separated each sampling location (49). On day 2, the 24-h
postexercise muscle biopsy was sampled from a single incision on the
opposite leg. Muscle tissue was immediately blotted and frozen in
liquid nitrogen and stored at ⫺80°C until analysis.
Phenylalanine and leucine concentrations. Blood and muscle in-
tracellular free concentrations of phenylalanine and leucine were
determined via the internal standard method, which includes mea-
surement of tracer enrichments for L-[ring-
13
C
6
]phenylalanine and
[1-
13
C]leucine and appropriate internal standards (L-[
13
C
9
,
15
N]phe-
nylalanine and [5,5,5-
2
H
3
]leucine). Measurements were determined
by gas chromatography-mass spectrometry (6890 Plus CG, 5973N
MSD, 7683 autosampler, Agilent Technologies, Palo Alto, CA) as
previously described (51).
RNA extraction and semiquantitative real-time PCR. Total RNA,
cDNA synthesis, and real-time PCR were conducted as previously
reported (12). The average RNA integrity number for 104 total
isolated RNA samples was 8.40 ⫾0.03 (1–10 scale; 10 highest) and
a 1.30 ⫾0.01 28S-to-18S ratio. All isolated RNA and cDNA samples
were stored at ⫺80°C until further analysis. A majority of the primer
sequences used in this experiment has been published previously (12).
Unpublished sequences and the accession number for CAT1/SLC7A1
(NM_003045) are the following: Fwd: CCCACTCTACTTAT-
CATCCC, Rev: ATACATTTAGCGACCTTTCTG. 
2
-Microglobu-
lin was used as a normalization gene, as it did not change over time
or between age groups. Relative fold changes were determined from
the Ct values using the 2
⫺⌬⌬Ct
method (35).
ATF4 and STAT3 nuclear and cytoplasmic protein isolation. Iso-
lation of protein subfractions was conducted using a commercially
available kit (NE-PER; Thermo Fisher Scientific). Muscle samples
were weighed and washed in 1⫻PBS. Samples were homogenized in
a cytoplasmic extraction reagent (I) that contained an appropriate
volume of protease inhibitors (Thermo-Scientific Halt Protease Inhib-
itor Cocktail). Muscle samples were then vortexed and incubated on
ice. A second cytoplasmic extraction reagent was added (II), the
sample was vortexed and then centrifuged, and the supernatant (cy-
toplasmic fraction) was collected. The pellet was resuspended in a
nuclear extraction reagent, then vortexed and incubated on ice for a
total of four times. Afterwards, the slurry was centrifuged, and the
supernatant collected (nuclear subfraction). Total protein was deter-
mined for both of the protein subfractions using the Bradford assay.
Nuclear and cytoplasmic protein was then subjected to Western
blotting. Effective separation of the protein subfraction was accom-
plished by minimal/undetectable levels of ␣-tubulin (a cytoplasmic
protein) in the nuclear protein compartment and lamin A (a nuclear
protein) in the cytoplasmic protein compartment (Fig. 1).
Western blotting. Protein expression analysis was performed as
previously reported (9). Western blot data were normalized to an
internal control (loaded on every gel to compare across blots). Anti-
bodies used were the following: LAT1 (cat. no. 5347; Cell Signaling),
CD98 (cat. no. sc-9160; Santa Cruz Biotechnology, Santa Cruz, CA),
SNAT2 (cat. no. sc-67081; Santa Cruz Biotechnology), ribosomal
protein S6 (rpS6) (S240/244; cat. no. 2215; Cell Signaling, Beverley,
136 AMINO ACID TRANSPORTER EXPRESSION AND RESISTANCE EXERCISE
J Appl Physiol •VOL 111 •JULY 2011 •www.jap.org
MA), eukaryotic initiation factor 2 ␣-subunit (eIF2␣) (S52; cat. no.
44728G; Invitrogen, Carlsbad, CA), total eIF2␣(cat. no. 9722; Cell
Signaling), ATF4 (cat. no. sc-200; Santa Cruz Biotechnology);
STAT3 (Y705; cat. no. 9131; Cell Signaling), total STAT3; (cat no.
9132; Cell Signaling), lamin A (cat. no. MAB3540; Millipore, Bil-
lerica, MA), and ␣-tubulin (cat. no. F2168; Sigma-Aldrich, St. Louis,
MO). Appropriate secondary antibodies were used, dependent on the
primary antibody of interest. Due to limited muscle tissue, only a
subset of subjects was used for Western analysis (see RESULTS and Fig.
1– 6 legends).
Statistical analysis. To measure differences across time and be-
tween age groups, data were statistically analyzed using a two-way
repeated-measures ANOVA. When a main effect existed, post hoc
tests (Fisher least significant difference) were conducted to assess
specific interactions. Subject characteristic analysis was conducted
using an unpaired t-test. Significance was set at P⬍0.05, unless
otherwise noted. All values are presented as means ⫾SE. All analyses
were performed with SigmaStat software (version 3.5).
RESULTS
Blood and muscle intracellular phenylalanine and leucine
concentrations. Phenylalanine and leucine concentrations from
young and older adults before and after resistance exercise are
presented in Table 1. Blood leucine concentrations were de-
creased at 3 h postexercise in young and older subjects com-
pared with basal levels (P⬍0.05). Muscle intracellular phe-
nylalanine concentrations decreased across all postexercise
time points in young and older adults compared with basal
levels (P⬍0.05). There was a main effect for time for blood
phenylalanine (P⬍0.05). When the age groups were col-
lapsed, there was a tendency for blood phenylalanine to de-
crease at3h(P⫽0.06), while there was a decrease at 6 h
postexercise compared with basal levels (P⬍0.05). There
were no changes in muscle intracellular leucine concentrations
following resistance exercise compared with basal levels (P⬎
0.05). There were no differences between the age groups for
any of the phenylalanine or leucine parameters (P⬎0.05).
Amino acid transporter mRNA and protein expression.
LAT1/SLC7A5 mRNA expression (Fig. 2A) was elevated at 3
h postexercise for both age groups compared with basal (P⬍
0.05). However, LAT1/SLC7A5 mRNA expression in the
older adults remained elevated and was higher at 6 h postex-
ercise than in the younger adults (P⬍0.05). LAT1/SLC7A5
protein expression (Fig. 2B) (young: n⫽13, old: n⫽11) was
elevated ⬃25% in the young adults at 6 and 24 h postexercise
compared with basal (P⬍0.05). There were no significant
changes in LAT1/SLC7A5 protein expression from basal at
any time point in the older adults (3 h, P⫽0.16; 6 h, P⫽0.19;
24 h, P⫽0.18). CD98/SLC3A2 mRNA expression (Fig. 2C)
(young: n⫽12, old: n⫽12) was elevated at 6 h postexercise
in young and older adults compared with basal levels (P⬍
0.05). CD98/SLC3A2 protein expression (Fig. 2D) was ele-
vated only in young adults at 24 h postexercise (⬃100%)
compared with basal (P⬍0.05). There were no changes in
CD98/SLC3A2 protein expression at any time point compared
with basal in the older adults (P⬎0.05). SNAT2/SLC38A2
mRNA expression (Fig. 3A) was elevated at 3 h postexercise
only for the older adults compared with basal levels (P⬍
0.05). SNAT2/SLC38A2 protein expression (Fig. 3B) was
unchanged in young and older adults postexercise compared
with basal levels (P⬎0.05). PAT1/SLC36A1 mRNA expres-
sion (Fig. 4A) tended to be higher at 3 h only in the older adults
(P⫽0.053) and was elevated at 6 h postexercise in both
younger and older adults compared with basal levels (P⬍
0.05). CAT1/SLC7A1 mRNA expression (Fig. 4B) was ele-
vated at 3 and 6 h postexercise in the older adults compared
with basal (P⬍0.05), while CAT1/SLC7A1 mRNA expres-
sion increased at 6 and 24 h postexercise in younger adults
compared with basal levels (P⬍0.05). There were no differ-
ences in basal amino acid transporter mRNA and protein
expression levels between young and older adults (P⬎0.05;
data not shown).
rpS6 (S240/244) and eIF2
␣
(S52) phosphorylation. rpS6
phosphorylation (S240/244) (Fig. 5A) tended to be elevated at
3h(P⫽0.056), but was elevated at 6 and 24 h postexercise
in the younger adults compared with basal levels (P⬍0.05).
rpS6 phosphorylation was elevated at 3 h postexercise in older
adults compared with basal levels (P⬍0.05). In addition,
younger adults exhibited a higher rpS6 phosphorylation at 24 h
compared with older adults (P⬍0.05). eIF2␣(S52) phosphor-
ylation (Fig. 5B) (young: n⫽13, old: n⫽12) was elevated
3–24 h postexercise in younger adults compared with basal
Fig. 1. Representative Western blot image from a young
and older adult at basal and 3, 6, and 24 h postexercise.
Gel was loaded alternately with nuclear (N) and cyto-
plasmic (C) subfractions and then probed with lamin A
(a nuclear protein) and ␣-tubulin (a cytoplasmic pro-
tein).
Table 1. Blood and muscle intracellular phenylalanine and
leucine concentration before and after resistance exercise in
young and older subjects
Basal 3 h Post 6 h Post 24 h Post
Blood phenylalanine, mol/l
Young 65 ⫾362⫾260⫾368⫾4
Old 66 ⫾460⫾360⫾366⫾4
Blood leucine, mol/l
Young 157 ⫾6 136 ⫾4* 147 ⫾5 157 ⫾9
Old 144 ⫾9 126 ⫾8* 144 ⫾9 138 ⫾5
Muscle intracellular
phenylalanine, mol/l
Young 65 ⫾554⫾3* 48 ⫾2* 50 ⫾4*
Old 70 ⫾459⫾3* 57 ⫾5* 57 ⫾4*
Muscle intracellular leucine,
mol/l
Young 170 ⫾18 153 ⫾15 150 ⫾18 165 ⫾18
Old 176 ⫾12 168 ⫾21 148 ⫾15 144 ⫾12
Values are means ⫾SE; n⫽13 young and n⫽13 older subjects, except
for muscle intracellular leucine (young, n⫽12; old, n⫽12). Post, after
resistance exercise. *Significantly different from basal (P⬍0.05). Time main
effect at 6 h for blood phenylalanine (P⬍0.05).
137AMINO ACID TRANSPORTER EXPRESSION AND RESISTANCE EXERCISE
J Appl Physiol •VOL 111 •JULY 2011 •www.jap.org
levels (P⬍0.05). eIF2␣(S52) phosphorylation tended to be
elevated at3h(P⫽0.059), but was elevated at6hintheolder
adults compared with basal levels (P⬍0.05). There were no
differences at baseline between young and older adults for rpS6
or eIF2␣phosphorylation (P⬎0.05; data not shown).
ATF4 and STAT3 (Y705) nuclear expression. Nuclear pro-
tein expression of ATF4 (Fig. 6A) (young: n⫽12, old: n⫽12)
was modestly higher in younger vs. the older adults [main
effect for age (P⬍0.05)]. There was a tendency for ATF4
nuclear expression to be higher in the younger adults 6 h
postexercise compared with basal levels (P⫽0.055). Phospho-
STAT3 (Y705) nuclear expression (Fig. 6B) (young: n⫽11,
old: n⫽12) was elevated at 6 h postexercise in the younger
adults (P⬍0.05) and elevated at 3, 6, and 24 h postexercise in
the older adults compared with basal levels (P⬍0.05). Older
adults tended to have higher STAT3 (Y705) nuclear expression
at 3 h postexercise compared with younger adults (P⫽0.054).
There were no differences at baseline between young and older
adults for nuclear ATF4 expression or STAT3 nuclear phos-
phorylation (P⬎0.05; data not shown).
DISCUSSION
The novel findings of this study were that 1) a single bout of
high-intensity resistance exercise increased the expression of several
skeletal muscle amino acid transporters (LAT1/SLC7A5, SNAT2/
SLC38A2, CD98/SLC3A2, PAT1/SLC36A1, and CAT1/SLC7A1)
in healthy men and women independent of age; 2) resistance
exercise increased rpS6 phosphorylation (S240/244) and the
nuclear protein expression of ATF4 primarily in young adults;
and 3) nuclear protein expression of STAT3 (Y705) was
increased to a greater extent in older adults. These data suggest
that, in young adults, the upregulation of amino acid trans-
porter mRNA and protein expression following exercise may
be associated with enhanced mTORC1 signaling and nuclear
ATF4 expression. However, in older adults, the increase in
amino acid transporter expression may be regulated by a
different mechanism, which may involve enhanced STAT3
phosphorylation. We propose that increased amino acid trans-
porter mRNA and protein expression following a bout of
resistance exercise may be an adaptive mechanism to increase
muscle amino acid sensitivity to increase amino acid import/
export during postexercise recovery or to amplify the muscle
protein synthesis response when combined with a subsequent
anabolic stimulus (i.e., protein-rich meal). Future work is
required to determine the precise role and contribution of the
exercise-induced upregulation of amino acid transporter ex-
pression on amino acid sensitivity and the regulation of intra-
cellular amino acid availability.
Our findings clearly show that amino acid transporter
mRNA expression (LAT1/SLC7A5, CD98/SLC3A2, SNAT2/
SLC38A2, and CAT1/SLC7A1) increased between 3 and 24 h
postexercise in fasted adults and, surprisingly, was not differ-
ent between the age groups (Figs. 2– 4). Although we noticed
an increase in LAT1 and CD98 protein expression that was
Fig. 2. Data (means ⫾SE) represent expression for L-type amino acid transporter 1 (LAT1)/solute-linked carrier (SLC) 7A5 mRNA (A), LAT1/SLC7A5 protein
(B), CD98/SLC3A2 mRNA (C), and CD98/SLC3A2 protein (D) before (basal) and after (3, 6, and 24 h) a single bout of resistance exercise in skeletal muscle
of young and older adults. *Significantly different than basal (P⬍0.05). #Significantly different between the age groups at indicated postexercise time point
(P⬍0.05). mRNA fold change was calculated using the 2
⫺⌬⌬Ct
method (35). Note: 13 young and 13 older adults were included in analysis, except for LAT1
(young, n⫽13; old, n⫽11) and CD98 protein expression (young, n⫽12; old, n⫽12).
138 AMINO ACID TRANSPORTER EXPRESSION AND RESISTANCE EXERCISE
J Appl Physiol •VOL 111 •JULY 2011 •www.jap.org
limited to young subjects, the inability to detect changes in
transporter protein expression in older adults may be due to an
underpowered sample size. In the present study design, we
were unable to gather information on amino acid kinetics to
clarify the direction of amino acid transport following exercise
(Table 1); however, we propose that increased amino acid
transporter expression may be a mechanism to increase the
sensitivity of muscle cells to amino acids. An increase in amino
acid transporters postexercise could be important for 1) acutely
increasing amino acid influx/efflux for muscle protein synthe-
sis/breakdown (4); 2) increasing amino acid sensitivity for a
protein-rich meal (5, 11); and/or 3) increasing amino acid
sensitivity in response to a second bout of exercise. Evidence
consistent with this “sensitizing” hypothesis is the observation
that a bout of exercise in rats improves insulin and IGF-I-
stimulated system A transport (i.e., SNAT2/SLC38A2) activity
during postexercise recovery (20, 52), while an anabolic mix-
ture provided after resistance exercise in humans enhances the
rate of muscle protein synthesis compared with exercise inde-
pendent of feeding (5, 8, 11). We have recently reported that
resistance exercise increased muscle protein synthesis in young
but not older adults over a 24-h postexercise period (15).
Therefore, our present data, in light of Fry et al. (15), would
infer that an upregulation of amino acid transporters may not
regulate muscle protein synthesis in the immediate postexer-
cise period when in the fasted state (at least in older adults), but
instead may influence amino acid sensitivity for a subsequent
anabolic stimulus. In support of this notion, Drummond et al.
(11) showed that a single bout of exercise when followed with
20 g of essential amino acids increased muscle protein synthe-
sis similarly in healthy, young and older adults 1– 6 h postex-
ercise. Regardless, whether or not increased amino acid trans-
porter expression is partly responsible for improving muscle
cell sensitization to amino acids, an increase in amino acid
transporter expression is an adaptive response to an acute bout
of unaccustomed resistance exercise and may be an important
player in the overall protein anabolic response following re-
sistance exercise.
Following resistance exercise, we found that rpS6 phosphor-
ylation (S240/244) (Fig. 5A) and nuclear ATF4 (Fig. 6A)
expression was significantly increased primarily in young but
not older subjects. ATF4 has been implicated in regulating
several genes associated with amino acid transport (i.e., LAT1/
SLC7A5, CD98/SLC3A2, SNAT2/SLC38A2, and CAT1/
SLC7A1) (1, 31, 37). Additionally, mTORC1 signaling has
Fig. 4. Data (means ⫾SE) represent mRNA expression for proton-assisted
amino acid transporter 1 (PAT1)/SLC36A1 (A) and cationic amino acid
transporter 1 (CAT1)/SLC7A1 (B) before (basal) and after (3, 6, and 24 h) a
single bout of resistance exercise in skeletal muscle of young (n⫽13) and
older (n⫽13) adults. *Significantly different than basal (Pⱕ0.05). mRNA
fold change was calculated using the 2
⫺⌬⌬Ct
method (35).
Fig. 3. Data (means ⫾SE) represent sodium-coupled neutral amino acid
transporter 2 (SNAT2)/SLC38A2 mRNA (A) and protein (B) expression before
(basal) and after (3, 6, and 24 h) a single bout of resistance exercise in skeletal
muscle of young (n⫽13) and older (n⫽13) adults. *Significantly different
than basal (P⬍0.05). mRNA fold change was calculated using the 2
⫺⌬⌬Ct
method (35).
139AMINO ACID TRANSPORTER EXPRESSION AND RESISTANCE EXERCISE
J Appl Physiol •VOL 111 •JULY 2011 •www.jap.org
been proposed to be a regulator of ATF4 and amino acid
transporter expression, as demonstrated by cell culture exper-
iments utilizing the chemical inhibitor rapamycin (1, 37, 43–
45). However, to the best of our knowledge, no data have been
reported identifying a regulatory role for mTORC1 signaling
on PAT1 expression. The possibility of mTORC1 playing a
part in driving ATF4 expression is modeled well in our present
findings and is in agreement with our previous report showing
robust increases in amino acid transporter expression following
amino acid ingestion in young subjects in association with
increased rpS6 phosphorylation and ATF4 expression (12).
ATF4 expression can also be positively regulated by eIF2␣
Ser52 phosphorylation although, paradoxically, eIF2␣(S52)
inhibits protein synthesis (22, 33). In our study, we show a
similar age-related increase in phosphorylation, whereas the
increase in ATF4 nuclear expression was limited to young
adults, suggesting that ATF4 may be regulated by eIF2␣
phosphorylation in young adults. Therefore, amino acid trans-
porter expression may be linked to mTORC1 signaling (e.g.,
Fig. 6. Data (means ⫾SE) represent protein expression for nuclear activating
transcription factor 4 (ATF4; A) and STAT3 phosphorylation at Thr705 (B)
before (basal) and after (3, 6, and 24 h) a single bout of resistance exercise in
skeletal muscle of young and older adults. Western blot insets are representa-
tive images corresponding to time points below (in duplicate) from a single
young and older adult. *Significantly different than basal (P⬍0.05). #Sig-
nificantly different between the age groups at indicated postexercise time point
(Pⱕ0.05). Note: a subset of the enrolled subjects was included for nuclear
ATF4 (young, n⫽12; old, n⫽12) and STAT3 (young, n⫽11; old, n⫽12)
analysis.
Fig. 5. Data (means ⫾SE) represent phosphorylation of ribosomal protein S6
(rpS6) at Ser240/244 (A) and eukaryotic initiation factor 2 ␣-subunit (eIF2␣)
at Ser52 (B) before (basal) and after (3, 6, and 24 h) a single bout of resistance
exercise in skeletal muscle of young and older adults. Western blot insets are
representative images corresponding to time points below (in duplicate) from
a single young and older adult. *Significantly different than basal (P⬍0.05).
#Significantly different between the age groups at indicated postexercise time
point (P⬍0.05). Note: 13 young and 13 older adults were included in analysis,
except for eIF2␣(S52) expression (young, n⫽13; old, n⫽12).
140 AMINO ACID TRANSPORTER EXPRESSION AND RESISTANCE EXERCISE
J Appl Physiol •VOL 111 •JULY 2011 •www.jap.org
rpS6), ATF4, and eIF2␣(S52) expression in young adults
following resistance exercise, while this relationship is less
obvious in older subjects.
We found that STAT3 (Y705) nuclear protein expression
was elevated to a greater extent in older subjects compared
with the young subjects postexercise (Fig. 6B). Recently, two
independent studies by Jones et al. (29, 30) showed that IL-6,
TNF-␣, and adiponectin increased system A transporter ex-
pression (i.e., SNAT2) in placental trophoblast cells, and this
was mediated through STAT3 transcriptional activity. Elevated
STAT3 phosphorylation (Y705) is in agreement with previous
resistance exercise studies in older subjects (46, 47) and
implies that stress-mediated damage may elevate nuclear
STAT3 levels and perhaps increase amino acid transporter
expression for amino acid export. Although the increase in
SNAT2/SLC38A2 expression was small in our experiment, we
cannot rule out that STAT3 (Y705) impacts the expression of
other amino acid transporters. Our initial data would suggest
that ATF4 may play a regulatory role (at least in the young
subjects) controlling amino acid transporter expression, but
clearly is not the only mechanism, as additional factors such as
the muscle stress response following resistance exercise may
also be involved.
We conclude that, following a single bout of resistance
exercise, 1) the mRNA and/or protein expression of several
amino acid transporters is elevated in both young and older
adults; and 2) amino acid transporter expression is likely
regulated differently in young and older subjects. The specific
mechanism(s) that regulates transporter expression remains to
be elucidated, but may involve mTORC1 signaling, ATF4,
eIF2␣, and STAT3 expression. An increase in amino acid
transporter expression following resistance exercise may be an
adaptive mechanism to increase amino acid sensitivity within
the cell during anabolic conditions of muscle growth and/or to
export amino acids when protein turnover increases during
muscle remodeling.
ACKNOWLEDGMENTS
We are grateful to the nursing staff at the Institute for Translational
Sciences Clinical Research Center and the Claude E. Pepper Older Americans
Independence Center recruitment coordinators, and Dr. Shaheen Dhanani for
assistance in screening, admitting, and assisting with the subjects during data
collection. We also thank Shelley Medina, Junfang Hao, and Ming Zheng for
help with the data analysis.
GRANTS
This study was supported in part by National Institutes of Health (NIH)
Grants R01-AR-049877 and P30-AG-024832. Additional support came from
the NIH/National Center for Research Resources (1UL1RR029876-01) and
NIH T32-HD007539.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
REFERENCES
1. Adams CM. Role of the transcription factor ATF4 in the anabolic actions
of insulin and the anti-anabolic actions of glucocorticoids. J Biol Chem
282: 16744 –16753, 2007.
2. Baird FE, Bett KJ, MacLean C, Tee AR, Hundal HS, Taylor PM.
Tertiary active transport of amino acids reconstituted by coexpression of
system A and L transporters in Xenopus oocytes. Am J Physiol Endocrinol
Metab 297: E822–E829, 2009.
3. Bevington A, Brown J, Butler H, Govindji S, KMK, Sheridan K,
Walls J. Impaired system A amino acid transport mimics the catabolic
effects of acid in L6 cells. Eur J Clin Invest 32: 590 –602, 2002.
4. Biolo G, Maggi SP, Williams BD, Tipton KD, Wolfe RR. Increased
rates of muscle protein turnover and amino acid transport after resistance
exercise in humans. Am J Physiol Endocrinol Metab 268: E514 –E520,
1995.
5. Burd NA, West DW, Moore DR, Atherton PJ, Staples AW, Prior T,
Tang JE, Rennie MJ, Baker SK, Phillips SM. Enhanced amino acid
sensitivity of myofibrillar protein synthesis persists for up to 24 h after
resistance exercise in young men. J Nutr 141: 568 –573, 2011.
6. Christie GR, Hajduch E, Hundal HS, Proud CG, Taylor PM. Intra-
cellular sensing of amino acids in Xenopus laevis oocytes stimulates p70
S6 kinase in a target of rapamycin-dependent manner. J Biol Chem 277:
9952–9957, 2002.
7. Cruz-Jentoft AJ, Baeyens JP, Bauer JM, Boirie Y, Cederholm T,
Landi F, Martin FC, Michel JP, Rolland Y, Schneider SM, Topinkova
E, Vandewoude M, Zamboni M. Sarcopenia: European consensus on
definition and diagnosis: report of the European Working Group on
Sarcopenia in Older People. Age Ageing 39: 412–423, 2010.
8. Dreyer HC, Drummond MJ, Pennings B, Fujita S, Glynn EL, Chinkes
DL, Dhanani S, Volpi E, Rasmussen BB. Leucine-enriched essential
amino acid and carbohydrate ingestion following resistance exercise
enhances mTOR signaling and protein synthesis in human muscle. Am J
Physiol Endocrinol Metab 294: E392–E400, 2008.
9. Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E, Rasmussen
BB. Resistance exercise increases AMPK activity and reduces 4E-BP1
phosphorylation and protein synthesis in human skeletal muscle. J Physiol
576: 613–624, 2006.
10. Drummond MJ, Dreyer HC, Fry CS, Glynn EL, Rasmussen BB.
Nutritional and contractile regulation of human skeletal muscle protein
synthesis and mTORC1 signaling. J Appl Physiol 106: 1374 –1384, 2009.
11. Drummond MJ, Dreyer HC, Pennings B, Fry CS, Dhanani S, Dillon
EL, Sheffield-Moore M, Volpi E, Rasmussen BB. Skeletal muscle
protein anabolic response to resistance exercise and essential amino acids
is delayed with aging. J Appl Physiol 104: 1452–1461, 2008.
12. Drummond MJ, Glynn EL, Fry CS, Timmerman KL, Volpi E, Ras-
mussen BB. An increase in essential amino acid availability upregulates
amino acid transporter expression in human skeletal muscle. Am J Physiol
Endocrinol Metab 298: E1011–E1018, 2010.
13. Evans K, Nasim Z, Brown J, Butler H, Kauser S, Varoqui H, Erickson
JD, Herbert TP, Bevington A. Acidosis-sensing glutamine pump SNAT2
determines amino acid levels and mammalian target of rapamycin signal-
ing to protein synthesis in L6 muscle cells. J Am Soc Nephrol 18:
1426 –1436, 2007.
14. Fried LP, Hadley EC, Walston JD, Newman AB, Guralnik JM,
Studenski S, Harris TB, Ershler WB, Ferrucci L. From bedside to
bench: research agenda for frailty. Sci Aging Knowledge Environ 2005:
pe24, 2005.
15. Fry CS, Drummond MJ, Glynn EL, Dickinson JM, Gundermann DM,
Timmerman KL, Walker DK, Dhanani S, Volpi E, Rasmussen BB.
Aging impairs contraction-induced human skeletal muscle mTORC1 sig-
naling and protein synthesis. Skeletal Muscle 1: ?–?, 2011.
16. Gjymishka A, Palii SS, Shan J, Kilberg MS. Despite increased ATF4
binding at the C/EBP-ATF composite site following activation of the
unfolded protein response, system A transporter 2 (SNAT2) transcription
activity is repressed in HepG2 cells. J Biol Chem 283: 27736 –27747,
2008.
17. Goberdhan DC. Intracellular amino acid sensing and mTORC1-regulated
growth: new ways to block an old target? Curr Opin Investig Drugs 11:
1360 –1367, 2010.
18. Goberdhan DC, Meredith D, Boyd CA, Wilson C. PAT-related amino
acid transporters regulate growth via a novel mechanism that does not
require bulk transport of amino acids. Development 132: 2365–2375,
2005.
19. Goberdhan DC, Ogmundsdottir MH, Kazi S, Reynolds B, Visval-
ingam SM, Wilson C, Boyd CA. Amino acid sensing and mTOR
regulation: inside or out? Biochem Soc Trans 37: 248 –252, 2009.
20. Henriksen EJ, Louters LL, Stump CS, Tipton CM. Effects of prior
exercise on the action of insulin-like growth factor I in skeletal muscle. Am
J Physiol Endocrinol Metab 263: E340 –E344, 1992.
21. Heublein S, Kazi S, Ogmundsdottir MH, Attwood EV, Kala S, Boyd
CA, Wilson C, Goberdhan DC. Proton-assisted amino-acid transporters
141AMINO ACID TRANSPORTER EXPRESSION AND RESISTANCE EXERCISE
J Appl Physiol •VOL 111 •JULY 2011 •www.jap.org
are conserved regulators of proliferation and amino-acid-dependent
mTORC1 activation. Oncogene 29: 4068 –4079, 2010.
22. Hinnebusch AG. Translational regulation of GCN4 and the general amino
acid control of yeast. Annu Rev Microbiol 59: 407–450, 2005.
23. Huang Y, Kang BN, Tian J, Liu Y, Luo HR, Hester L, Snyder SH. The
cationic amino acid transporters CAT1 and CAT3 mediate NMDA recep-
tor activation-dependent changes in elaboration of neuronal processes via
the mammalian target of rapamycin mTOR pathway. J Neurosci 27:
449 –458, 2007.
24. Hundal HS, Taylor PM. Amino acid transceptors: gate keepers of
nutrient exchange and regulators of nutrient signaling. Am J Physiol
Endocrinol Metab 296: E603–E613, 2009.
25. Hyatt SL, Aulak KS, Malandro M, Kilberg MS, Hatzoglou M. Adap-
tive regulation of the cationic amino acid transporter-1 (Cat-1) in Fao
cells. J Biol Chem 272: 19951–19957, 1997.
26. Hyde R, Christie GR, Litherland GJ, Hajduch E, Taylor PM, Hundal
HS. Subcellular localization and adaptive up-regulation of the System A
(SAT2) amino acid transporter in skeletal-muscle cells and adipocytes.
Biochem J 355: 563–568, 2001.
27. Hyde R, Hajduch E, Powell DJ, Taylor PM, Hundal HS. Ceramide
down-regulates system A amino acid transport and protein synthesis in rat
skeletal muscle cells. FASEB J 19: 461–463, 2005.
28. Hyde R, Taylor PM, Hundal HS. Amino acid transporters: roles in
amino acid sensing and signalling in animal cells. Biochem J 373: 1–18,
2003.
29. Jones HN, Jansson T, Powell TL. Full-length adiponectin attenuates
insulin signaling and inhibits insulin-stimulated amino acid transport in
human primary trophoblast cells. Diabetes 59: 1161–1170, 2010.
30. Jones HN, Jansson T, Powell TL. IL-6 stimulates system A amino acid
transporter activity in trophoblast cells through STAT3 and increased
expression of SNAT2. Am J Physiol Cell Physiol 297: C1228 –C1235,
2009.
31. Kilberg MS, Shan J, Su N. ATF4-dependent transcription mediates
signaling of amino acid limitation. Trends Endocrinol Metab 20: 436 –
443, 2009.
32. King PA. Effects of insulin and exercise on amino acid transport in rat
skeletal muscle. Am J Physiol Cell Physiol 266: C524 –C530, 1994.
33. Kubica N, Jefferson LS, Kimball SR. Eukaryotic initiation factor 2B and
its role in alterations in mRNA translation that occur under a number of
pathophysiological and physiological conditions. Prog Nucleic Acid Res
Mol Biol 81: 271–296, 2006.
34. Kumar V, Selby A, Rankin D, Patel R, Atherton P, Hildebrandt W,
Williams J, Smith K, Seynnes O, Hiscock N, Rennie MJ. Age-related
differences in the dose-response relationship of muscle protein synthesis to
resistance exercise in young and old men. J Physiol 587: 211–217, 2009.
35. Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2[-delta delta C(T)] method.
Methods 25: 402–8, 2001.
36. Lopez AB, Wang C, Huang CC, Yaman I, Li Y, Chakravarty K,
Johnson PF, Chiang CM, Snider MD, Wek RC, Hatzoglou M. A
feedback transcriptional mechanism controls the level of the arginine/
lysine transporter cat-1 during amino acid starvation. Biochem J 402:
163–173, 2007.
37. Malmberg SE, Adams CM. Insulin signaling and the general amino acid
control response. Two distinct pathways to amino acid synthesis and
uptake. J Biol Chem 283: 19229 –19234, 2008.
38. Mayhew DL, Kim JS, Cross JM, Ferrando AA, Bamman MM.
Translational signaling responses preceding resistance training-mediated
myofiber hypertrophy in young and old humans. J Appl Physiol 107:
1655–1662, 2009.
39. McDowell HE, Christie GR, Stenhouse G, Hundal HS. Leucine acti-
vates system A amino acid transport in L6 rat skeletal muscle cells. Am J
Physiol Cell Physiol 269: C1287–C1294, 1995.
40. Narici MV, Maffulli N. Sarcopenia: characteristics, mechanisms and
functional significance. Br Med Bull 95: 139 –159, 2010.
41. Palii SS, Kays CE, Deval C, Bruhat A, Fafournoux P, Kilberg MS.
Specificity of amino acid regulated gene expression: analysis of genes
subjected to either complete or single amino acid deprivation. Amino Acids
37: 79 –88, 2009.
42. Palii SS, Thiaville MM, Pan YX, Zhong C, Kilberg MS. Characteriza-
tion of the amino acid response element within the human sodium-coupled
neutral amino acid transporter 2 (SNAT2) System A transporter gene.
Biochem J 395: 517–527, 2006.
43. Peng T, Golub TR, Sabatini DM. The immunosuppressant rapamycin
mimics a starvation-like signal distinct from amino acid and glucose
deprivation. Mol Cell Biol 22: 5575–5584, 2002.
44. Roos S, Kanai Y, Prasad PD, Powell TL, Jansson T. Regulation of
placental amino acid transporter activity by mammalian target of rapamy-
cin. Am J Physiol Cell Physiol 296: C142–C150, 2009.
45. Roos S, Lagerlof O, Wennergren M, Powell TL, Jansson T. Regulation
of amino acid transporters by glucose and growth factors in cultured
primary human trophoblast cells is mediated by mTOR signaling. Am J
Physiol Cell Physiol 297: C723–C731, 2009.
46. Thalacker-Mercer AE, Dell’Italia LJ, Cui X, Cross JM, Bamman
MM. Differential genomic responses in old vs. young humans despite
similar levels of modest muscle damage after resistance loading. Physiol
Genomics 40: 141–149, 2010.
47. Trenerry MK, Carey KA, Ward AC, Farnfield MM, Cameron-Smith
D. Exercise-induced activation of STAT3 signaling is increased with age.
Rejuvenation Res 11: 717–724, 2008.
48. Verrey F, Closs EI, Wagner CA, Palacin M, Endou H, Kanai Y. CATs
and HATs: the SLC7 family of amino acid transporters. Pflügers Arch
447: 532–542, 2004.
49. Volpi E, Chinkes DL, Rasmussen BB. Sequential muscle biopsies during
a 6-h tracer infusion do not affect human mixed muscle protein synthesis
and muscle phenylalanine kinetics. Am J Physiol Endocrinol Metab 295:
E959 –E963, 2008.
50. Wolfe RR. Regulation of muscle protein by amino acids. J Nutr 132:
3219S–3224S, 2002.
51. Wolfe RR, Chinkes DL. Isotope Tracers in Metabolic Research Princi-
ples and Practice of Kinetic Analysis. Hobokon, NJ: Wiley-Liss, 2005.
52. Zorzano A, Balon TW, Garetto LP, Goodman MN, Ruderman NB.
Muscle alpha-aminoisobutyric acid transport after exercise: enhanced
stimulation by insulin. Am J Physiol Endocrinol Metab 248: E546–E552,
1985.
142 AMINO ACID TRANSPORTER EXPRESSION AND RESISTANCE EXERCISE
J Appl Physiol •VOL 111 •JULY 2011 •www.jap.org
