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J Biol Chem, Vol. 273, Issue 43, 28178-28184, October 23, 1998
From the Amino acids have been identified as important
signaling molecules involved in pancreatic Nutrients, in particular, amino acids have been recognized as
important signaling mediators in pancreatic Insulin and other growth factors induce increases in protein synthesis,
in part, by stimulating mRNA translation. The regulation of protein
synthesis is believed to be central to cell replication since re-entry
of cells into the cell cycle in response to growth factors and
nutrients is accompanied by an approximate doubling of protein
synthesis before the onset of DNA synthesis and cell division (3, 4).
This obligatory role for an increase in the rate of protein synthesis
for cells to enter and progress through the cell cycle has suggested
that activation of protein translation is an important initial event in
the mitogenic response.
The initiation phase of mRNA translation is generally rate-limiting
for protein synthesis (see Refs. 3-6 for reviews). Initiation is
mediated in part by the eIF-4F complex, which is composed of three
subunits, eIF-4 Recent studies have implicated a signaling pathway involving the
mammalian target of rapamycin (mTOR) as an upstream regulator of PHAS-I
phosphorylation. Rapamycin is a potent immunosuppressant and
antiproliferative agent that mediates its effects through formation of
an active complex with its intracellular receptor, FK506-binding
protein of Mr = 12,000 (9). This rapamycin
complex then binds to members of the TOR (target of rapamycin) family which include TOR1p and TOR2p in yeast and a mammalian homologue, mTOR,
also designated as FRAP or RAFT1 (10, 11). In yeast and mammalian
cells, activation of TOR is required for G1 progression and
cell proliferation (12). Recent studies indicate that mTOR directly
phosphorylates PHAS-I, although how mTOR is regulated is not yet
defined (13-15). In insulin-sensitive cells, activation of the insulin
receptor is believed to result in phosphorylation of insulin receptor
substrate-1 (IRS-1) and insulin receptor substrate-2 (IRS-2) which then
activates phosphoinositide (PI) 3-kinase (16). New evidence supports
the conclusion that insulin-stimulated phosphorylation and activation
of mTOR is mediated by the protein kinase B signaling pathway (17).
PHAS-I and p70s6k are located downstream in this pathway
and are proposed to be regulated by mTOR in a parallel manner (11, 18,
19). This signaling pathway can be blocked by inhibiting PI 3-kinase
with wortmannin, although higher concentrations of wortmannin also inhibit mTOR. Phosphorylation and activation of p70s6k
mediates ribosomal protein S6 phosphorylation and may be essential during the G1 phase of the cell cycle (18, 19). Overall,
these studies suggest that PHAS-I and p70s6k exert
important roles in growth-regulated protein translation in a variety of
cells.
Our studies have recently shown that incubation of pancreatic islets
with elevated glucose levels results in rapid and
concentration-dependent phosphorylation of PHAS-I (20).
This effect is due, in part, to insulin released into the incubation
medium, since exogenous insulin increases whereas conditions that block
insulin exocytosis from the Materials--
Male Sprague-Dawley rats were purchased from
Harlan Sprague-Dawley (Indianapolis, IN). Collagenase type P was
obtained from Boehringer Mannheim. CMRL-1066 and RPMI 1640 tissue
culture media, penicillin, streptomycin, Hanks' balanced salt
solution, L-glutamine, MEM amino acids solution, and MEM
non-essential amino acids solution were obtained from Life
Technologies, Inc. Fetal bovine serum was from HyClone (Logan, UT).
Rapamycin was from Biomol (Plymouth Meeting, PA). Ficoll was from
Sigma. PHAS-I antibody was generated in rabbit with recombinant
His-tagged rat PHAS-I (7). The secondary antibody was
peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA). The antibody for p70s6k was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other
chemicals were from commercially available sources.
Amino Acid Composition--
Krebs-Ringer bicarbonate buffer
(KRBB: 25 mM HEPES, 115 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, and 1 mM
MgCl2, pH 7.4, 0.1% bovine serum albumin) was supplemented
with MEM amino acids solution, MEM non-essential amino acids solution,
and L-glutamine. For these experiments, the 1×
concentration of amino acids was defined as the following in
mM: L-arginine 0.73, L-cystine 0.2, L-glutamine 2.0, L-histidine·HCl·H2O 0.2, L-isoleucine 0.4, L-leucine 0.4, L-lysine HCl 0.5, L-methionine 0.1, L-phenylalanine 0.2, L-threonine 0.4, L-tryptophan 0.05, L-tyrosine 0.2, L-valine 0.4, L-alanine 0.1, L-asparagine 0.1, L-aspartic acid 0.1, L-glutamic acid 0.1, glycine 0.1, L-proline
0.1, L-serine 0.1. The osmolarity of each solution was
measured using a Wescor Vapor Pressure Osmometer (Logan, UT) and found
to be between 300 and 310 mOsm/kg.
Pancreatic Islet Isolation and Culture--
Islets were isolated from male
Sprague-Dawley rats (200-250 g) by collagenase digestion as described
previously (23). Briefly, pancreata were inflated with Hanks' balanced
salt solution, and the tissue was isolated, minced, and digested with 7 mg of collagenase/pancreas for 7 min at 39 °C. Islets were separated
on a Ficoll step density gradient and then selected with a
stereomicroscope to exclude any contaminating tissues. Islets were
cultured overnight in an atmosphere of 95% air, 5% CO2 in
cCMRL.
PHAS-I and p70s6k Assays--
To achieve a quiescent
state, RINm5F cells (1 × 106 cells/3 ml) or islets
(200/1 ml) were washed free of culture media and fetal bovine serum and
preincubated for 2 h at 24 or 37 °C in KRBB in the absence of
glucose and amino acids. KRBB was replaced as described in the figure
legends. Following experimental treatments, cells or islets were washed
with phosphate-buffered saline and solubilized in 300 or 30 µl,
respectively, of Laemmli sample buffer, heated at 100 °C for 5 min,
and centrifuged at 10,000 × g for 15 min to remove
insoluble materials. The supernatants were processed for
SDS-polyacrylamide gel electrophoresis and Western blotting of PHAS-I
or p70s6k as described previously (20). Detection was
performed using ECL reagents from Amersham Pharmacia Biotech. PHAS-I
Essential Amino Acids Induce Phosphorylation of PHAS-I in RINm5F
Cells--
Previous studies have demonstrated that phosphorylation of
PHAS-I, a necessary step for the release of the rate-limiting
initiation factor, eIF-4E, results in a decrease in electrophoretic
mobility of this protein when separated on SDS-polyacrylamide gels
(24). Thus, the hyperphosphorylated
Branched-chain Amino Acids Are Essential in the Regulation of
PHAS-I and p70 S6 Kinase by Pancreatic 
-Cells
A POSSIBLE ROLE IN PROTEIN TRANSLATION AND MITOGENIC
SIGNALING*
,,,,¶
Department of Pathology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
§ Department of Pharmacology, University of Virginia School
of Medicine, Charlottesville, Virginia 22908
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-cell proliferation,
although the cellular mechanism responsible for this effect is not well
defined. We previously reported that amino acids are required for
glucose or exogenous insulin to stimulate phosphorylation of PHAS-I
(phosphorylated heat- and acid-stable protein regulated by insulin), a
recently discovered regulator of translation initiation during cell
mitogenesis. Here we demonstrate that essential amino acids, in
particular branched-chain amino acids (leucine, valine, and
isoleucine), are largely responsible for mediating this effect. The
transamination product of leucine, 
-ketoisocaproic acid, also
stimulates PHAS-I phosphorylation although the transamination products
of isoleucine and valine are ineffective. Since amino acids are
secretagogues for insulin secretion by -cells, we investigated
whether endogenous insulin secreted by -cells is involved.
Interestingly, branched-chain amino acids stimulate phosphorylation of
PHAS-I independent of endogenous insulin secretion since genistein (10 µM) and herbimycin A (1 µM), two
tyrosine kinase inhibitors in the insulin signaling pathway, exert no
effect on amino acid-induced phosphorylation of PHAS-I. Furthermore,
branched-chain amino acids retain their ability to induce
phosphorylation of PHAS-I under conditions that block insulin secretion
from -cells. In exploring the signaling pathway responsible for
these effects, we find that rapamycin (25 nM) inhibits the
ability of branched-chain amino acids to stimulate the phosphorylation
of PHAS-I and p70s6 kinase, suggesting that the mammalian
target of rapamycin signaling pathway is involved. The branched-chain
amino acid, leucine, also exerts similar effects on PHAS-I
phosphorylation in isolated pancreatic islets. In addition, we find
that amino acids are necessary for insulin-like growth factor (IGF-I)
to stimulate the phosphorylation of PHAS-I indicating that a
requirement for amino acids may be essential for other -cell growth
factors in addition to insulin and IGF-I to activate this signaling
pathway. We propose that amino acids, in particular branched-chain
amino acids, may promote -cell proliferation either by stimulating
phosphorylation of PHAS-I and p70s6k via the mammalian
target of rapamycin pathway and/or by facilitating the proliferative
effect mediated by growth factors such as insulin and IGF-I.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-cell function. In
addition to modulating insulin secretion by -cells and glucagon secretion by -cells, amino acids have been identified as important signaling agents in the initiation and modulation of -cell
proliferation. Previous studies by Swenne (1, 2) have demonstrated that increases in essential amino acids significantly enhance
[3H]thymidine incorporation into DNA by pancreatic
islets. Although amino acids alone and in combination with insulin,
insulin-like growth factor
(IGF-I),1 and other growth
factors are well documented to exert a mitogenic effect on both adult
and fetal islets, the cellular mechanism responsible for this effect is
not well defined.
, eIF-4A, and eIF-4E (6). eIF-4 is a large subunit
(Mr = 220,000) that binds eIF-4A
(Mr = 45,000) and eIF-4E
(Mr = 25,000). eIF-4A is an
ATP-dependent helicase, and eIF-4E is the mRNA
cap-binding protein. The initiation factor, eIF-4E, is the least
abundant of the eIF-4F subunits, and it is generally believed that the
amount of eIF-4E is limiting for translation initiation. eIF-4E is
involved in recognition of the mRNA cap, a structure of
m7GpppN (N represents any nucleotide) located at the
5'-end of almost all eukaryotic messages. The availability
of eIF-4E is regulated by PHAS-I (also designated as 4E-BP1), a heat-
and acid-stable eIF-4E-binding protein first identified in rat
adipocytes (7, 8). PHAS-I inhibits the binding of eIF-4E to eIF-4G (also designated as eIF-4), thus preventing formation of a complex necessary for efficient binding and proper positioning of the 40 S
ribosomal subunit of mRNA. When phosphorylated in the appropriate sites after exposure of responsive cells to insulin, PHAS-I dissociates from eIF-4E, thus allowing eIF-4E to participate in translation initiation.
-cell inhibit phosphorylation of PHAS-I.
In addition, conditions that stimulate insulin secretion from the
-cell lines, TC6-F7 and TC3, increase phosphorylation of
PHAS-I, suggesting that -cells of the islet, in part, mediate this
effect. These findings indicate that glucose stimulates PHAS-I
phosphorylation via insulin interacting with its own receptor on the
-cell which may serve as an important mechanism for autoregulation
of protein synthesis by translation. During the course of these
studies, we discovered that amino acids are required for glucose or
exogenous insulin to stimulate the phosphorylation of PHAS-I.
Furthermore, amino acids alone dose-dependently stimulate
phosphorylation of PHAS-I in a rapamycin-sensitive manner which is
further enhanced by insulin. We have therefore examined in the present
study the signal transduction pathways whereby amino acids, in
particular branched-chain amino acids, alone and in synergy with
insulin and other growth factors activate the mTOR signaling
pathway.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Cell Lines--
RINm5F cells, an
insulin-secreting -cell line (21, 22), were cultured by the
Washington University Tissue Culture Support Center in RPMI 1640 containing 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin, 0.1 mM non-essential amino acids,
16.8 mM glucose, 1 mM sodium pyruvate, 10 mM HEPES, pH 7.4. Cells were subcultured in "complete"
CMRL-1066 tissue culture medium (cCMRL) containing 5.5 mM
glucose, 2 mM L-glutamine, 10% (v/v)
heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in Petri dishes (60 × 15 mm) at a
concentration of 1 × 106 cells/3 ml and incubated at
37 °C under an atmosphere of 95% air, 5% CO2 for
24 h before initiating experiments. Where "CMRL" is stated,
this culture media does not contain fetal bovine serum.
, , , and 
were quantitated by densitometry using a
Molecular Dynamics Personal Densitometer Scanning Instrument
(Sunnyvale, CA). Data were expressed as percent of PHAS-I and ,
over total PHAS-I. Insulin in the culture media was assayed by the
radioimmunoassay core facility of the Washington University Diabetes
Research Training Center.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-form of PHAS-I migrates most
slowly, followed by the intermediary -form and the nonphosphorylated -form of PHAS-I. Under some experimental conditions in the present studies, PHAS-I is detected which represents a more
hyperphosphorylated form of PHAS-I than PHAS-I.
. Essential amino acids (1×,
lane 3) were as effective in mediating phosphorylation of
PHAS-I (69%) compared with a complete complement of amino acids
(73%, lane 2). It is noted in Fig. 1 that the
nonphosphorylated form of PHAS-I, PHAS-I, is not detected. PHAS-I
does occur in RINm5F cells and intact islets, although occasionally it
is not detected dependent on the degree of quiescence of the cells.
Although non-essential amino acids (lane 4) and glutamine
(lane 5) did cause some PHAS-I phosphorylation, 30 and
40% respectively, the effect was not nearly as significant as that
produced by either essential or a complete complement of amino acids.
Therefore, our present studies have focused specifically in defining
the role of essential amino acids in mitogenic signaling.
View larger version (28K):
[in a new window]
Fig. 1.
Essential amino acids induce phosphorylation
of PHAS-I in RINm5F cells. RINm5F cells were preincubated in KRBB
in the absence of glucose and amino acids for 2 h. Following
preincubation, buffer was replaced with KRBB containing total amino
acids (1×), essential amino acids (1×), non-essential amino acids
(1×), or glutamine (1×) as indicated for 30 min. Cells were processed
for immunoblotting of PHAS-I as described under "Experimental
Procedures." Results are representative of three separate
experiments.
-cells (25-27). As shown in Fig.
2 (lane 1), in the absence of amino acids 30% of PHAS-I was in the - and -form. A complete mixture of amino acids (1×, lane 2) resulted in 72% of
PHAS-I to be in the - and -form. All 12 of the minimal essential
amino acids caused some increase in the percent of PHAS-I and -
compared with the absence of amino acids. The branched-chain amino
acids, leucine (Leu, 72%), isoleucine (Ile, 65%), and valine (Val,
77%) were among the most effective in stimulating formation of
PHAS-I and (lanes 3-5), compared with the absence of
amino acids (lane 1) and a complete complement of amino
acids (lane 2). It is noted that valine (10 mM)
also results in a significant appearance of the hyperphosphorylated
form of PHAS-I, PHAS-I (lane 5). Of the remaining
essential amino acids (methionine, phenylalanine, tryptophan, cystine,
threonine, tyrosine, arginine, histidine, and lysine), only tyrosine
(65%) was as effective as the branched-chain amino acids (leucine,
isoleucine, and valine) to induce the formation of PHAS-I
(lanes 6-14). We therefore decided to limit the remainder of this study to the branched-chain amino acids, although these data do
not negate a role for the other amino acids in the regulation of
protein translation.
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-ketoisocaproic acid (KIC),
dose-dependently mediated the increased formation of
PHAS-I. Leucine at concentrations of 2 mM and greater
resulted in enhanced phosphorylation of PHAS-I, whereas this same
effect was observed at a 10-fold lower concentration for KIC. The
effects of valine, isoleucine, and their metabolites,
-ketoisovalerate (KIV) and -keto--methylvalerate (KMV),
respectively, were also examined on PHAS-I phosphorylation. Although
both valine and isoleucine dose-dependently induced PHAS-I
phosphorylation (Fig. 4, A and B), the transamination products of valine, KIV, and
isoleucine, KMV, were ineffective in stimulating the phosphorylation of
PHAS-I (Fig. 4C, lanes 4 and 6). These latter
findings suggest that unlike the metabolite of leucine, KIC, either the
transport of KIV and KMV into -cells is impaired and/or their
metabolism is not sufficient to induce phosphorylation of PHAS-I.
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Branched-chain Amino Acids Stimulate Phosphorylation of PHAS-I
Independent of Insulin Secreted by -Cells--
Since amino acids
are secretagogues for insulin secretion by -cells, we next
determined whether endogenous insulin secreted by RINm5F cells is
involved in amino acid-induced phosphorylation of PHAS-I. In the first
approach, RINm5F cells were treated with KRBB-containing branched-chain
amino acids (leucine, isoleucine, and valine at 3.3 mM for
each amino acid) ± genistein (10 µM) or herbimycin A (1 µM), two tyrosine kinase inhibitors, with different mechanisms of action, in the upstream insulin signaling pathway (28,
29). As shown in Fig. 5A,
branched-chain amino acids stimulated phosphorylation of PHAS-I
(lane 3), in comparison to the absence of amino acids
(lane 1) and a complete complement of amino acids
(lane 2). This effect was not prevented by either genistein
or herbimycin A (lanes 4 and 5). Similar results
were obtained with genistein at concentrations of 1 and 100 µM (data not shown). Genistein and herbimycin A are
effective tyrosine kinase inhibitors in islets and RINm5F cells.
Previously we have shown that these inhibitors block
interleukin-1-stimulated tyrosine kinase activation, resulting in the
inhibition of NF
B translocation to the nucleus and blockade of
subsequent transcription and translation of the inducible nitric oxide
synthase gene by rat islets and RINm5F cells (30, 31). These data
suggest that branched-chain amino acids induce phosphorylation of
PHAS-I independent of the upstream insulin signaling pathway by
-cells.
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-cells by reducing the incubation temperature
from 37 °C (lanes 1-3) to 24 °C (lanes
4-6) did not attenuate the ability of branched-chain amino acids
to induce PHAS-I formation (compare lanes 5 and
6 with lanes 2 and 3). In these
studies (Fig. 5B), insulin secretion levels in the absence
of amino acids were 3.2 ± 0.1 nM at 37 °C
(lane 1) and 2.1 ± 0.2 nM at 24 °C
(lane 4). At a concentration of 1× amino acids, insulin
levels were 6.1 ± 0.9 nM at 37 °C (lane
2) and 2.4 ± 0.7 nM at 24 °C (lane
5). Insulin secretion levels with branched-chain amino acids (10 mM) were 8.6 ± 2.2 nM at 37 °C
(lane 3) and 2.5 ± 0.3 nM at 24 °C (lane 6). These results suggest that endogenous insulin
secretion exerted no effect on amino acid-induced phosphorylation of
PHAS-I. Taken together, these findings support the concept that
phosphorylation of PHAS-I stimulated by amino acids is independent of
endogenous insulin secretion by -cells.
Branched-chain Amino Acids Induce Phosphorylation of PHAS-I via the
mTOR Pathway--
Although several studies suggest that PHAS-I is
phosphorylated in cell-free preparations by a variety of kinases such
as casein kinase II, protein kinase C, and mitogen-activated protein
kinase (24), recent studies suggest that mTOR (mammalian target of rapamycin) is the predominant PHAS-I kinase (13-15). In addition, mTOR
appears to regulate phosphorylation of PHAS-I and p70s6k in
a parallel manner (11, 18, 19). In exploring the signaling pathway
involved in amino acid-induced phosphorylation of PHAS-I, RINm5F cells
were exposed to branched-chain amino acids (leucine, isoleucine, and
valine at 3.3 mM for each amino acid) in the presence or
absence of the mTOR inhibitor, rapamycin (25 nM), and the
PI-3 kinase inhibitor, wortmannin (100 nM), for 30 min. As
shown in Fig. 6, branched-chain amino
acids induced the formation of PHAS-I (lane 3), in
comparison to the absence of amino acids (lane 1) and a
complete complement of amino acids (lane 2). PHAS-I is also observed in lane 3 which may be attributed to the
presence of valine in the branched-chain amino acid mixture. This
increased phosphorylation of PHAS-I by branched-chain amino acids was
blocked by both rapamycin and wortmannin (lanes 4 and
5).
|
Amino Acid-induced Phosphorylation of PHAS-I in Pancreatic
Islets--
Studies were performed next to determine if amino
acid-induced phosphorylation of PHAS-I in isolated pancreatic islets
was similar to that described in the -cell line, RINm5F. This
experimental protocol was performed over an incubation period of 3 h as shown in Fig. 7A to allow
for more complete uptake and oxidation of leucine by isolated islets
(25, 26). Under these conditions, an increase in glucose concentration
from 3 to 20 mM (Fig. 7A, lanes 1 and
2) in the absence of amino acids resulted in a shift of
PHAS-I from to and to some extent formation of PHAS-I. This
small increase in the formation of PHAS-I in the absence of added
amino acids is believed to result from proteolysis and release of amino
acids intracellularly due to the total incubation period of 5 h
without exogenous amino acids. In all previous studies conducted for 30 min following a 2-h incubation without amino acids, the formation of
PHAS-I was not observed by islets or -cells. The presence of 10 mM leucine in combination with 20 mM glucose
facilitated a significant shift of PHAS-I to the -form which was
also accompanied by enhanced insulin secretion (Fig. 7, A
and B, lanes 5). These findings indicate that amino acids synergize with insulin similar to RINm5F cells to fully activate this
signal transduction pathway. Furthermore, leucine alone stimulates some
PHAS-I formation under conditions of only basal insulin secretion
(Fig. 7, A and B, lanes 4), suggesting that
leucine alone also can stimulate PHAS-I phosphorylation similar to that observed with RINm5F cells.
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and loss of PHAS-I (Fig.
7A, lanes 6 and 7), confirming the above findings
that leucine alone independent of insulin secretion can stimulate
PHAS-I phosphorylation in islets. Interestingly, leucine in the
presence of 20 mM glucose at 24 °C (Fig. 7A, lane
8), conditions where enhanced basal insulin secretion is observed
(Fig. 7B, lane 8), resulted in significant formation of
PHAS-I. The explanation for the ability of leucine to convert PHAS-I
to fully phosphorylated PHAS-I under these conditions is believed to
be due to the presence of a threshold level of insulin accumulated,
approximately 3-fold over basal levels, over this 3-h incubation period
which synergizes with amino acids to shift PHAS-I to PHAS-I.
These findings indicate the following: 1) both isolated islets and the
-cell line, RINm5F, require amino acids to facilitate
glucose-stimulated phosphorylation of PHAS-I; 2) amino acids mediate
PHAS-I phosphorylation independent of endogenous insulin secretion; and
3) amino acids also synergize with insulin to further enhance PHAS-I
phosphorylation.
IGF-I Requires Amino Acids to Stimulate Phosphorylation of
PHAS-I--
Our previous studies indicated that amino acids are
essential for insulin to induce phosphorylation of PHAS-I, suggesting a
critical role for amino acids in facilitating this effect (20). To
define further whether the requirement for amino acids to facilitate PHAS-I phosphorylation may represent a general phenomenon for other
growth factors, we evaluated the effect of insulin-like growth factor I
(IGF-I) on PHAS-I phosphorylation. In this design, RINm5F cells were
incubated with KRBB ± IGF-I (1-100 nM) supplemented with amino acids (0. 1×) for 30 min. As shown in
Fig. 8, IGF-I (100 nM) alone
is ineffective in inducing phosphorylation of PHAS-I (lane
3) in comparison to the absence of amino acids (lane 1) and a complete complement of amino acids (lane 2). In this
case, the absence of amino acids results in PHAS-I and some being detected. The amount of PHAS-I detected, as stated earlier
(see Fig. 1), can vary dependent on the level of quiescence of the cells. However, IGF-I (1-100 nM) plus amino acids (0. 1×)
caused a dose-dependent enhancing effect on formation of
PHAS-I (lanes 5-8). These results indicate that a
requirement for amino acids in PHAS-I phosphorylation may be essential
for other -cell growth factors in addition to insulin and IGF-I to
activate this signaling pathway. These results further raise the
possibility that amino acids facilitate the proliferative effect of
growth factors such as insulin and IGF-I by inducing the
phosphorylation of PHAS-I.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Our previous studies indicated that a complete complement of amino
acids normally present in tissue culture medium stimulated the
phosphorylation of PHAS-I in -cells (20). An initial focus of the
present study indicated that essential amino acids in comparison to
non-essential amino acids were most effective in inducing the phosphorylation of PHAS-I. However, these findings do not necessarily preclude the possibility that non-essential amino acids also mediate to
a lesser extent the phosphorylation of PHAS-I since some shift of
PHAS-I to more phosphorylated forms was observed in RINm5F cells
treated with non-essential amino acids in comparison to the absence of
amino acids. This possibility is in agreement with a recent report
indicating that non-essential amino acids (alanine and glycine)
significantly increased cell proliferation in bovine embryos cultured
in a chemically defined medium (34).
Among all essential amino acids tested, the branched-chain amino acids
(leucine, valine, and isoleucine), as a group, were the most effective
in stimulating the phosphorylation of PHAS-I. To define the mechanism
by which branched-chain amino acids stimulated the phosphorylation of
PHAS-I, the effects of their metabolites on PHAS-I phosphorylation were
evaluated. These studies demonstrated that the transamination product
of leucine, -ketoisocaproic acid (KIC), dose-dependently
mediated the increased phosphorylation of PHAS-I. This suggested that
the transport and metabolism of leucine is required although the
possibility exists that leucine independent of its metabolism may also
contribute to the phosphorylation of PHAS-I. However, unlike KIC, the
transamination products of valine, -ketoisovalerate (KIV), and
isoleucine, -keto--methylvalerate (KMV), were ineffective in
stimulating the phosphorylation of PHAS-I, indicating that either the
transport of KIV and KMV into -cells and/or their metabolism is
insufficient to induce phosphorylation of PHAS-I. Even though it is
likely that if isoleucine and valine were transported into -cells,
these branched-chain amino acids would be converted to their -keto
transamination products in a manner similar to leucine. In support of
this possibility, previous reports have indicated that a single enzyme,
branched-chain aminotransferase, catalyzes the reversible
transamination of all the branched-chain amino acids, leucine,
isoleucine, and valine to their respective -keto acids (35, 36).
Since valine and to a lesser extent isoleucine stimulated PHAS-I
phosphorylation as shown in Fig. 4, it is reasonable to assume that
exogenous KIV and KMV probably did not gain adequate entry into
-cells in this in vitro experimental design.
Amino acids are potent stimuli for insulin secretion from -cells.
For example, leucine as previously discussed serves as an insulin
secretagogue under some conditions and also potentiates glucose-stimulated insulin secretion from -cells (37, 38). Although
the amino acids arginine, lysine, and isoleucine alone are not insulin
secretagogues, these amino acids potentiate glucose-stimulated insulin
secretion by -cells (39). Amino acids also stimulate glucagon
secretion from -cells which may in turn trigger insulin secretion
from -cells (40). Multiple approaches were employed in the present
study to assess whether amino acid-induced phosphorylation of PHAS-I
was due to endogenous insulin secretion by -cells. Both inhibitors
of tyrosine kinase activity and reduction of the incubation temperature
from 37 to 24 °C, conditions which block almost completely
secretagogue-induced insulin exocytosis without significantly
inhibiting -cell metabolism, failed to attenuate the ability of
branched-chain amino acids to induce PHAS-I phosphorylation. These
findings support the concept that amino acid-induced phosphorylation of
PHAS-I is independent of insulin secretion by -cells. Moreover, a
recent study by Patti et al. (41) has also demonstrated that high physiologic concentrations of amino acids stimulate PHAS-I phosphorylation in cultured hepatoma cells.
In insulin-sensitive cells, insulin and other growth factors including
IGF-I mediate PHAS-I phosphorylation via a signaling pathway involving
activation of IRS-I and IRS-2 and also PI 3-kinase (16). Both PHAS-I
and p70s6k are located downstream in this insulin signaling
pathway and are regulated by mTOR in a parallel manner. More recently,
studies have indicated that the serine/threonine kinase, Akt (protein kinase B), is required for insulin and growth factors to mediate the
phosphorylation of PHAS-I (42, 43). Akt is proposed to be located
downstream of PI 3-kinase and upstream of mTOR and functions in concert
with mTOR to phosphorylate PHAS-I (17). In the present study, the
signaling pathway involved in amino acid-induced phosphorylation of
PHAS-I was evaluated by treating RINm5F cells with branched-chain amino
acids in the absence or presence of the mTOR inhibitor, rapamycin (25 nM), and PI 3-kinase inhibitor, wortmannin (100 nM). These studies indicated that rapamycin (25 nM) effectively blocked phosphorylation of both PHAS-I and p70s6k based on gel mobility shift assays following
exposure to branched-chain amino acids. In a similar manner, the PI
3-kinase inhibitor, wortmannin (100 nM), also prevented
amino acid-induced phosphorylation of PHAS-I and p70s6k.
Although the ability of rapamycin to block amino acid-induced phosphorylation of PHAS-I and p70s6k is predicted by its
inhibitory effect on mTOR, an explanation for similar inhibitory
effects produced by the PI 3-kinase inhibitor, wortmannin, is more
complex. Although it is possible that wortmannin also inhibits mTOR
under these conditions, the concentration of wortmannin (100 nM) used in the present studies is significantly lower than
that required for inhibition of mTOR activity (12). Alternatively,
Patti et al. (41) recently reported that the PI 3-kinase
inhibitor, wortmannin, also abolished amino acid activation of
p70s6k in hepatoma cells at concentrations well below that
required for inhibition of mTOR. These authors concluded that both mTOR and other wortmannin-sensitive kinases including PI 3-kinase are involved in amino acid-induced phosphorylation of PHAS-I in this hepatoma cell line. These latter findings are also consistent with our
results indicating that branched-chain amino acids induce PHAS-I
phosphorylation in a rapamycin- and wortmannin-sensitive manner in
-cells. Although neither the addition of amino acids with cultured
hepatoma cells (41) nor their removal with CHO-IR cells (44) altered
protein kinase B/Akt activity, the effects of branched-chain amino
acids on Akt activity in -cells has not yet been evaluated.
Since amino acids have been shown to exert a mitogenic effect on
-cells (1, 2) and to also increase the release of insulin and
insulin-like growth factors in vivo, we evaluated the
ability of insulin-like growth factor I (IGF-I) to mediate the
phosphorylation of PHAS-I. These studies demonstrated that IGF-I
similar to insulin stimulated PHAS-I phosphorylation in an amino
acid-dependent manner. This ability of IGF-I to stimulate PHAS-I phosphorylation is consistent with a recent study indicating that IGF-I-induced DNA synthesis in RINm5F cells is associated with the
phosphorylation of IGF-I receptors and IRS-2 (45). The specific
mechanism whereby amino acids facilitate both insulin and IGF-I-induced
phosphorylation of PHAS-I in -cells is not presently known. Our
studies also indicated that IGF-I-like insulin synergizes with amino
acids to enhance further PHAS-I phosphorylation in -cells. It is
possible that this synergistic effect of insulin and IGF-I in
combination with amino acids is due to an increase in the transport
and/or metabolism of amino acids by -cells.
These findings clearly emphasize the importance of branched-chain amino
acids alone and in combination with insulin, IGF-I, and possibly other
growth factors to mediate protein synthesis, -cell growth, and
proliferation under different nutritional and disease states, including
diabetes mellitus (35). These results may explain, in part, the ability
of branched-chain amino acids, in particular leucine to increase
protein turnover in muscle (46, 47). A novel aspect of the present
study is the demonstration that both insulin-secreting -cells and
classical insulin target cells (skeletal muscle, adipocytes, and liver)
utilize similar if not identical signaling pathways proposed to
up-regulate protein translation, cell cycle progression, and cell
proliferation. A more fundamental understanding of the striking
similarities in these critical signal transduction pathways in
-cells and insulin target cells may aid in defining common signaling
defects responsible for impaired insulin secretion and the development
of insulin resistance associated with diabetes mellitus.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Joan Fink for excellent technical assistance; the Washington University Tissue Culture Support Center; and the Radioimmunoassay Core Facility of the Washington University Diabetes Research and Training Center.
| |
FOOTNOTES |
|---|
* This study was supported by an American Diabetes Association Research Grant (to M. L. M.), an American Diabetes Association Mentor-based Fellowship (to G. X.), National Institutes of Health Grants DK06181 (to M. L. M.), DK52753, DK28312, and AR41180 (to J. C. L., Jr.).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.
¶ To whom correspondence should be addressed: Dept. of Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110-1093. Tel.: 314-362-7435; Fax: 314-362-4096; E-mail: mcdaniel{at}pathology.wustl.edu.
The abbreviations used are:
IGF-I, insulin-like
growth factor-I; eIF, eukaryotic initiation factor; PI 3-kinase, phosphoinositide 3-kinase; TOR, target of rapamycin; mTOR, mammalian
target of rapamycin; KRBB, Krebs-Ringer bicarbonate buffer; KIC, -ketoisocaproic acid; KIV, -ketoisovalerate; KMV, -keto--methylvalerate; MEM, minimal essential medium; IRS, insulin receptor substrate; PHAS-I, phosphorylated heat- and
acid-stable protein regulated by insulin.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
- Swenne, I. (1992) Diabetologia 35, 193-201[CrossRef][Medline] [Order article via Infotrieve]
- Swenne, I. (1985) Diabetes 34, 803-807[Abstract]
- Morley, S. J. (1997) Biochem. Soc. Trans. 25, 503-509[Medline] [Order article via Infotrieve]
- Lawrence, J. C., Jr., and Abraham, R. T. (1997) Trends Biochem. Sci. 22, 345-349[CrossRef][Medline] [Order article via Infotrieve]
- Pain, V. M. (1996) Eur. J. Biochem. 236, 747-771[Medline] [Order article via Infotrieve]
- Proud, C. G. (1992) Curr. Top. Cell Regul. 32, 243-369[Medline] [Order article via Infotrieve]
-
Lin, T-A.,
Kong, X,
Haystead, T. A. J.,
Pause, A.,
Belsham, G.,
Sonenberg, N.,
and Lawrence, J. C., Jr.
(1994)
Science
266,
653-656
[Abstract/Free Full Text] - Pause, A., Belsham, G. J., Gingras, A-C., Donze, O., Lin, T-A., Lawrence, J. C., Jr., and Sonenberg, N. (1994) Nature 371, 762-767[CrossRef][Medline] [Order article via Infotrieve]
- Abraham, R. T., and Wiederrecht, G. J. (1996) Annu. Rev. Immunol. 14, 483-510[CrossRef][Medline] [Order article via Infotrieve]
-
Zheng, X-F.,
and Schreiber, S. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3070-3075
[Abstract/Free Full Text] -
Hara, K.,
Yonezawa, K.,
Kozlowski, M. T.,
Sugimoto, T.,
Andrabi, K.,
Weng, Q-P.,
Kasuga, M.,
Nishimoto, I.,
and Avruch, J.
(1997)
J. Biol. Chem.
272,
26457-26463
[Abstract/Free Full Text] - Brunn, G. J., Williams, J., Sabers, C., Wiederrecht, G., Lawrence, J. C., Jr., and Abraham, R. T. (1996) EMBO J. 15, 5256-5267[Medline] [Order article via Infotrieve]
-
Brunn, G. J.,
Hudson, C. C.,
Sekulic, A.,
Williams, J. M.,
Hosoi, H.,
Houghton, P. J.,
Lawrence, J. C., Jr.,
and Abraham, R. T.
(1997)
Science
277,
99-101
[Abstract/Free Full Text] -
Brunn, G. J.,
Fadden, P.,
Haystead, T. A. J.,
and Lawrence, J. C., Jr.
(1997)
J. Biol. Chem.
272,
32547-32550
[Abstract/Free Full Text] -
Burnett, P. E.,
Barrow, R. K.,
Cohen, N. A.,
Snyder, S. H.,
and Sabatini, D. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1432-1437
[Abstract/Free Full Text] - Myers, M. G., Jr., and White, M. F. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 615-658[CrossRef][Medline] [Order article via Infotrieve]
-
Scott, P. H.,
Brunn, G. J.,
Kohn, A. D.,
Roth, R. A.,
and Lawrence, J. C., Jr.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7772-7777
[Abstract/Free Full Text] -
Kawasome, H.,
Papst, P.,
Webb, S.,
Keller, G. M.,
Johnson, G. L.,
Gelfand, E. W.,
and Terada, N.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5033-5038
[Abstract/Free Full Text] - von Manteuffel, S. R., Dennis, P. B., Pullen, N., Gingras, A-C., Sonenberg, N., and Thomas, G. (1997) Mol. Cell. Biol. 17, 5426-5436[Abstract]
-
Xu, G.,
Marshall, C. A.,
Lin, T-A.,
Kwon, G.,
Munivenkatappa, R. B.,
Hill, J. R.,
Lawrence, J. C., Jr.,
and McDaniel, M. L.
(1998)
J. Biol. Chem.
273,
4485-4491
[Abstract/Free Full Text] -
Gazdar, A. F.,
Chick, W. L.,
Oie, H. K.,
Sims, H. L.,
King, D. L.,
Weir, G. C.,
and Lauris, V.
(1980)
Proc. Natl. Acad. Sci. U. S. A.
77,
3519-3523
[Abstract/Free Full Text] - Bhathena, S. J., Oie, H. K., Gazdar, A. F., Voyles, N. R., Wilkins, S. D., and Recant, L. (1982) Diabetes 31, 521-531[Abstract]
- McDaniel, M. L., Colca, J. R., Kotagal, N., and Lacy, P. E. (1983) Methods Enzymol. 98, 182-200[Medline] [Order article via Infotrieve]
-
Haystead, T. A. J.,
Haystead, C. M. M.,
Hu, C.,
Lin, T-A.,
and Lawrence, J. C., Jr.
(1994)
J. Biol. Chem.
269,
23185-23191
[Abstract/Free Full Text] -
Hellman, B.,
Sehlin, J.,
and Täljedal, I-B.
(1971)
Endocrinol.
89,
1432-1439
[Abstract/Free Full Text] - Hellman, B., Sehlin, J., and Täljedal, I-B. (1971) Biochem. J. 123, 513-521[Medline] [Order article via Infotrieve]
-
Prentki, M.,
and Renold, A. E.
(1983)
J. Biol. Chem.
258,
14239-14244
[Abstract/Free Full Text] -
Akiyama, T.,
Ishida, J,
Nakagawa, S.,
Ogawara, H.,
Watanabe, S.,
Itoh, N.,
Shibuya, M.,
and Fukami, Y.
(1987)
J. Biol. Chem.
262,
5592-5595
[Abstract/Free Full Text] -
Rao, G. N.,
Delafontaine, P.,
and Runge, M. S.
(1995)
J. Biol. Chem.
270,
27871-27875
[Abstract/Free Full Text] -
Corbett, J.,
Kwon, G. K.,
Misko, T. P.,
Rodi, C. P.,
and McDaniel, M. L.
(1994)
Am. J. Physiol.
267,
C48-C54
[Abstract/Free Full Text] - Kwon, G., Corbett, J. A., Rodi, C. P., Sullivan, P., and McDaniel, M. L. (1995) Endocrinology 136, 4790-4795[Abstract]
-
Escolar, J. C.,
Hoo-Paris, R.,
Castex, C.,
and Sutter, B. C. J.
(1987)
J. Endocrinol.
115,
225-231
[Abstract/Free Full Text] -
Renström, E.,
Eliasson, L.,
Bokvist, K.,
and Rorsman, P.
(1996)
J. Physiol. (Lond.)
494,
41-52
[Abstract/Free Full Text] - Lee, E-S., and Fukui, Y. (1996) Biol. Reprod. 55, 1383-1389[Abstract]
- May, M. E., and Buse, M. G. (1989) Diabetes Metab. Rev. 5, 227-245[Medline] [Order article via Infotrieve]
-
Davoodi, J.,
Drown, P. M.,
Bledsoe, R. K.,
Wallin, R.,
Reinhart, G. D.,
and Hutson, S. M.
(1998)
J. Biol. Chem.
273,
4982-4989
[Abstract/Free Full Text] - Tsuruzoe, K., Araki, E., Furukawa, N., Shirotani, T., Matsumoto, K., Kaneko, K., Motoshima, H., Yoshizato, K., Shirakami, A., Kishikawa, H., Miyazaki, J-I., and Shichiri, M. (1998) Diabetes 47, 621-631[Abstract]
- Milner, R. D. G. (1969) Lancet 1, 1075-1076[Medline] [Order article via Infotrieve]
- Floyd, J. C., Jr., Fajans, S. S., Conn, J. W., Knopf, R. F., and Rull, J. (1966) J. Clin. Invest. 45, 1487-1502
-
Kuhara, T.,
Ikeda, S.,
Ohneda, A.,
and Sasaki, Y.
(1991)
Am. J. Physiol.
260,
E21-E26
[Abstract/Free Full Text] - Patti, M-E., Brambilla, E., Luzi, L., Landaker, E. J., and Kahn, C. R. (1998) J. Clin. Invest. 101, 1519-1529[Medline] [Order article via Infotrieve]
- Mendez, R., Kollmorgen, G., White, M. F., and Rhoads, R. E. (1997) Mol. Cell Biol. 17, 5184-5192[Abstract]
-
Gingras, A. C.,
Kennedy, S. G.,
O'Leary, M. A.,
Sonenberg, N.,
and Hay, N.
(1998)
Genes Dev.
12,
502-513
[Abstract/Free Full Text] -
Hara, K.,
Yonezawa, K.,
Weng, Q-P.,
Kozlowski, M. T.,
Belham, C.,
and Avruch, J.
(1998)
J. Biol. Chem.
273,
14484-14494
[Abstract/Free Full Text] - Zhang, Q., Berggren, P-O., Hansson, A., and Tally, M. (1998) J. Endocrinol. 156, 573-581[Abstract]
- Buse, M. G., and Reid, S. S. (1975) J. Clin. Invest. 56, 1250-1261
- Li, J. B., and Jefferson, L. S. (1978) Biochim. Biophys. Acta 544, 351-359[Medline] [Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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V. C. Arantes, V. P. A. Teixeira, M. A. B. Reis, M. Q. Latorraca, A. R. Leite, E. M. Carneiro, A. T. Yamada, and A. C. Boschero Expression of PDX-1 Is Reduced in Pancreatic Islets from Pups of Rat Dams Fed a Low Protein Diet during Gestation and Lactation J. Nutr., October 1, 2002; 132(10): 3030 - 3035. [Abstract] [Full Text] [PDF]
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C. J. Lynch, S. M. Hutson, B. J. Patson, A. Vaval, and T. C. Vary Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E824 - E835. [Abstract] [Full Text] [PDF]
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C. J. Lynch, B. J. Patson, J. Anthony, A. Vaval, L. S. Jefferson, and T. C. Vary Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E503 - E513. [Abstract] [Full Text] [PDF]
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I.-H. Park, R. Bachmann, H. Shirazi, and J. Chen Regulation of Ribosomal S6 Kinase 2 by Mammalian Target of Rapamycin J. Biol. Chem., August 23, 2002; 277(35): 31423 - 31429. [Abstract] [Full Text] [PDF]
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 G. Xu, W. Zhang, M. K. Ma, and H. L. McLeod Human Carboxylesterase 2 Is Commonly Expressed in Tumor Tissue and Is Correlated with Activation of Irinotecan Clin. Cancer Res., August 1, 2002; 8(8): 2605 - 2611. [Abstract] [Full Text] [PDF]
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S. R. Kimball Regulation of Global and Specific mRNA Translation by Amino Acids J. Nutr., May 1, 2002; 132(5): 883 - 886. [Abstract] [Full Text] [PDF]
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M. K. Lingohr, L. M. Dickson, J. F. McCuaig, S. R. Hugl, D. R. Twardzik, and C. J. Rhodes Activation of IRS-2--Mediated Signal Transduction by IGF-1, but not TGF-{alpha} or EGF, Augments Pancreatic {beta}-Cell Proliferation Diabetes, April 1, 2002; 51(4): 966 - 976. [Abstract] [Full Text] [PDF]
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H. Tang, E. Hornstein, M. Stolovich, G. Levy, M. Livingstone, D. Templeton, J. Avruch, and O. Meyuhas Amino Acid-Induced Translation of TOP mRNAs Is Fully Dependent on Phosphatidylinositol 3-Kinase-Mediated Signaling, Is Partially Inhibited by Rapamycin, and Is Independent of S6K1 and rpS6 Phosphorylation Mol. Cell. Biol., December 15, 2001; 21(24): 8671 - 8683. [Abstract] [Full Text] [PDF]
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 J. Chillaron, R. Roca, A. Valencia, A. Zorzano, and M. Palacin Heteromeric amino acid transporters: biochemistry, genetics, and physiology Am J Physiol Renal Physiol, December 1, 2001; 281(6): F995 - F1018. [Abstract] [Full Text] [PDF]
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J. S. Greiwe, G. Kwon, M. L. McDaniel, and C. F. Semenkovich Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E466 - E471. [Abstract] [Full Text] [PDF]
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L. S. Jefferson and S. R. Kimball Amino Acid Regulation of Gene Expression J. Nutr., September 1, 2001; 131(9): 2460S - 2466. [Abstract] [Full Text] [PDF]
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 A.-C. Gingras, B. Raught, and N. Sonenberg Regulation of translation initiation by FRAP/mTOR Genes & Dev., April 1, 2001; 15(7): 807 - 826. [Full Text]
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J. C. Anthony, T. G. Anthony, S. R. Kimball, and L. S. Jefferson Signaling Pathways Involved in Translational Control of Protein Synthesis in Skeletal Muscle by Leucine J. Nutr., March 1, 2001; 131(3): 856S - 860. [Abstract] [Full Text]
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C. J. Lynch Role of Leucine in the Regulation of mTOR by Amino Acids: Revelations from Structure-Activity Studies J. Nutr., March 1, 2001; 131(3): 861S - 865. [Abstract] [Full Text]
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G. Xu, G. Kwon, W. S. Cruz, C. A. Marshall, and M. L. McDaniel Metabolic Regulation by Leucine of Translation Initiation Through the mTOR-Signaling Pathway by Pancreatic {beta}-Cells Diabetes, February 1, 2001; 50(2): 353 - 360. [Abstract] [Full Text]
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 E. HORNSTEIN, H. TANG, and O. MEYUHAS Mitogenic and Nutritional Signals Are Transduced into Translational Efficiency of TOP mRNAs Cold Spring Harb Symp Quant Biol, January 1, 2001; 66(0): 477 - 484. [Abstract] [PDF]
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L. J. Van Winkle Amino Acid Transport Regulation and Early Embryo Development Biol Reprod, January 1, 2001; 64(1): 1 - 12. [Abstract] [Full Text]
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J. E. Kim and J. Chen Cytoplasmic-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin-sensitive signaling and translation initiation PNAS, December 8, 2000; (2000) 11511898. [Abstract] [Full Text]
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S. R. Kimball, L. S. Jefferson, H. V. Nguyen, A. Suryawan, J. A. Bush, and T. A. Davis Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process Am J Physiol Endocrinol Metab, November 1, 2000; 279(5): E1080 - E1087. [Abstract] [Full Text] [PDF]
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O. J. Shah, J. C. Anthony, S. R. Kimball, and L. S. Jefferson 4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle Am J Physiol Endocrinol Metab, October 1, 2000; 279(4): E715 - E729. [Abstract] [Full Text] [PDF]
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J. C. Anthony, F. Yoshizawa, T. G. Anthony, T. C. Vary, L. S. Jefferson, and S. R. Kimball Leucine Stimulates Translation Initiation in Skeletal Muscle of Postabsorptive Rats via a Rapamycin-Sensitive Pathway J. Nutr., October 1, 2000; 130(10): 2413 - 2419. [Abstract] [Full Text]
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C. Jousse, A. Bruhat, M. Ferrara, and P. Fafournoux Evidence for Multiple Signaling Pathways in the Regulation of Gene Expression by Amino Acids in Human Cell Lines J. Nutr., June 1, 2000; 130(6): 1555 - 1560. [Abstract] [Full Text]
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B. K. Law, P. Norgaard, and H. L. Moses Farnesyltransferase Inhibitor Induces Rapid Growth Arrest and Blocks p70s6k Activation by Multiple Stimuli J. Biol. Chem., April 6, 2000; 275(15): 10796 - 10801. [Abstract] [Full Text] [PDF]
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T. C. Vary, L. S. Jefferson, and S. R. Kimball Role of eIF4E in stimulation of protein synthesis by IGF-I in perfused rat skeletal muscle Am J Physiol Endocrinol Metab, January 1, 2000; 278(1): E58 - E64. [Abstract] [Full Text] [PDF]
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 M. C. Lorenz, N. S. Cutler, and J. Heitman Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae Mol. Biol. Cell, January 1, 2000; 11(1): 183 - 199. [Abstract] [Full Text]
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M. E. Cardenas, N. S. Cutler, M. C. Lorenz, C. J. Di Como, and J. Heitman The TOR signaling cascade regulates gene expression in response to nutrients Genes & Dev., December 15, 1999; 13(24): 3271 - 3279. [Abstract] [Full Text]
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T. C. Vary, L. S. Jefferson, and S. R. Kimball Amino acid-induced stimulation of translation initiation in rat skeletal muscle Am J Physiol Endocrinol Metab, December 1, 1999; 277(6): E1077 - E1086. [Abstract] [Full Text] [PDF]
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R. E. Rhoads Signal Transduction Pathways That Regulate Eukaryotic Protein Synthesis J. Biol. Chem., October 22, 1999; 274(43): 30337 - 30340. [Full Text] [PDF]
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 J. Petrik, B. Reusens, E. Arany, C. Remacle, C. Coelho, J. J. Hoet, and D. J. Hill A Low Protein Diet Alters the Balance of Islet Cell Replication and Apoptosis in the Fetal and Neonatal Rat and Is Associated with a Reduced Pancreatic Expression of Insulin-Like Growth Factor-II Endocrinology, October 1, 1999; 140(10): 4861 - 4873. [Abstract] [Full Text]
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G. Kwon, G. Xu, C. A. Marshall, and M. L. McDaniel Tumor Necrosis Factor alpha -induced Pancreatic beta -Cell Insulin Resistance Is Mediated by Nitric Oxide and Prevented by 15-Deoxy-Delta 12,14-prostaglandin J2 and Aminoguanidine. A ROLE FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR gamma ACTIVATION AND iNOS EXPRESSION J. Biol. Chem., June 25, 1999; 274(26): 18702 - 18708. [Abstract] [Full Text] [PDF]
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A.-C. Gingras, S. P. Gygi, B. Raught, R. D. Polakiewicz, R. T. Abraham, M. F. Hoekstra, R. Aebersold, and N. Sonenberg Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism Genes & Dev., June 1, 1999; 13(11): 1422 - 1437. [Abstract] [Full Text]
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S. R. Kimball, L. M. Shantz, R. L. Horetsky, and L. S. Jefferson Leucine Regulates Translation of Specific mRNAs in L6 Myoblasts through mTOR-mediated Changes in Availability of eIF4E and Phosphorylation of Ribosomal Protein S6 J. Biol. Chem., April 23, 1999; 274(17): 11647 - 11652. [Abstract] [Full Text] [PDF]
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L. M. Dickson, M. K. Lingohr, J. McCuaig, S. R. Hugl, L. Snow, B. B. Kahn, M. G. Myers Jr., and C. J. Rhodes Differential Activation of Protein Kinase B and p70S6K by Glucose and Insulin-like Growth Factor 1 in Pancreatic beta -Cells (INS-1) J. Biol. Chem., June 8, 2001; 276(24): 21110 - 21120. [Abstract] [Full Text] [PDF]
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J. Rohde, J. Heitman, and M. E. Cardenas The TOR Kinases Link Nutrient Sensing to Cell Growth J. Biol. Chem., March 23, 2001; 276(13): 9583 - 9586. [Abstract] [Full Text] [PDF]
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J. E. Kim and J. Chen Cytoplasmic-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin-sensitive signaling and translation initiation PNAS, December 19, 2000; 97(26): 14340 - 14345. [Abstract] [Full Text] [PDF]
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