Metabolic Regulation by Leucine of Translation Initiation Through the mTOR-Signaling Pathway by Pancreatic ß-Cells
Guang Xu,
Guim Kwon,
Wilhelm S. Cruz,
Connie A. Marshall, and
Michael L. McDaniel
From the Department of Pathology and Immunology (G.X., G.K., W.C.,
C.A.M., M.L.M.), Washington University School of Medicine, St. Louis,
Missouri.
Address correspondence and reprint requests to Michael L. McDaniel, PhD,
Department of Pathology and Immunology, Washington University School of
Medicine, Box 8118, 660 South Euclid Ave., St. Louis, MO 63110. E-mail:
mcdaniel{at}pathology.wustl.edu
.
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ABSTRACT
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Recent findings have demonstrated that the branched-chain amino acid
leucine can activate the translational regulators, phosphorylated heat- and
acid-stable protein regulated by insulin (PHAS-I) and p70 S6 kinase
(p70s6k), in an insulin-independent and rapamycin-sensitive manner
through mammalian target of rapamycin (mTOR), although the mechanism for this
activation is undefined. It has been previously established that
leucine-induced insulin secretion by ß-cells involves increased
mitochondrial metabolism by oxidative decarboxylation and allosteric
activation of glutamate dehydrogenase (GDH). We now show that these same
intramitochondrial events that generate signals for leucine-induced insulin
exocytosis are required to activate the mTOR mitogenic signaling pathway by
ß-cells. Thus, a minimal model consisting of leucine and glutamine as
substrates for oxidative decarboxylation and an activator of GDH,
respectively, confirmed the requirement for these two metabolic components and
mimicked closely the synergistic interactions achieved by a complete
complement of amino acids to activate p70s6k in a
rapamycin-sensitive manner. Studies using various leucine analogs also
confirmed the close association of mitochondrial metabolism and the ability of
leucine analogs to activate p70s6k. Furthermore, selective
inhibitors of mitochondrial function blocked this activation in a reversible
manner, which was not associated with a global reduction in ATP levels. These
findings indicate that leucine at physiological concentrations stimulates
p70s6k phosphorylation via the mTOR pathway, in part, by serving
both as a mitochondrial fuel and an allosteric activator of GDH.
Leucine-mediated activation of protein translation through mTOR may contribute
to enhanced ß-cell function by stimulating growth-related protein
synthesis and proliferation associated with the maintenance of ß-cell
mass.
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INTRODUCTION
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A number of recent studies have described synergistic interactions between
insulin and amino acids to regulate protein synthesis by modulating the mRNA
binding step in translation initiation. Insulin and amino acids have been
shown to exert their effects through phosphorylation of the translational
regulators phosphorylated heat-and acid-stable protein regulated by insulin
(PHAS-I) and p70 S6 kinase (p70s6k) via the serine and threonine
protein kinase, mammalian target of rapamycin (mTOR)
(1,2,3,4,5,6,7).
mTOR-mediated activation of p70s6k results in the phosphorylation
of ribosomal protein S6, which correlates with the translation of mRNAs that
encode both ribosomal proteins and translational elongation factors
(8,9).
The phosphorylation of PHAS-I through mTOR facilitates the release of eIF-4E
and allows its participation in translation initiation, especially of mRNAs
with high 5'-UTR secondary structures
(10,11,12,13).
In support of this mechanism, rapamycin, a potent inhibitor of mTOR, blocks
the phosphorylation of PHAS-I and p70s6k that results in inhibition
of translation initiation in response to insulin and amino acids. This
rapamycin-sensitive pathway is believed to preferentially stimulate
growth-related protein synthesis, leading to cell-cycle progression and
proliferation
(14,15,16).
More recently, the role of amino acids in regulating translation initiation
by nutrient signaling rather than in serving solely as precursors for protein
synthesis has become an important area of investigation
(17,18).
Thus, amino acids have been shown to be obligatory for insulin and growth
factorsignaling through mTOR
(19,20,21,22,23,24,25,26).
Furthermore, amino acids, in particular the branched-chain amino acids,
leucine, isoleucine, and valine, have been shown to independently activate the
mTOR pathway. Of these branched-chain amino acids, leucine has generated
significant interest due to its unique ability to regulate PHAS-I and
p70s6k at physiological concentrations, stimulate protein
synthesis, and inhibit lysosomal autophagy
(23,26,27,28,29,30).
Leucine has also been reported to enhance pancreatic ß-cell replication
in the fetal rodent pancreas, although the cellular mechanism for this effect
has not been defined (31).
Our previous studies with pancreatic ß-cells have demonstrated that
amino acids are required for glucose or exogenous insulin to stimulate the
phosphorylation of PHAS-I
(25). Amino acids alone also
dose-dependently stimulate the phosphorylation of PHAS-I, which is enhanced by
insulin. We have further shown that branched-chain amino acids retained their
ability to induce phosphorylation of PHAS-I and p70s6k in a
rapamycin-sensitive manner under conditions that block insulin secretion by
ß-cells (26). These
findings indicated that branched-chain amino acids modulate protein
translation by ß-cells via the mTOR-signaling pathway in an
insulin-independent manner. Similar findings have been documented with regard
to the ability of leucine to activate the mTOR-signaling pathway in other
cellular models
(20,21,22,23,24).
The cellular mechanisms whereby leucine activates translational regulators
through mTOR remain undefined. Studies designed to address the mechanism for
the requirement of leucine for mTOR activation have focused on the
intracellular metabolism of leucine
(23), tRNA aminoacylation
(32), structural
characteristics of the leucine molecule for a putative recognition site
(22,33),
kinase(s) (7), or
phosphatase(s) (20). It has
been previously established that leucine-induced insulin secretion by
ß-cells is mediated by the metabolism of leucine via the mitochondria by
oxidative decarboxylation in combination with the ability of leucine to
allosterically activate glutamate dehydrogenase (GDH) as shown in
Fig. 1
(34,35).
Although the metabolically linked secondary signals generated by the
metabolism of leucine to facilitate insulin secretion are unknown, it is
probable that these same intramitochondrial events, unique to the metabolism
of leucine, result in the generation of other signals independent of insulin
that are directed toward enhanced protein translation and cell proliferation.
In the present study, we have evaluated the hypothesis that this same
metabolic-signaling pathway involving the metabolism of leucine in combination
with its allosteric activation of GDH is necessary to activate the mTOR
mitogenicsignaling pathway in ß-cells.

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FIG. 1. Proposed model for leucine's role in mitogenic signaling. -KG,
-ketoglutarate; AOAA, aminooxyacetic acid; AT, aminotransferase; BCKDH,
branched-chain keto-acid dehydrogenase; GDH, glutamate
dehydrogenase; IR, insulin receptor; IRS, insulin receptor substrate;
p70s6k, p70 S6 kinase; PHAS-I, phosphorylated heat- and acid-stable
protein regulated by insulin; PKB, protein kinase B.
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EXPERIMENTAL PROCEDURES
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Materials. CellTiter 96 AQueous One Solution Reagent
([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
[MTS] reagent) was purchased from Promega (Madison, WI). Rotenone and
b(±) 2-amino-bicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH) were
obtained from Sigma (St. Louis, MO). Leucine analogs, leucine-amide
hydrochloride (H-Leu-NH2HCl), N-acetyl leucine
N-methyl-amide (Ac-Leu-NHMe),
-methyl DL-leucine, and
N-acetyl leucine amide (Ac-Leu-NH2), were purchased from
Bachem Feinchemikalien AG (Bubendorf, Switzerland). CMRL-1066 and RPMI-1640
tissue culture media, penicillin, streptomycin, L-glutamine, minimum essential
medium (MEM) amino acids solution, and MEM nonessential amino acids solution
were obtained from Life Technologies (Gaithersburg, MD). Rapamycin was from
Biomol (Plymouth Meeting, PA). The antibody for p70s6k was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibody was
peroxidase-conjugated donkey anti-rabbit IgG from Jackson ImmunoResearch
Laboratories (West Grove, PA). Enhanced chemiluminescence (ECL) reagents and
L-[U-14C]leucine were from Amersham Pharmacia (Piscataway, NJ). All
of the other chemicals were obtained from commercially available sources.
Amino acid composition. Krebs-Ringer bicarbonate buffer (KRBB) (in
mmol/l: 25 HEPES, 111 NaCl, 25 NaHCO3,5 KCl, 2.5 CaCl2,
and 1 MgCl2) was supplemented with MEM amino acids solution, MEM
nonessential amino acids solution, and L-glutamine to make 1x, or
complete amino acids, in which composition was described previously
(25). Basal amino acids were
defined the same as 1x, or complete amino acids, but excluding the
branched-chain amino acids (leucine, isoleucine, and valine). Our defined
physiological concentrations (mmol/l) of leucine, isoleucine, and valine were
0.4, 0.4, and 0.2, respectively, as in RPMI tissue culture media. This
compares with the concentrations (mmol/l) of leucine, isoleucine, and valine
(0.20, 0.11, and 0.25, respectively) found in the basal plasma of rats by
Mortimore et al. (30) and
0.25, 0.10, and 0.18, respectively, found in the portal vein of fasted rats by
Blommaart et al. (18).
Pancreatic ß-cell line. RINm5F cells, an insulin-secreting
ß-cell line (36), were
maintained and cultured as described previously
(25).
MTS assay. RINm5F cells were cultured in 96-well plates at a density
of 2.0 x 105 cells/200 µl culture media. To achieve a
quiescent state, cells were washed free of culture media and fetal bovine
serum three times with KRBB and preincubated for 1 h at 37°C in KRBB in
the absence of glucose and amino acids. KRBB was replaced as described in the
figure legends. The MTS assay reagent (20 µl/well) and treatment solution
(100 µl/well) were added simultaneously to the cells and mixed well by
gentle shaking. Cells were further incubated for 2 h at 37°C. The 450-nm
absorbance values were measured at 2 h.
p70s6k Assay. RINm5F cells (1.0 x
106cells/ml) were preincubated for 2 h at 37°C in KRBB in the
absence of glucose and amino acids. KRBB was replaced as described in the
figure legends. After experimental treatments, cells were processed for
SDS-PAGE and Western blotting of p70s6k
(26). Detection was performed
using ECL reagents. The activation of p70s6k occurs through
multisite phosphorylation that results in a slower migrating band compared
with nonphosphorylated p70s6k, which is detected by gel shift
mobility assays (37). The
insulin in the culture media was assayed by the radioimmunoassay core facility
of the Washington University Diabetes Research Training Center.
L-[U-14C]leucine oxidation assay. RINm5F cells (2.0
x 107) were preincubated in T-25 culture flasks in KRBB (5
ml) in the absence of glucose and amino acids for 2 h. Buffer was then
replaced with KRBB (5 ml) containing basal amino acids, 0.4 mmol/l leucine
± aminooxyacetic acid (AOAA) (5 mmol/l) ± BCH (5mmol/l) + 1
µCi L-[U-14C]leucine for 2 h. The amount of
14CO2 produced was trapped and quantitated by
scintillation counting.
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RESULTS
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In the present experimental design, RINm5F cells are initially incubated
for 2 h in the complete absence of amino acids in KRBB to dephosphorylate
p70s6k to basal levels. Subsequently, ß-cells were incubated
under the following conditions: 1) complete amino acids, including
the branched-chain amino acids, as a positive control; 2) basal amino
acids, which are devoid of leucine, isoleucine, and valine; and 3)
amino acids in the presence of either leucine, isoleucine, or valine as a test
condition to evaluate the ability of each branched-chain amino acid separately
and/or in combination to mediate p70s6k phosphorylation. The
multisite phosphorylation of p70s6k is detected by the appearance
of a slower migrating or upper band compared with nonphosphorylated
p70s6k as determined by gel shift mobility assays.
Effects of
leucine, isoleucine, and valine on p70s6k activation. As shown
in Fig. 2A, leucine at
0.4 mmol/l promotes the phosphorylation of p70s6k (lane
4), with the appearance of a slower migrating band (upper band) compared
with basal conditions, but isoleucine (0.4 mmol/l), valine (0.2 mmol/l), or
the combination of isoleucine and valine (lanes 5-7) do not mimic
this effect. However, leucine in the presence of valine or isoleucine
(lanes 8 and 9) again restores phosphorylation of
p70s6k. To determine if leucine under these conditions also
increases cellular metabolism by ß-cells, the MTS assay was used as a
measure of NAD(P)H production. As shown in
Fig. 2B, only leucine
(0.4 mmol/l) in the presence of glutamine (2 mmol/l), as a precursor for
glutamate that serves as a substrate for glutamate dehydrogenase (GDH),
significantly enhances ß-cell metabolism, suggesting that mitochondrial
metabolism is important for leucine to activate p70s6k.

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FIG. 2A : Leucine-induced phosphorylation of p70s6k at a
physiological concentration in RINm5F cells. Cells were preincubated in KRBB
in the absence of glucose and amino acids for 2 h. Media were then replaced
with KRBB containing either complete amino acids as a positive control or
basal amino acids, which excluded leucine, isoleucine, or valine. Leucine,
isoleucine, or valine was then added as indicated for 30 min. Cells were
processed for immunoblotting of p70s6k. Results are representative
of four separate experiments. The multisite phosphorylation of
p70s6k in this and all subsequent immunoblots is detected by a
slower migrating or upper band compared with nonphosphorylated
p70s6k as determined by gel-shift mobility assays.
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FIG. 2B : Effects of glutamine on branched-chain amino acid-mediated metabolism
by RINm5F cells. Cells were preincubated in KRBB in the absence of glucose and
amino acids for 1 h. Media were replaced with KRBB containing basal amino
acids ± glutamine (2 mmol/l), leucine, isoleucine, or valine as
indicated for 2 h with the MTS reagent. Results are representative of five
separate experiments.
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Although leucine activates p70s6k at a physiological
concentration of 0.4 mmol/l, isoleucine and valine require greater than
physiological concentrations to produce this same effect. As shown in
Fig. 3, leucine at 2, 5, and 10
mmol/l causes complete activation of p70s6k (lanes 4-6),
whereas isoleucine and valine mimic these effects only at 10 mmol/l (lanes
9 and 12). Leucine is metabolized in ß-cells exclusively by
the mitochondria, initially by its transamination to
-ketoisocaproic
acid (KIC) and subsequently by oxidative decarboxylation of KIC to acetyl-CoA
(see Fig. 1). KIC also
dose-dependently activates p70s6k over a concentration range of
0.4-4 mmol/l (data not shown).

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FIG. 3. Isoleucine and valine-induced phosphorylation of p70s6k at
greater than physiological concentrations in RINm5F cells. Cells were
preincubated in KRBB in the absence of glucose and amino acids for 2 h. Media
were then replaced with KRBB containing either complete amino acids as a
positive control or basal amino acids, which excluded leucine, isoleucine, and
valine. Leucine, isoleucine, or valine was then added as indicated for 30 min.
Cells were processed for immunoblotting of p70s6k. Results are
representative of five separate experiments.
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As shown in Fig. 4, the
ability of leucine to promote phosphorylation of p70s6k is blocked
by rapamycin (25 nmol/l), an inhibitor of mTOR, and wortmannin (100 nmol/l),
an inhibitor of phosphoinositide 3-kinase (PI 3-K). Because PI 3-K activity is
not required for amino acid stimulation of mTOR
(23), inhibition of
p70s6k by wortmannin may be due to decreased amino acid transport,
a direct effect on mTOR, or nonspecific inhibition of other
phosphatidylinositol kinases
(23,38).
Rapamycin (25 nmol/l) also did not inhibit leucine-induced cellular metabolism
as determined by the MTS assay or the metabolism of
L-[U-14C]leucine by oxidative decarboxylation to
14CO2 (data not shown).

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FIG. 4. Effects of rapamycin and wortmannin on leucine-induced phosphorylation
of p70s6k in RINm5F cells. Cells were preincubated in KRBB in the
absence of glucose and amino acids for 2 h. During the last hour of
preincubation, rapamycin or wortmannin was added to cells. Media were then
replaced with KRBB containing either complete amino acids as a positive
control or basal amino acids, which excluded leucine, isoleucine, or valine.
Leucine ± inhibitors were then added as indicated for 30 min. Cells
were processed for immunoblotting of p70s6k. Results are
representative of three separate experiments.
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Role for leucine metabolism by oxidative decarboxylation and allosteric
activation of GDH to promote p70s6k phosphorylation. To
determine if both oxidative decarboxylation and GDH activation are essential
in leucinemediated activation of p70s6k, experiments were performed
in the absence of glutamine and glutamate as shown in
Fig. 5 (lanes 3-10).
This removal did not prevent the ability of leucine to promote activation of
p70s6k (lane 4). This unexpected result may be explained
by the endogenous production of glutamate, which is mediated, in part, by the
availability of
-ketoglutarate as an acceptor for the amino-transferase
reaction that results in glutamate formation as shown in
Fig. 1. In fact, removing
glutamine and glutamate from the complete amino acids resulted in a 2.8-fold
increase in the conversion of 14C-leucine to
14CO2 by the oxidative decarboxylation pathway (data not
shown), which would increase the generation of glutamate from
-ketoglutarate by this same aminotransferase reaction. Thus, removing
glutamine and glutamate from the incubation media stimulates the catabolism of
leucine by enhancing the transamination of leucine to KIC with the
concomittant generation of endogenous glutamate
(39).

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FIG. 5. Phosphorylation of p70s6k induced by leucine requires its
metabolism and allosteric activation of GDH in RINm5F cells. Cells were
preincubated in KRBB in the absence of glucose and amino acids for 2 h. During
the last 30 min of preincubation, AOAA was added to cells. Media were then
replaced with KRBB containing either complete amino acids as a positive
control or basal amino acids, which excluded leucine, isoleucine, and valine.
Lanes 3-10 do not contain glutamine and glutamate. Leucine or KIC
± AOAA was added as indicated for 30 min. Cells were processed for
immunoblotting of p70s6k. Results are representative of three
separate experiments.
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To block the formation of endogenous glutamate and to evaluate a role for
the metabolism of leucine via oxidative decarboxylation to mediate
p70s6k phosphorylation, we next used AOAA, an inhibitor of the
transamination of leucine to KIC
(22,40).
As shown in Fig. 5 (lanes
5-8), leucine-induced p70s6k phosphorylation is
dose-dependently inhibited by AOAA (1-10 mmol/l). In a similar manner, the
ability of KIC to activate p70s6k (lane 9) is also
prevented by AOAA (lane 10). This latter effect is explained both by
the metabolism of KIC by oxidative decarboxylation to acetyl-CoA and also by
the rapid conversion of KIC to leucine through the reversible aminotransferase
reaction, which allows leucine to also allosterically activate GDH
(34,35).
Minimal model including only leucine and glutamate. Our next
approach was to determine if leucine and glutamine are sufficient to promote
phosphorylation of p70s6k in the absence of all other amino acids.
In this minimal model, leucine is required to provide substrate for its
metabolism by the oxidative decarboxylation pathway and also serve as an
allosteric activator of GDH. Glutamine is necessary to provide a source of
glutamate for the GDH-mediated production of
-ketoglutarate and its
subsequent metabolism by the mitochondria. In addition, glutamine is
extensively converted to glutamate, whereas glutamate is poorly transported
into ß-cells (34). As
shown in Fig. 6A,
leucine (0.4 mmol/l) or glutamine (2 mmol/l) (lanes 3 and 4)
alone produced a small increase in p70s6k phosphorylation above
basal values, whereas a combination of leucine and glutamine markedly
activated p70s6k (lane 5). This full activation of
p70s6k is comparable to that produced by a complete complement of
amino acids (lane 2). This ability of a combination of leucine and
glutamine to mediate full activation of p70s6k was also
dose-dependently inhibited by AOAA (lanes 6-8).
Figure 6B also
illustrates that this same minimal model consisting of leucine and glutamine
that results in full activation of p70s6k (lane 5) is
blocked by rapamycin (lane 6).

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FIG. 6A Leucine and glutamine are sufficient to induce phosphorylation of
p70s6k in RINm5F cells. Cells were preincubated in KRBB in the
absence of glucose and amino acids for 2 h. During the last 30 min of
preincubation, AOAA was added to cells. Media were then replaced with KRBB
containing either complete amino acids as a positive control or leucine alone,
glutamine alone, or both ± AOAA, as indicated for 30 min. Cells were
processed for immunoblotting of p70s6k. Results are representative
of five separate experiments.
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FIG. 6B: Leucine and glutamine-induced phosphorylation of p70s6k is
rapamycin sensitive. Cells were preincubated in KRBB in the absence of glucose
and amino acids for 2 h. During the last hour of preincubation, rapamycin (25
nmol/l) was added to cells. Media were then replaced with KRBB containing
leucine alone, glutamine alone, or both ± rapamycin as indicated for 30
min. Cells were processed for immunoblotting of p70s6k. Results are
representative of four separate experiments.
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Effects of structural analogs of leucine on p70s6k
activation. A recent report by Shigemitsu et al.
(33) evaluated the structural
requirements of leucine that are necessary to activate p70s6k by a
rat hepatoma cell line, H4IIE. In this structural analysis, it was found that
leucine with a modification of its carboxyl group, H-Leu-NH2HCl,
and leucine with a modified
-hydrogen atom, H-
-Me-DL-Leu-OH,
stimulated p70s6k activity. In contrast, leucine with modifications
of its amino and carboxyl groups, Ac-Leu-NH2 and Ac-Leu-NHMe,
lacked activity and also inhibited the ability of leucine to activate
p70s6k. As shown in Fig.
7A, we also observed that H-Leu-NH2HCl
(lane 6) and H-
-Me-DL-Leu-OH (lane 7) activated
p70s6k, whereas Ac-Leu-NH2 (lane 5) and
Ac-Leu-NHMe (lane 8) are inactive. In our experimental model, the
ability of the most active leucine analog, H-Leu-NH2HCl, correlated
with its ability to stimulate ß-cell metabolism in the presence of
glutamine as determined by the MTS assay. As shown in
Fig. 7B,
H-Leu-NH2HCl stimulates an increase in ß-cell metabolism
similar to leucine, whereas the partial agonist, H-
-Me-DL-Leu-OH,
resulted in no detectable increase. It is presumed that our inability to
demonstrate an enhancement of ß-cell metabolism by the partial agonist,
H-
-Me-DL-Leu-OH (Fig.
7B), is due to the limits of detection by the MTS
assay.

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FIG. 7A: Phosphorylation of p70s6k induced by leucine and leucine
analogs. Cells were preincubated in KRBB in the absence of glucose and amino
acids for 2 h. Media were then replaced with KRBB containing either complete
amino acids as a positive control or basal amino acids, which excluded
leucine, isoleucine, or valine. Leucine or leucine analogs were added as
indicated for 30 min. Cells were processed for immunoblotting of
p70s6k. Results are representative of four separate
experiments.
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FIG. 7B : Effects of leucine and leucine analogs on RINm5F cell metabolism.
Cells were preincubated in KRBB in the absence of glucose and amino acids for
1 h. Media were then replaced with KRBB containing basal amino acids ±
glutamine (2 mmol/l), ± leucine or leucine analogs (4 mmol/l) as
indicated for 2 h with the MTS reagent. Results are means ± SE of three
separate experiments.
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Effects of the nonmetabolized leucine analog BCH. It has been
demonstrated previously that leucine and a nonmetabolized leucine analog, BCH,
activate GDH in pancreatic ß-cells
(34,35).
The ability of this nonmetabolized leucine analog to activate
p70s6k in our model system was next evaluated. As shown in
Fig. 8A, BCH over a
concentration range of 0.2-10 mmol/l failed to activate p70s6k.
Because leucine-induced p70s6k activation is proposed based on our
studies to require the metabolism of leucine by oxidative decarboxylation and
allosteric activation of GDH, we evaluated the effects of BCH and AOAA on the
metabolism of L-[U-14C]leucine by oxidative decarboxylation to
14CO2 by the ß-cell mitochondria
(Fig. 8B). As
anticipated, AOAA, an inhibitor of the amino-transferase reaction that
converts leucine to KIC, completely blocked the metabolism of
14C-leucine to 14CO2. Unexpectedly, BCH also
almost completely blocked the metabolism of 14C-leucine by
oxidative decarboxylation to 14CO2. Although the failure
of BCH alone to activate p70s6k is consistent with our hypothesis,
the inhibitory effects produced by BCH on the oxidative decarboxylation
pathway of leucine by ß-cells limited its use in elucidating the
mechanism responsible for leucine-induced activation of p70s6k.

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FIG. 8A: Activation of GDH by nonmetabolized BCH does not induce phosphorylation
of p70s6k in RINm5F cells. Cells were preincubated in KRBB in the
absence of glucose and amino acids for 2 h. Media were then replaced with KRBB
containing either complete amino acids as a positive control, basal amino
acids, which excluded leucine, isoleucine, or valine, or basal amino acids +
BCH as indicated for 30 min. Cells were processed for immunoblotting of
p70s6k. Results are representative of three separate
experiments.
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FIG. 8B: Inhibition of L-[U-14C]leucine decarboxylation by AOAA and
BCH. Cells (2.0 x 107) were preincubated in KRBB in the
absence of glucose and amino acids for 2 h. Media were then replaced with KRBB
containing basal amino acids supplemented with 0.4 mmol/l leucine + 1 µCi
L-[U-14C]leucine. AOAA or BCH was added as indicated for 2 h. The
amount of 14CO2 produced was trapped and quantitated by
scintillation counting. Results are means ± SE of three separate
experiments.
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Role for the mitochondria in mediating leucine-induced activation of
p70s6k. To further confirm a role for the ß-cell
mitochondria to mediate leucine-induced activation of p70s6k, a
series of studies were performed to inhibit mitochondrial function. In this
experimental design, ß-cells were exposed to a complete complement of
amino acids to stimulate p70s6k activation in the presence of 3
mmol/l glucose. As shown in Fig.
9A, exposure of ß-cells to azide, an inhibitor of
mitochondrial cytochrome c oxidase
(41) dose-dependently (1-10
mmol/l) inhibited the ability of a complete complement of amino acids to
promote activation of p70s6k (lanes 3-5). This ability of
azide to block amino acid-induced activation of p70s6k was not the
result of cytotoxicity because its inhibitory effect was readily reversed, as
shown in lanes 6-8. Also, as shown in
Fig. 9B, azide, under
these identical conditions, did not inhibit glucose metabolism through
glycolysis indicating that a global reduction in cellular ATP is not
responsible for its inhibitory effects on p70s6k activation.
Furthermore, additional inhibitors of the mitochondrial electron-transfer
chain including rotenone, an inhibitor of complex 1, and antimycin A, an
inhibitor of complex 3 (42),
produced similar dose-dependent and reversible inhibition of amino
acid-induced activation of p70s6k (data not shown).

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FIG. 9A : Phosphorylation of p70s6k induced by amino acids requires
mitochondrial oxidation in RINm5F cells. Cells were preincubated in KRBB in
the absence of glucose and amino acids for 2 h. During the last hour of
preincubation, azide was added to cells. Media were then replaced with KRBB
containing 3 mmol/l glucose and complete amino acids ± azide for 30
min. In addition, cells in lanes 6-8 were washed three times with PBS
and buffer was replaced with KRBB containing complete amino acids and 3 mmol/l
glucose without azide for 30, 60, or 90 min as indicated for recovery. Cells
were processed for immunoblotting of p70s6k. Results are
representative of three separate experiments.
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|

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FIG. 9B : Effects of azide on RINm5F cell metabolism. Cells were preincubated in
KRBB in the absence of glucose and amino acids for 1 h. During the
preincubation, azide was added to cells. Media were then replaced with KRBB
containing complete amino acids + glucose (3 mmol/l) ± azide as
indicated for 2 h with the MTS assay reagent. Results are means ± SE of
three separate experiments.
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|
 |
DISCUSSION
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In the present study, we have attempted to determine the mechanism
responsible for the unique ability of leucine to activate the mTOR-signaling
pathway relative to other amino acids. These studies have focused on the
ability of leucine to be metabolized exclusively by the mitochondria in
combination with its unique ability to allosterically activate GDH that
further energizes the mitochondria by the production of the Krebs Cycle
intermediate,
-ketoglutarate. The removal of leucine from a complete
complement of amino acids inhibits p70s6k activation and, at
corresponding concentrations from fasting to postprandial, dose-dependently
restores the ability of the complete complement of amino acids to activate
p70s6k.
Although isoleucine and valine can activate p70s6k, this is only
achieved at higher concentrations as compared with leucine. This large
difference in the concentration dependency of isoleucine and valine to
activate p70s6k may relate to the reduced ability of isoleucine and
valine to activate GDH compared with leucine because similar pathways
involving oxidative decarboxylation also metabolize isoleucine and valine
(34).
The specific mechanisms responsible for leucine activation of these
translational regulators are unknown. Both the intracellular metabolism of
leucine and its interaction with a recognition site or other unknown proteins
requiring the unique structure of leucine have been proposed
(7,20,22,23,32,33).
The ability of KIC to activate p70s6k has been used to support the
hypothesis that leucine metabolism is required for p70s6k
activation (23). However, the
observation that AOAA, an inhibitor of the amino transaminase that blocks the
rapid conversion of KIC to leucine, prevents KIC-mediated p70s6k
activation has suggested that the specific structure of leucine is important
(22). In the present study, we
show that both leucine metabolism and its allosteric interaction with GDH
requiring its specific structure or recognition site are important in
p70s6k activation. Studies using various leucine analogs also
support the close association of mitochondrial metabolism and the ability of
leucine to activate p70s6k. The unique ability of leucine to
stimulate insulin secretion by its metabolism and its interaction with GDH has
been previously documented
(34,35,43).
The unique ability of leucine to activate p70s6k via its
metabolism and its interaction with GDH has been further studied using a
minimal model system consisting of only leucine and glutamine. This minimal
model mimicked closely the synergistic interactions achieved by a complete
complement of amino acids and provided full activation of p70s6k in
a rapamycin and AOAA-inhibited manner (Fig.
6A). The small increases in basal p70s6k
observed with leucine alone in this minimal model are believed to be mediated,
in part, by endogenous glutamate formation as a consequence of the conversion
of leucine to KIC by the aminotransferase reaction. An additional factor
believed to contribute to elevated basal levels of p70s6k
activation in this in vitro model is our inability to regulate the endogenous
production of leucine by ß-cells under these nutrient-deprived culture
conditions.
Our attempts to assess the effects of activation of GDH with the
nonmetabolized leucine analog, BCH, were problematic. BCH was clearly
ineffective in activating p70s6k over a concentration range of
0.2-10 mmol/l in the absence of branched amino acids, which is consistent with
our overall hypothesis. However, it was determined subsequently that BCH
blocked L-[U-14C]leucine metabolism by oxidative decarboxylation to
14CO2. Plausible explanations for this latter effect are
that BCH inhibits the conversion of leucine to KIC by the aminotransferase
reaction and/or that BCH competes with leucine entry into ß-cells at the
level of the L-system amino acid transporter due to its structural similarity
to leucine. In either case, our data support the hypothesis that the ability
of leucine to activate the mTOR pathway is not only structural but also
requires the metabolism of leucine.
Because our studies have indicated that leucine's ability to activate mTOR
is linked to its metabolism by the ß-cell mitochondria, attempts were
made to further establish this correlation by perturbing mitochondrial
function. As demonstrated with azide, its ability to inhibit leucine
activation of p70s6k was rapidly reversed after its removal and was
not associated with a global reduction in ATP levels because glucose
metabolism by glycolysis was unaltered
(Fig. 9B). It has
also been recently proposed that increases in mitochondrial-derived ATP by
ß-cells may provide localized or privileged elevations of ATP essential
for sustained closure of KATP channels
(44). In addition, a
ß-cell line depleted of mitochondrial DNA displayed defects in cytochrome
c oxidase activity, glucose, and leucine-induced increases in cellular ATP
content and respiratory chain-driven ATP synthesis
(45).
In summary, as illustrated schematically in
Fig. 1, leucine has been
previously shown to stimulate insulin secretion by ß-cells due to its
metabolism by oxidative decarboxylation and the ability of leucine to
allosterically activate GDH by the ß-cell mitochondria. Both acetyl-CoA
and
-ketoglutarate appear to be necessary as Kreb's Cycle substrates to
fully activate the ß-cell mitochondria. Although these metabolically
linked secondary signals originate from the mitochondria, the identity of
these mediators and the mechanism leading to insulin exocytosis are unknown.
Our findings show that leucine also uses these same metabolic pathways to
activate mTOR mitogenic signaling in ß-cells. This mechanism appears to
involve other metabolically linked secondary signals because leucine-induced
activation of protein translation is rapamycin sensitive and insulin
independent. Future studies will explore the metabolic links between the
metabolism of leucine by the ß-cell mitochondria and mTOR activation.
 |
ACKNOWLEDGMENTS
|
---|
This study was supported by National Institutes of Health Grant DK55024
(M.L.M.), an American Diabetes Association Mentor-Based Fellowship (G.X.), and
an American Diabetes Association Research Grant (M.L.M.)
The authors thank Joan Fink for her excellent technical assistance.
 |
FOOTNOTES
|
---|
Ac-Leu-NH2, N-acetyl leucine amide; Ac-Leu-NHMe,
N-acetyl leucine N-methyl-amide; AOAA, aminooxyacetic acid;
BCH, b(±) 2-amino-bicyclo [2,2,1] heptane-2-carboxylic acid; ECL,
enhanced chemiluminescence; GDH, glutamate dehydrogenase;
H-Leu-NH2HCl, leucine-amide hydrochloride; KIC,
-ketoisocaproic acid; KRBB, Krebs-Ringer bicarbonate buffer; MEM,
minimal essential medium; mTOR, mammalian target of rapamycin; MTS,
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt; p70s6k, p70 S6 kinase; PHAS-I, phosphorylated heat- and
acid-stable protein regulated by insulin; PI 3-K, phosphoinositide
3-kinase.
Received for publication July 27, 2000
and accepted in revised form October 11, 2000
 |
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