Tissue-specific effects of chronic dietary leucine and
norleucine supplementation on protein synthesis in rats
Christopher J.
Lynch1,
Susan
M.
Hutson2,
Brian J.
Patson1,
Alain
Vaval1, and
Thomas C.
Vary1
1 Department of Cellular and Molecular Physiology,
Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033; and 2 Department of
Biochemistry, Wake Forest University Medical School, Winston-Salem,
North Carolina 27157
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ABSTRACT |
Acute
administration of leucine and norleucine activates the mammalian
target of rapamycin (mTOR) cell-signaling pathway and increases rates
of protein synthesis in a number of tissues in fasted rats. Although
persistent stimulation of mTOR signaling is thought to increase protein
synthetic capacity, little information is available concerning the
effects of chronic administration of these agonists on protein
synthesis, mTOR signal transduction, or leucine metabolism. Hence, we
developed a model of chronic leucine/norleucine supplementation via
drinking water and examined the effects of chronic (12 days)
supplementation on protein synthesis in adipose tissue, kidney, heart,
liver, and skeletal muscle from ad libitum-fed rats. The relative
concentration of proteins involved in mTOR signaling and the two
initial steps in leucine oxidation were also examined. Leucine or
norleucine supplementation was accompanied by increased rates of
protein synthesis in adipose tissue, liver, and skeletal muscle, but
not in heart or kidney. Supplementation was not associated with
increases in the anabolic hormones insulin or insulin-like growth
factor I. Chronic supplementation did not cause apparent adaptation in
either components of the mTOR cell-signaling pathway that respond to
leucine (mTOR, ribosomal protein S6 kinase, and eukaryotic initiation
factor 4E-binding protein-1) or the first two steps in leucine
metabolism (the mitochondrial isoform of branched-chain amino acid
transaminase, branched-chain keto acid dehydrogenase, and
branched-chain keto acid dehydrogenase kinase), which may be involved
in terminating the signal from leucine. These results suggest that
provision of leucine or norleucine supplementation via the drinking
water results in stimulation of postprandial protein synthesis in
adipose tissue, skeletal muscle, and liver without notable adaptive
changes in signaling proteins or metabolic enzymes.
mammalian target of rapamycin; adipose tissue; eukaryotic
initiation factor 4E-binding protein-1; branched-chain keto acid
dehydrogenase kinase
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INTRODUCTION |
POSTPRANDIAL INCREASES in the plasma
concentration of amino acids after a mainly protein-containing meal may
provide a signal for accelerating protein synthesis (9, 10, 25,
37, 38, 51). The mammalian target of rapamycin (mTOR)-signaling
pathway has been proposed as one potential target for mediating these effects. In adipocytes, the efficacy of amino acids in activating mTOR
signaling appears to be related to their structural similarity to
leucine. Thus leucine and norleucine are posited to be agonists at a
common leucine recognition site in adipocytes, LeuRa
(48, 49). Short-term administration of leucine stimulates
protein synthesis by enhancing mRNA translation initiation through an increase in the number of polysomes and an increased rate of formation of the 40S initiation complex (24). These actions improve
the efficiency of the mRNA translation initiation cycle.
Insulin and branched-chain amino acids (BCAA) influence protein
synthesis by activating the serine/threonine kinase mTOR, which then
stimulates downstream targets such as the translation repressor,
eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1, or
PHAS-I) and the 70-kDa ribosomal protein kinase, S6K1 [for review see
Gingras et al. (30)]. Leucine stimulates the hyperphosphorylation of 4E-BP1 (27, 57, 71), resulting in its release from eIF4E, thus allowing the initiation cycle to proceed
more efficiently. Multisite phosphorylation of S6K1 is associated with
acute changes in synthesis of a subset of proteins that may lead to
subsequent changes in global protein synthesis. Notably,
phosphorylation of S6 is associated with increased translation of
messenger RNA species with terminal oligopyrimidine (TOP) tracts at the
5'-cap. Because many TOP-containing mRNAs encoded for proteins are
components of the protein synthetic machinery, it is expected that
persistent activation of mTOR would lead to increases in protein
synthetic capacity (55); however, this hypothesis has not
been rigorously tested in vivo. In fact, although leucine regulates
protein synthesis acutely, it is not known whether or not chronic oral
supplementation of leucine stimulates rates of protein synthesis.
The need to better understand leucine metabolism arises from studies
that suggest a leucine metabolite or leucine metabolism, rather than
leucine itself, may be the signal for activation of mTOR (27, 49, 57 and compare 63, 70). The first step in leucine metabolism is
reversible, transamination of leucine to
-ketoisocaproate catalyzed
by the branched-chain aminotransferase isoenzymes [mitochondrial (BCATm) and cytosolic (BCATc)]. BCATm is expressed ubiquitously (3, 15, 32, 42-44), whereas BCATc is found primarily
in neural tissue (40). The next step is irreversible
oxidative decarboxylation of the branched-chain
-keto acids to
produce the corresponding branched-chain acyl-CoA derivatives catalyzed by the mitochondrial branched-chain
-keto acid dehydrogenase (BCKD)
enzyme complex. The mammalian BCKD complex contains multiple copies of
three enzymes: a branched-chain
-keto acid decarboxylase (E1)
composed of 2
and 2
subunits, a dihydrolipoyl transacylase (E2),
and a dihydrolipoyl dehydrogenase (E3) (35). The activity of the complex within a tissue is regulated by
phosphorylation-dephosphorylation catalyzed by a specific kinase and
phosphatase. The phosphorylation state of the complex is controlled
primarily by the activity of the BCKD kinase; phosphorylation of S293
on the E1-
subunit results in inactivation (22, 35,
58). Depending on the tissue, activity state is influenced by
hormones, diabetes, exercise, starvation, acidosis, or low dietary
protein feeding (for review see Ref. 54). The kinase can
be inhibited directly in vitro by the keto acid of leucine, which in
turn results in activation of the BCKD complex. This may explain the
activation of BCKD in skeletal muscle after leucine injection
(28, 35, 36). Although it is apparent that the enzymes
involved in the initial steps in leucine metabolism are present in
adipose tissue, their relative level of expression compared with other
tissues is not known.
It is important to understand the effects of chronic elevations in
leucine, because concentrations of BCAAs are chronically elevated in
human and animal forms of obesity and adipose tissue appears to be
highly responsive to leucine (18, 23). For example, it is
not known whether chronic exposure to excess leucine or leucine
mimetics (norleucine) results in changes in protein synthetic capacity.
Alternatively, the levels and activity of enzymes involved in either
the cell-signaling response to leucine or the metabolism of leucine
might adapt to a chronic increase in plasma leucine concentrations.
Therefore, in this study, we have examined the effects of chronic,
continuous elevations in plasma leucine by use of a new model of
chronic leucine or norleucine supplementation. Norleucine was used
because it is a structural analog of leucine that we have shown can
stimulate mTOR signaling and protein synthesis in vitro and in vivo
(48-50). In contrast to leucine, acutely administered norleucine does not stimulate insulin secretion and is not incorporated into protein. A continuous supply of these amino acids was provided in
the drinking water. Using this model, we have determined the effect of
chronic leucine or norleucine supplementation on postprandial protein
synthesis in adipose tissue as well as in muscle, heart, liver, and
kidney. Plasma hormone concentrations and tissue RNA levels were
examined as potential mediators of the effects on protein synthesis.
The tissue-dependent expression of proteins involved in the
mTOR-signaling pathway and leucine catabolism in adipose tissue were
compared with expression of these proteins in the other tissues. The
results show that chronic supplementation of leucine or norleucine
stimulates postprandial protein synthesis in responsive tissues without
affecting levels of signaling proteins or BCAA catabolic enzymes. The
protein synthesis responses displayed a higher degree of tissue
specificity compared with the acute effects of leucine on protein
synthesis [e.g., in the preceding study (50)]. The
likelihood that this may reflect differences in the mechanisms
mediating the acute effects of leucine administered to a fasting animal
and the chronic effects of leucine in ad libitum-fed animals reported
in the present communication is discussed.
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EXPERIMENTAL PROCEDURES |
Animals and treatment protocol.
The Institutional Animal Care and Use Committee approved the animal
protocol. Male Sprague-Dawley rats were purchased from Charles River
and maintained at our facility for
7 days before the start of the
treatment protocol. The light cycle began at 7 AM and the dark cycle
began at 7 PM, with rats fed ad libitum with measurements made 2-4
h after the beginning of the light cycle. Two identical studies with
rats were conducted in which animals were allocated to one of the
following three groups: control (6 rats), leucine supplemented (8 rats), and norleucine supplemented (6 rats). Animals were caged in
pairs to reduce anxiety-induced changes in food intake. Two experiments
were conducted that initially had a planned experimental design of six
animals per group. Two extra rats were placed in the leucine group. The
mean starting body weights of animals in each group were not
statistically different (control: 96.2 ± 2.6 g,
n = 12; leucine supplemented: 97.8 ± 1.8, n = 16; norleucine supplemented: 96.2 ± 2.4, n = 12).
Each cage had two ceramic food containers in separate corners of the
cage to facilitate ad libitum feeding. Food remaining in dishes and
crumbs that fell through the cage mesh were weighed daily starting
1-2 days before day 0. Food consumption was calculated as food consumed per cage and divided by two. Water or leucine analog-containing water (114 mM leucine or norleucine) was provided starting on day 0. The amount of fluid consumed was measured
daily. A drinking bottle was also always hung on an empty cage per cage rack. This allowed an estimate of the amount of water lost each day
from dripping due to placing of the drinking bottle or handling of the
wheeled cage rack (generally this was 1-2 ml). This amount lost
was subtracted from the water consumption measurements. Net water
consumption was measured per rat cage and divided by two.
Protein synthesis.
Protein synthesis measurements were made on the morning of the 12th day
of dietary supplementation. Food and water/supplement were provided
until the time of anesthetization. Thus, in contrast to the previous
study on fasted rats (50), these measurements were made in
ad libitum-fed rats. The animals were judged to be in the postprandial
phase on the basis of the presence of food in their stomachs and
elevated insulin concentrations. Rates of protein synthesis in vivo
were estimated using the flooding-dose method to measure the
incorporation of radioactive phenylalanine into protein. This method
has been described previously and characterized in our laboratory
(65-67). Briefly, an incision was made in the neck of
anesthetized animals (Nembutal, 50 mg/kg body wt) for the placement of
PE-50 catheters in the carotid artery. A bolus of
L-[3H]phenylalanine (0.2 mCi · ml
1 · µmol
1, 30 µCi/100 g body wt, 1 ml/100 g body wt) was infused as a bolus intravenously. Ten minutes after injection of the radioisotope, an
arterial blood sample (3 ml) was taken for measurement of phenylalanine concentrations and radioactivity. The concentration of phenylalanine and other amino acids was determined by HPLC analysis of supernatants from trichloroacetic acid extracts of plasma (19). In
addition, the radioactivity in the phenylalanine peak was measured to
determine the specific activity of
L-[3H]phenylalanine in the blood.
Gastrocnemius muscle, heart, kidney, liver, and epididymal adipose
tissue were excised and frozen between clamps precooled in liquid
nitrogen, weighed, and stored at
84°C. The frozen tissue was
powdered under liquid nitrogen and then stored at
84°C for later
measurements as described in the following section.
Measurements of incorporation of radioactivity in proteins.
Approximately 0.3-0.5 g of frozen powdered tissue was homogenized
in 2 ml of ice-cold 3.6% (wt/vol) perchloric acid (HClO4) and centrifuged. The supernatant was decanted, and the pellet was
washed a minimum of five times with 3.6% (wt/vol) HClO4 to remove any acid-soluble radioactivity. The pellet was washed with acetone, followed by a mixture of chloroform-methanol (1:1, vol/vol) and then water. The pellet was then dissolved in 0.1 M NaOH, and aliquots were assayed for protein by the biuret method with crystalline bovine serum albumin as a standard. Another aliquot was assayed for
radioactivity by liquid scintillation spectrometry using the proper
corrections for quenching (dpm). Rates of protein synthesis were
calculated by dividing the amount of radioactivity incorporated into
protein by the specific radioactivity of phenylalanine in the plasma.
The assumption in the use of this technique to estimate the rate of
protein synthesis in vivo is that the tissue phenylalanine concentration is elevated to high concentration, thereby limiting any
dilution effect of nonradioactive phenylalanine derived from proteolysis on the intracellular specific radioactivity. Under the
condition of elevated plasma phenylalanine concentrations (~1.3 ± 0.9 mM), the specific radioactivity of the plasma phenylalanine is
assumed to be equal to the specific radioactivity of the tRNA-bound phenylalanine. Studies by McKee et al. (52) and Williams
et al. (69) have shown that, at a perfusate phenylalanine
concentration of 0.4 mM, the perfusate and intracellular and tRNA-bound
phenylalanine have the same specific radioactivity within 10 min of the
start of perfusion with radioisotopes.
BCKD complex activity.
Extraction of the BCKD complex from tissues (50-100 mg tissue) was
performed essentially as described by Block et al.
(5) by use of the modification in Ref.
15. BCKD activity was measured by release of
14CO2 from
-keto-[1-14C]isocaproate. Total BCKD complex activity,
which is an estimate of enzyme amount, was measured after activation of
a separate aliquot of the same sample in the presence of
MnCl2 and lambda protein phosphatase (4). The
activity state of BCKD is the ratio of actual activity before
activation to total activity obtained after activation by phosphatase
treatment. A unit of activity was defined as 1 nmol
14CO2 formed/min at 37°C.
Hormone assays.
Insulin and leptin concentrations were measured by RIA with a kit from
Linco Research (St. Charles, MO). Liver and serum concentrations of
insulin-like growth factor (IGF) were assayed according to Fan et al.
(21).
Western blot analysis.
For Western blotting of cytosolic proteins, the frozen, powdered tissue
was homogenized in 7 vol of homogenization buffer (in mM: 20 HEPES, pH
7.4, 2 EGTA, 50 NaF, 100 KCl, 0.2 EDTA, 50
-glycerophosphate, 1 DTT,
0.1 PMSF, 1 benzamidine, 0.5 sodium vanadate, and 1 µM microcystin
LR) with a Polytron homogenizer. For mitochondrial proteins and mTOR,
0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was included in the homogenization buffer to release
mitochondrial proteins. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the pellet was discarded. An
aliquot of the supernatant was reserved for protein assay, and the rest
was added to an equal volume of 2× Laemmli sodium dodecyl sulfate
(SDS) sample buffer. The mixtures were boiled for 3 min and centrifuged
at 16,000 g for 4 min.
To detect mTOR, proteins were separated on a 5% Bio-Rad (Hercules, CA)
Criterion Tris-glycine gel and transferred to PVDF for 3 h at 50 V
in transfer buffer (10 mM CAPS, pH 11.0, 10% methanol, and 0.1%
SDS) by use of a platinum electrode Bio-Rad Criterion blotter.
Positive controls for mTOR in Western blots were rat brain lysates (100 µg/lane) and a recombinant FLAG-tagged version of human TOR [aka
FK506 rapamycin-associated protein (FRAP)] expressed in Sf-9 insect
cells. The FRAP/FLAG 1392 baculovirus transfer vector described
previously (7, 8) was obtained as a generous gift from Dr.
Stuart L. Schreiber (Boston, MA). Immunoblotting was performed using an
antibody (MTAB5), produced in our laboratory, directed against a
keyhole limpet hemocyanin-linked peptide, (C)-QREPKEMQKPQWYRHT FEE,
representing an NH2-terminal sequence (residues
221-40) of RAFT, a rat form of mTOR. PVDF membranes were incubated
in a 1:1,000 dilution for 1 h at room temperature and then
overnight at 4°C. Bands were detected with an enhanced
chemiluminescence ECL Western Blotting Kit from Amersham Pharmacia
(Piscataway, NJ).
To examine 4E-BP1 concentration and phosphorylation, cytosolic proteins
(100 µg) were separated on 15% acrylamide gels containing a reduced
bisacrylamide concentration that allows the electrophoretic resolution
of 4E-BP1 into three bands: least phosphorylated and fastest migrating,
; intermediate,
; and slowest migrating and most extensively
phosphorylated,
(45, 49). Formation of the most highly
phosphorylated form, which migrates as the
-band, correlates with
decreased binding to eIF4E. For detection of total S6K1 and
phosphorylation of S6K1 on T389, cytosolic proteins were separated on a
7.5% Bio-Rad Criterion Tris-glycine gel. After transfer to PVDF, the
blots were probed using an antibody to S6K1 (Cell Signaling Technology,
Beverly, MA).
BCAT isoenzyme-specific antiserum was raised in rabbits as described in
Wallin et al. (68). Purified recombinant human BCATm (13) was used as antigen. For preparation of the
affinity-purified BCATm antibodies, human BCATm-Sepharose was prepared
by coupling the purified human recombinant BCAT isoenzyme to AfFigel 10 support (Bio-Rad, Richmond, CA) according to the manufacturer's
directions. The BCKD antiserum generated against E1 of the purified rat
liver BCKD complex was a gift from Dr. Yoshi Shimomura (Nagoya, Japan). This antiserum recognizes the E1
, E1
, and E2 BCKD subunits.
BCKD kinase-specific antiserum was raised in rabbits with the use of
purified recombinant human BCKD kinase as antigen. Affinity-purified antibodies were obtained by chromatography on a recombinant BCKD kinase-AH-Sepharose 4B column resin and prepared as recommended by the
supplier (Amersham Pharmacia Biotech). Serum was saturated with 50%
ammonium sulfate and the precipitate harvested by centrifugation. The
precipitate was dissolved in PBS and applied to the column. After
extensive washing of the column with PBS, the anti-BCKD kinase
antibodies were eluted with 4 M urea and 0.5 M NaCl in 0.1 M sodium
acetate buffer, pH 4.0. The affinity-purified antibodies were dialyzed
against 50% glycerol-water and stored in aliquots at
85°C. These
procedures were carried out at 4°C.
The BCKD E1-
, the BCKD kinase, and the BCATm proteins in the tissue
supernatant were separated on 10% Bio-Rad Criterion Tris-glycine gels
and then electrophoretically transferred to PVDF membrane at 100 V for
45 min in transfer buffer (10 mM CAPS, pH 11.0, and 10% methanol). The
resulting PVDF membranes were blocked with 5% (wt/vol) skim milk in
Tris-buffered saline-Tween-20 and incubated with their respective
antibodies as follows: rabbit anti-BCKD E1-
(1:1,000 dilution),
rabbit anti-BCKD kinase serum (1:1,000 dilution), or rabbit anti-BCATm
(1:1,000 dilution) for 1 h at room temperature. Specific bands
were detected using an ECL Western Blotting Kit from Amersham
Pharmacia. NIH Image 1.61 was used to perform densitometry of
ECL-exposed X-ray films.
Statistical analysis.
The feeding study was performed twice with similar results. At least
six animals were examined per condition for each of the Western
blotting studies and other studies. ANOVA statistical analysis was
performed using the INSTAT program and a Student-Newman-Keuls post test
where appropriate.
 |
RESULTS |
The early work of Harper and colleagues (33, 34)
demonstrated adverse effects on growing animals of a low-protein diet containing an inordinately large amount of a single amino acid. These
adverse effects included decreased food intake when norleucine or
leucine was added directly to the chow. However, it has also been shown
that, when fed a normal diet, rats prefer norleucine (62)
or leucine (59) solutions over water. Therefore, in this study, young growing rats were fed a commercial diet containing 24.5%
protein, and leucine or norleucine supplements (114 mM) were supplied
in the drinking water.
Figure 1 shows the total daily sum of the
leucine and norleucine consumed from both food and liquid over the
course of the experiment. Only the norleucine-supplemented animals
received significant amounts of norleucine. The leucine and norleucine in the rat chow were 2.04 and 0%, respectively. Before
supplementation, the amount of the leucine consumed was similar in each
group (~0.28-0.30 g · 100 g body
wt
1 · day
1). Leucine intake per
100 g body wt stayed about the same for control rats over the
course of the experiment. In contrast, the total amount of leucine plus
norleucine consumed from water and food in the two supplemented groups
doubled within a day of adding leucine or norleucine to the drinking
water. This difference in the leucine plus norleucine intake between
the two experimental groups and the control group was maintained over
the entire protocol period (Fig. 1).

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Fig. 1.
Sum of leucine and norleucine consumed in food and
liquid. The daily intake (g) of leucine and norleucine consumed due to
chow intake or liquid intake was determined starting 1 day before
supplementation began (1st point on graph) and thereafter. Body weights
were also measured daily. The figure shows the sum of the leucine and
norleucine consumed adjusted to 100 g body wt. Note that only 1 group actually received both leucine and norleucine, the
norleucine-supplemented group. The norleucine group received leucine
from rat chow and, starting on day 0 (after the 1st point on
the graph), 114 mM norleucine from their water. Control rats received
rat chow ad libitum and water throughout (i.e., leucine from rat chow
only). Leucine-supplemented animals received leucine from their rat
chow and, starting on day 0, from their water supplemented
with 114 mM leucine.
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Effects of dietary supplementation on body weight, food intake, and
water consumption.
All groups gained weight at similar rates over the course of the
experiment (Fig. 2, top). A
trend of increased body weight in the leucine- and
norleucine-supplemented groups was noted over the last few days of the
study; however, this was not statistically significant. Figure 2
(middle) shows that, overall, neither leucine nor norleucine
supplementation had adverse effects on food intake; indeed, food intake
was similar in the three groups over the protocol period. The fact that
food intake was equivalent among the groups allows us to evaluate the
independent effects of leucine and norleucine supplementation, even
though the rats were in the postprandial phase when the measurements
were made. In agreement with previous palatability studies (59,
62), adding leucine or norleucine to the water did not
significantly diminish fluid intake (Fig. 2, bottom).
Although there were statistically significant differences in the amount
of fluid consumed by rats provided one of the amino acid solutions on
some individual days, these differences were not consistent (Fig. 2,
bottom).

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Fig. 2.
Effect of leucine and norleucine supplementation on body
weight (top), dry food intake (middle), and fluid
intake (bottom). Daily body weights were determined, and the
amount of food and fluid consumed was measured in rats receiving
distilled water (Control, n = 6) or distilled water
containing 114 mM leucine (n = 8) or norleucine
(n = 6) as indicated. Results are means ± SE from
a single experiment representative of 2 such studies.
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Plasma hormone and amino acid concentrations.
To determine whether leucine or norleucine supplementation affected
hormones associated with energy metabolism or growth, circulating serum
concentrations of insulin, IGF-I, and leptin were measured at the time
of death (postprandial state), and the results are summarized in Table
1. Chronic amino acid supplementation had
no significant effect on serum hormone concentrations, nor were hepatic
tissue levels of IGF-I affected significantly by leucine or norleucine
supplementation (control, 203 ± 14; leucine, 216 + 7.7;
norleucine, 186 ± 9 ng/mg wet wt liver tissue).
Serum amino acid concentrations in the control, leucine-supplemented,
and norleucine-supplemented rats were determined, and the results are
shown in Table 2. Differences in the
effects of leucine and norleucine supplementation were observed. Serum leucine (47%) and Tyr (72%) concentrations were significantly higher
in the leucine-supplemented group compared with the control group. The
rises in leucine are equivalent to increases observed in obesity
(18, 23). Although the results did not reach statistical significance, plasma Ala, Asp, Gln, Ile, Pro, and Val were elevated compared with control animals in both the leucine- and
norleucine-supplemented groups. The sum of the BCAA concentrations and
the total amino acid concentrations were both significantly higher in
the leucine-supplemented group compared with control animals, whereas
differences did not reach statistical significance in the norleucine
group.
Protein synthesis.
Next, we measured rates of protein synthesis in selected rat tissues
after chronic dietary supplementation. In contrast to the previous
study (50), which utilized fasted rats, these animals were
in the postprandial phase, as judged by the food present in their small
bowel and food in their stomachs. Consequently, the measured rates of
protein synthesis in the control group were higher than those observed
in fasted rats (Fig. 3; compare with Ref.
50). In the norleucine- and leucine-supplemented groups, rates of protein synthesis were significantly higher than these already
elevated (i.e., due to feeding) control rates in adipose tissue,
gastrocnemius muscle, and liver (Fig. 3). In terms of percent increase,
the effect of leucine or norleucine supplementation on protein
synthesis was most dramatic on adipose tissue (277 and 377%,
respectively). Although measured rates of protein synthesis also
appeared higher in heart and kidney than those in the control group,
the data are not statistically different (Fig. 3). Supplementation did
not affect tissue total RNA concentrations (data not shown); therefore,
the mechanism of the increase in protein synthesis seems to involve an
increase in translational efficiency.

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Fig. 3.
Effect of leucine and norleucine supplementation on
protein synthesis. The rates of protein synthesis in heart
(A), epididymal adipose tissue (Adipose; B),
white gastrocnemius muscle (Gastroc.; B), kidney
(C), and liver (C) were measured in rats, as
described in EXPERIMENTAL PROCEDURES. White gastrocnemius
is a mixed fiber containing the ratio of slow oxidative, fast oxidative
glycolytic. and fast glycolytic fibers 0:10:90. The rats had received
either distilled water (Control) or distilled water containing 100 mM
leucine or norleucine for 10 days as indicated. Means ± SE are
shown. *Effects of leucine supplementation were significantly different
from control (P < 0.05); **the same for
norleucine-supplemented rats.
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Tissue distribution and effects of leucine and norleucine
supplementation on the mTOR-signaling pathway.
Little information is available on the relative tissue distribution of
components of the mTOR cell-signaling pathway or the effect of chronic
leucine supplementation on these. Therefore, tissue concentrations of
mTOR, 4E-BP1, and S6K1 as well as the level of 4E-BP1 and S6K1
phosphorylation were determined. For mTOR, we used both a commercial
anti-FRAP antibody from Stress Gen Biotechnologies (not shown) and a
newly developed MTAB5 antibody from our laboratory (Fig.
4). Both detected the same ~240-kDa band in tissue lysates from baculovirus-infected Sf-9 cells expressing a recombinant human mTOR with an amino-terminal epitope tag MDYKDDDDK (Fig. 4 top, lane R). Furthermore, both
antibodies detected a band with similar electrophoretic mobility in
lysates from rat brain (known to contain high concentrations of mTOR)
as well as the tissues pertinent to this study (Fig. 4, top,
lane B). Because these antibodies were directed against two
entirely different regions in mTOR, it is likely that the
immunoreactive band represents mTOR. In Fig. 4 (bottom), the
relative amount of mTOR per milligram of soluble tissue lysate protein
is presented. Each of the lanes in Fig. 4 (top) was loaded
with either 50 or 100 µg of tissue, and the loading differences were
equalized to prepare the bar graphs shown in Fig. 4 (bottom)
and in subsequent figures. Most tissues, with the exception of adipose
tissue, expressed similar amounts of mTOR per milligram of soluble
tissue protein (Fig 4, bottom). Adipose tissue contained the
most mTOR per milligram of soluble tissue protein compared with other
peripheral tissues examined. Neither leucine nor norleucine
supplementation had any significant effect on the content of mTOR
(Table 3).

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Fig. 4.
Tissue distribution of mammalian target of rapamycin
(mTOR) in control animals. Soluble tissue lysate protein (50 or 100 µg of protein, as indicated above blots) from control animals: kidney
(K), liver (L), heart (H), adipose tissue (A), gastrocnemius (G),
recombinant FLAG-FRAP (R), or brain (B) were solubilized in SDS-PAGE
sample buffer and separated on 5% polyacrylamide Tris-glycine gels.
After transfer to PVDF membrane, the blots were probed with
affinity-purified mTAB5 antibody.
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As shown in Fig. 5, the content of 4E-BP1
in heart, liver, gastrocnemius, and adipose tissue was approximately
the same. However, renal content was approximately five times lower
than the other peripheral tissues examined (Fig. 5). This is in
agreement with the preceding study (50) in younger rats,
in which 4E-BP1 could not be detected in kidney. There were no
significant differences among the total (i.e.,
-,
-, and
-forms combined) amounts of 4E-BP1 in any tissue (Table 3). The
percentages of 4E-BP1 in the
-form are shown in Table
4. The percentage of 4E-BP1 in the
-form in the control tissues was higher than in the preceding study,
again consistent with the animals being in the postprandial rather than
the fasted state. No further increase in 4E-BP1 phosphorylation was
caused by the supplements at the time these measurements were taken
(Table 4). Compared with the other tissues, adipose tissue had the
highest percentage of 4E-BP1 in the
-form. This is consistent with
the observation that adipose tissue also had the highest concentration
of mTOR per milligram of solubilized tissue protein.

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Fig. 5.
Tissue distribution of eukaryotic initiation factor
4E-binding protein-1 (4E-BP1). Soluble control tissue lysate proteins
(100 µg of protein) from kidney (K), liver (L), heart (H), adipose
tissue (A) or gastrocnemius (G) were solubilized in SDS-PAGE sample
buffer and separated on 15% polyacrylamide SDS-PAGE gels. After
transfer to PVDF membrane, the blots were probed with affinity-purified
4E-BP1 antibody.
|
|
Figure 6 shows a representative Western
blot and a graph of the tissue distribution of S6K1, corrected for the
amount of protein loaded. Multiple electrophoretic forms corresponding
to multisite phosphorylation were observed as previously reported
(e.g., Ref. 26). The concentration of S6K1 was ~25%
higher in kidney, liver, and adipose tissue compared with heart and
gastrocnemius. The amounts of total S6K1 (Table 3) or S6K1
phosphorylated on T389 (elevated consistent with postprandial state,
data not shown) in the tissues were the same in the control and
leucine- and norleucine-supplemented groups.

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Fig. 6.
Tissue distribution of ribosomal protein S6 kinase-1
(S6K1). Soluble control tissue lysate proteins (50 or 100 µg of
protein, as indicated) from kidney (K), liver (L), heart (H), adipose
tissue (A) or gastrocnemius (G) were solubilized in SDS-PAGE sample
buffer and separated on 7.5% polyacrylamide SDS-PAGE gels. After
transfer to PVDF membrane, the blots were probed with affinity-purified
S6K1 antibody.
|
|
Effects of chronic leucine and norleucine supplementation on
enzymes involved in leucine metabolism.
Leucine injection and dietary protein content have been shown to affect
BCATm and/or BCKD activity and expression in several tissues (1,
5, 16, 35, 56, 60, 64). Therefore, the effect of chronic
administration of excess leucine or norleucine on the key enzymes
involved in the initial steps of leucine metabolism was examined using
immunoblotting to determine levels of BCATm, BCKD subunit, and BCKD
kinase proteins. BCKD activity was also measured.
Figure 7 shows a representative Western
blot and the tissue distribution of BCATm in control tissues. As
reported previously (43), BCATm is not found in adult rat
liver. The pattern of BCATm enzyme protein levels agrees with the
reported distribution of BCATm activity in heart, kidney, and
gastrocnemius (2, 39, 43). Measurement of BCATm in adipose
tissue (corrected for protein loading) reveals levels of BCATm
equivalent to those observed in kidney. This result is significant,
because kidney is one of the tissues with high BCATm activity
(64). Dietary supplementation with leucine or norleucine
had no statistically significant effect on the tissue BCATm
concentrations (Table 5). Representative blots showing the tissue distribution of BCKD subunits and BCKD kinase
are shown in Figs. 8 and
9. The
levels of BCKD subunits were examined using an antibody to the
E1-
-subunit that also recognizes E1
and E2 subunits. Relative
levels of E1
protein were similar in liver and kidney (Table 5).
Adipose tissue had nearly the same level of E1
protein as that found
in heart muscle. BCKD subunit proteins were lowest in gastrocnemius
(Fig. 8). Interestingly, the blots also revealed that levels of E2 and
E1
relative to E1
exhibit tissue-specific differences.

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Fig. 7.
Tissue distribution of the mitochondrial isoform of
branched-chain aminotransferase (BCATm). Control tissue lysate proteins
(50 or 100 µg of detergent solubilized tissue protein as indicated)
from kidney (K), liver (L), heart (H), adipose tissue (A) or
gastrocnemius (G) were solubilized in SDS-PAGE sample buffer and
separated on 10% polyacrylamide SDS-PAGE gels. After transfer to PVDF
membrane, the blots were probed with affinity-purified BCATm
antibody.
|
|

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Fig. 8.
Tissue distribution of branched-chain keto-acid
dehydrogenase E1- catalytic subunit (BCKD E1- ). Control tissue
lysate proteins (50 or 100 µg of detergent solubilized tissue protein
as indicated) from kidney (K), liver (L), heart (H), adipose tissue (A)
or gastrocnemius (G) were solubilized in SDS-PAGE sample buffer and
separated on 15% polyacrylamide SDS-PAGE gels. After transfer to PVDF
membrane, the blots were probed with BCKD E1- antibody.
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Fig. 9.
Tissue distribution of BCKD kinase (BCKDK). Detergent
solubilized control tissue lysate proteins (50 or 100 µg of protein
as indicated) from kidney (K), liver (L), heart (H), adipose tissue (A)
or gastrocnemius (G) were diluted in SDS-PAGE sample buffer and
separated on 10% polyacrylamide SDS-PAGE gels. After transfer to PVDF
membrane, the blots were probed with affinity-purified BCKDK
antibody.
|
|
The comparison of tissue levels of BCATm and BCKD subunits and BCKD
kinase in the controls and leucine- and norleucine-supplemented groups
is summarized in Table 5. As observed with proteins of the
mTOR-signaling pathway, dietary supplementation with leucine or
norleucine had no statistically significant effect on the tissue BCATm
concentrations (Fig. 7) or levels of E1-
subunit (Fig. 8) or BCKD
kinase (Fig. 9). Consistent with results from Western blotting, no
statistically significant differences in BCAT or BCKD activity and
activity state were found between control and leucine-supplemented or
control and norleucine-supplemented groups (data not shown). Adipose
tissue activity was not measured, because we did not have sufficient
quantities of tissue.
The activity of BCKD kinase is thought to control the activity state of
the BCKD complex (35). As suggested by measurements of
BCKD activity state and kinase mRNA levels in rat and other species
(61), skeletal muscle contained the highest levels of BCKD
kinase protein, whereas liver had the lowest levels of BCKD kinase
protein. Relatively lower concentrations of BCKD kinase were also found
in adipose tissue. In some tissues, Western blotting of whole tissue
lysates for the kinase revealed "doublets" around 43-46 kDa
(Fig. 9). Relatively few Western blots of the kinase are available in
the literature, but in mitochondria extracts, only a single band has
been observed (e.g., Ref. 46), although a doublet was
detected in the recombinant kinase preparation reported by Popov et al.
(58) and transgene studies reported by Doering and Danner
(17). Because protease inhibitors were present in the
extraction buffer, the higher molecular weight band may represent BCKD
kinase that still contains the mitochondrial targeting signal.
 |
DISCUSSION |
In this study, we have developed a model for chronic
supplementation in rats with leucine or norleucine that does not affect food intake or growth. Using this model, we have shown that leucine or
norleucine stimulates protein synthesis in a tissue-selective manner
and that the tissue responses differ from those reported in
food-deprived rats orally administered leucine or norleucine (50). In our chronic model, animals were fed the
experimental diets for 12 days, and protein synthesis was measured in
ad libitum-fed animals in the postprandial phase. With chronic
supplementation, protein synthesis was elevated in adipose tissue,
liver, and skeletal muscle, but not in kidney or heart. In the
preceding, acute study (50), leucine and norleucine had
different effects on protein synthesis: leucine administration
stimulated protein synthesis in adipose tissue, muscle, and kidney,
whereas norleucine was effective in all tissues (50). The
tissue-specific differences in effects of leucine and norleucine
supplementation in these two models suggest that there may be varied
pathways by which amino acids such as leucine affect protein synthesis
in body tissues and/or that the mechanisms involved in leucine's acute
and chronic effects on protein synthesis occur by different mechanisms.
This idea is in agreement with recent studies showing that the acute affects of leucine are mediated by both rapamycin-sensitive and rapamycin-insensitive pathways (12, 53). The
rapamycin-sensitive pathway involves 4E-BP1, S6K1, and mTOR; however,
little is known about the rapamycin-insensitive pathway.
In the present study, insulin concentrations were high in the control
animals and in the supplemented animals, as was the degree of 4E-BP1
and S6K1 phosphorylation. These findings are consistent with the
postprandial state of the animals. These parameters were much lower in
controls from the preceding acute study, in which the animals were food
deprived. Thus, in the chronic study, leucine and norleucine were able
to stimulate protein synthesis above the already high levels of protein
synthesis caused by ad libitum feeding alone. These effects were not
associated with further increases in plasma insulin or IGF
concentrations and therefore probably represent direct effects of the
supplements on the affected target tissues. The apparent lack of effect
of chronic supplementation on mTOR-signaling proteins may be related to
the time at which the measurements were made (i.e., maximally stimulated by the postprandial state). Presumably, differences would be
seen at other times of day, when the animals were drinking but not yet
eating; however, further studies are required to determine the exact
mechanism responsible for the increase in protein synthesis we observed.
Tissue specificity and comparison of adipose tissue to other
tissues.
Persistent activation of mTOR and downstream targets of mTOR have been
linked to an increased protein synthetic capacity (for reviews see
Refs. 20, 55). It is anticipated, therefore,
that the cumulative effects of consuming leucine and norleucine in the
water may be an increase in protein synthetic capacity in certain
tissues. The stimulation of protein synthesis by chronic leucine
administration was surprisingly tissue specific. Thus protein synthesis
in heart and kidney was unaffected by leucine or norleucine
supplementation, in contrast to the effects in other tissues. There are
at least four possible explanations for this tissue specificity, that
is, for the lack of response in heart and kidney. The first possible
explanation for the tissue specificity is that the supplementation for
12 days may lead to downregulation of an important component(s) of the
leucine-signaling pathway in heart and or kidney. If such adaptation
does occur, it seems unlikely that it is due either to changes in the
components of mTOR signaling or to the leucine metabolic pathways that
we examined, because these did not change appreciably. Further studies
will be required to evaluate this possibility once more information develops about how leucine activates mTOR signaling and as more information develops on the rapamycin-insensitive pathway. The second
possibility is that either heart or kidney may already be maximally
stimulated by ad libitum feeding. This seems particularly likely in
heart, because we observed that S6K1 was stimulated strongly by the
carbohydrate feeding in the preceding study (50), in
contrast to other tissues where the control carbohydrate meal had no
effect on S6K1. Third, heart and kidney may be poor responders, because
they do not express the proteins coupling the presence of
leucine to activation of the mTOR-signaling pathway or overexpress proteins antagonizing the signaling, such as phosphatases. In particular, kidney was noted to have comparably low levels of 4E-BP1.
We also noted a rather different pattern of S6K1 responses in kidney
and heart compared with gastrocnemius and adipose tissue in our
previous, acute study (50). Although both heart and kidney showed an acute protein synthesis response to leucine and norleucine in
fasted rats, no S6K1 response to either carbohydrate, leucine, or
norleucine gavage was observed in kidney. Thus kidney may become an
ideal tissue in which to examine the rapamycin-insensitive effects of
leucine on protein synthesis. Similarly, S6K1 from heart did not show
increased phosphorylation in response to oral leucine administration,
as did S6K1 from muscle and adipose tissue. Last, leucine metabolism
should also be considered. Kidney and heart express a high
concentration of both BCATm and BCKD relative to other tissues. The
resulting high flux through leucine metabolic pathways might diminish
the ability of leucine to regulate mTOR signaling.
Adipose tissue gave the most robust response in protein synthesis to
chronic leucine or norleucine supplementation. Thus the role of dietary
amino acids as metabolic substrates in adipose tissue may be
underappreciated. Adipose tissue also had the greatest level of
4E-BP1 phosphorylation. This may possibly be related to the finding
that, compared with other peripheral tissues, adipose tissue expressed
the greatest amount of mTOR per milligram of solubilized tissue protein.
Per gram tissue wet weight, adipose tissue, and skeletal muscle had
equivalent capacities for protein synthesis. However, skeletal muscle
represents 35-40% of body weight, so it has a larger impact on
whole body protein synthesis compared with adipose tissue. Although
there was not sufficient adipose tissue to make BCKD activity
measurements, previous studies in our laboratory (49) and
earlier studies (31) have demonstrated the capacity of
adipocytes to transaminate leucine, and the results in Fig. 7 show that
BCATm levels per milligram of detergent-solubilized lysate protein are
high in adipose tissue. Furthermore, studies by Goodman's group
[Frick and colleagues (28, 29)] showed that the fat cell
dehydrogenase is readily regulated by insulin and the ketoacid of
leucine, presumably through BCKD kinase. Thus, although lower
concentrations of the kinase and dehydrogenase are generally observed
in adipose tissue, they seem to be in an appropriate ratio to allow
nutritional regulation. Thus adipocytes may be an excellent model
system in which to elucidate the mechanism of mTOR regulation by leucine.
Tissue-specific expression of enzymes involved in leucine
catabolism and leucine as a potential nutrient signal.
It is not entirely clear whether leucine or the transamination
metabolite
-ketoisocaproate mediates the effects on mTOR signaling. We (27, 49) and others (63) found leucine to
be more efficacious than
-ketoisocaproate in skeletal muscle and
adipocytes. On the other hand, Patti et al. (57) and Xu et
al. (70) reported that
-ketoisocaproate was more
efficacious in different cell lines. Attempts have been made to address
this question by inhibiting BCATm (27, 49, 57, 70). A
limiting factor is that specific inhibitors of the first reversible
step in leucine metabolism are not available, and the available
inhibitors can affect ATP concentrations within the cells because they
are least potent against BCATm (75, 86). This is
important, because ATP concentration may affect mTOR activity due to
its relatively low Km for ATP (14).
Although it is recognized that this is still an open question, the
tissue-dependent expression of BCATm, BCKD, and BCKD kinase would seem
ideally suited to allow leucine to operate as a nutritional signal in
liver and peripheral tissues. BCATm is not found in adult rat liver
(39). Becasue mTOR signaling is regulated by leucine in
freshly isolated rat hepatocytes (6), the absence of BCATm
would facilitate its role as a nutritional signal there. The first step
in leucine metabolism takes place primarily in extrahepatic tissues
such as skeletal muscle, which releases considerable amounts of
-ketoisocaproate (41, 44). In kidney, muscle, or
adipose tissue, either dietary leucine or
-ketoisocaproate may serve
as a nutrient signal, as all possess considerable BCAT activity.
Skeletal muscle expresses a disproportionately large amount of BCKD
kinase relative to the amount of BCKD, thus limiting oxidation and
promoting
-ketoisocaproate release (15). In liver, there is a disproportionately high concentration of the dehydrogenase relative to the BCKD kinase. Thus liver may be important for oxidizing circulating ketoacid that escapes extrahepatic metabolism and removing
the leucine/
-ketoisocaproate signal. A second benefit of having
little or no hepatic BCATm activity but very active BCKD activity in
the liver is to ensure that dietary leucine reaches peripheral tissues
in sufficiently high concentrations to perform its function as a
nutrient signal for the presence of amino acids in a meal. Thus,
without BCATm in liver, leucine will be spared from so-called
"first-pass" metabolism. As mentioned in the previous studies
(47, 50), the lack of effect of norleucine on insulin secretion suggests that different mechanisms may be responsible for the
effects of BCAA/
-keto acids in islet cells [e.g., described by Xu
et al. (70)] and other peripheral tissues. Future studies will be required to determine whether leucine and/or
-ketoisocaproate mediate these effects and for a complete
understanding of the complex signaling pathways that mediate the
effects of the nutritional signaling molecules.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Sue Grigson, Josh Anthony, Jim Jefferson, and Scot
Kimball for helpful information. Dr. Grigson is also appreciated for
lending us the animal watering cylinders, as is Dr. Charles Lang for
generously providing the IGF assays. We also thank Trisha Garges,
Maggie McNitt, Jingjing Liu, Gina Deiter, Don Trapolsi, Mandi Fratini,
Diane Watts, and Mac Wood for technical assistance.
 |
FOOTNOTES |
This work was supported by grants from the Penn State Equal Opportunity
Planning Committee (A. Vaval), the National Institutes of Health
(DK-53843, C. J. Lynch), (GM-39277, AA-12814, T. C. Vary) and
(DK-34738, S. M. Hutson), and the US Department of Agriculture (98-35200-6067).
Address for reprint requests and other correspondence:
C. J. Lynch, Dept. of Cellular & Molecular Physiology, The
Pennsylvania State Univ. College of Medicine, 500 Univ. Dr., Hershey,
PA 17033 (E-mail: clynch{at}psu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 25, 2002;10.1152/ajpendo.00085.2002
Received 26 February 2002; accepted in final form 19 June 2002.
 |
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