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J. Biol. Chem., Vol. 277, Issue 16, 13628-13634, April 19, 2002
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From the Division of Molecular Physiology, Medical Sciences
Institute/Wellcome Trust Biocentre Complex, Dow Street, University of
Dundee, Dundee DD1 5EH, United Kingdom
Received for publication, September 6, 2001, and in revised form, January 16, 2002
SAT1-3 comprise members of the recently cloned
family of System A transporters that mediate the sodium-coupled uptake
of short chain neutral amino acids, and their activity is regulated
extensively by stimuli such as insulin, growth factors, and amino acid
availability. In skeletal muscle, insulin stimulates System A activity
rapidly by a presently ill-defined mechanism. Here we demonstrate that insulin induces an increase in the plasma membrane abundance of SAT2 in
a phosphatidylinositol 3-kinase-dependent manner
and that this increase is derived from an endosomal compartment that is required for the hormonal activation of System A. Chloroquine, an
acidotropic weak base that impairs endosomal recycling of membrane proteins, induced a complete inhibition in the insulin-mediated stimulation of System A, which was associated with a loss in SAT2 recruitment to the plasma membrane. The failure to stimulate System A
and recruit SAT2 to the cell surface could not be attributed to a block
in insulin signaling, as chloroquine had no effect on the
insulin-mediated phosphorylation of protein kinase B or glycogen
synthase kinase 3 or upon insulin-stimulated GLUT4 translocation and
glucose transport. Our data indicate strongly that insulin increases
System A transport in L6 cells by stimulating the exocytosis of SAT2
carriers from a chloroquine-sensitive endosomal compartment.
The plasma membranes of mammalian cells possess multiple transport
systems for the cellular exchange of amino acids (1), and of the
classical amino acid transport systems described the best studied has
been the System A amino acid carrier, which mediates the sodium-coupled
uptake of short-chain neutral amino acids (e.g. alanine).
System A is expressed in many cell types. Its ability to mediate the
uptake of amino acids with N-methyl substitutions has
enabled the discrimination of System A from other amino acid transporters using N-methylaminoisobutyric acid
(Me-AIB)1 as a paradigm
substrate. Functional studies using this substrate have shown that
System A activity is highly pH-sensitive and that the carrier is
subject to both long- and short-term modulation (1). Numerous
functional studies have demonstrated, for example, that System A
activity is subject to adaptive up-regulation by amino acid deprivation
in multiple cell types and that this response is dependent upon gene
expression, since it is inhibited by cycloheximide and actinomycin D,
inhibitors of translation and transcription, respectively (recently
reviewed by us (2)). Whether such modulation results from the increased
expression of System A carriers or that of regulatory molecule(s)
capable of effecting changes in transporter activity has remained
unclear. However, the recent cloning of three isoforms of the
System A transporter (SAT, also known as amino acid transporter A
(ATA)), termed SAT1-3/ATA1-3 (3-6), has meant that the molecular
regulation of this transport system can now be investigated in detail.
The availability of molecular probes to System A has allowed us to
demonstrate recently that the adaptive increase in transport activity
triggered by amino acid deprivation stems from a selective
up-regulation in the expression of SAT2 protein in the plasma membrane
of muscle and fat cells (7). This finding is fully consistent with the observation of others that depriving human fibroblasts of amino acids
induces an increase in SAT2 mRNA levels (8).
In addition to adaptive regulation, System A can also be stimulated
acutely by hormones such as insulin (9-11), growth factors (12), and
cell stresses (11-13). These stimuli induce a rapid increase in the
Vmax of System A transport that does not rely upon synthesis of new carriers, but which is thought to involve the
acute modulation of carrier function effected by molecules participating in early insulin and growth factor signaling. We have
shown previously that the stimulatory effects of insulin and IGF-1 on
System A can be blocked by inhibitors of phosphatidylinositol 3-kinase
(PI3K) (10-11) and that expression of a constitutively active form of
protein kinase B (PKB), which lies downstream of PI3K in the insulin
signaling cascade, mimics the effect of insulin on this amino acid
transporter (14). Precisely how the hormonal activation of PI3K and PKB
are linked to an increase in functional System A activity is not yet
understood, but it is plausible that signaling from these molecules
either stimulates the activity of SAT proteins resident in the plasma
membrane or that additional SAT carriers are recruited to the cell
surface from an intracellular compartment. The concept of carrier
recruitment from intracellular compartments in response to insulin is
well established for the GLUT4 glucose transporter (15). However, it
remains currently unknown whether SAT proteins can be recruited to the
plasma membrane in a manner analogous to GLUT4. In an attempt to
address this issue, we have investigated the effects of the acidotropic
weak base, chloroquine (CQ), which impairs endosomal function and hence protein recycling (16, 17), and cytochalasin D (CD), which disrupts the
actin-dependent endocytosis of cell surface proteins (18,
19) on the insulin-mediated activation of System A and upon the
subcellular distribution of SAT2. We demonstrate, for the first time,
that disrupting endosomal trafficking results in the loss of
insulin-stimulated System A transport and that this correlates with a
failure to recruit the SAT2 System A carrier from endosomal membranes.
Materials--
Culture media ( Cell Culture--
Monolayers of L6 muscle cells were cultured to
the stage of myotubes as described previously (14-20) in Subcellular Fractionation of L6 Myotubes--
Subcellular
membranes from L6 myotubes were isolated as described previously
(21-24). Briefly, 2 h prior to fractionation, cells were washed,
and growth medium (containing 5 mM glucose) was
supplemented with chloroquine (100 µM). In some dishes,
insulin (100 nM) was added 30 min prior to cell harvesting;
control dishes received vehicle alone. At the end of the incubation
period, cells from five (15 cm) dishes were scraped off the plates with
a rubber policeman, pooled, and gently pelleted. The cell pellet was
resuspended in ice-cold buffer (250 mM sucrose, 20 mM Hepes, 5 mM NaN3, 2 mM EGTA, pH 7.4 plus 1 protease inhibitor tablet/50 ml) and
homogenized. The cellular homogenate was subjected to a series of
differential centrifugation steps to isolate crude cell membranes that
were subsequently fractionated on a discontinuous sucrose gradient (32, 40, and 50% sucrose by mass) at 210,000 × g for
2.5 h. Membranes from on top of the 32% sucrose cushion and those
at the 32/40% and 40/50% sucrose interfaces were recovered and their
protein content determined using the Bradford assay with bovine serum albumin as standard (25).
Subcellular Fractionation of Rat Skeletal Muscle--
Rat
skeletal muscle was excised from the hind limbs of male Sprague-Dawley
rats (250-300 g) and fractionated based on a procedure established by
Klip and co-workers (26). The procedure involves skeletal muscle
homogenization and a series of differential centrifugation steps that
allow isolation of crude muscle membranes. These were subsequently
fractionated on a discontinuous sucrose density gradient (25, 30, and
35%) as described previously (26, 27). This procedure resulted in the
separation of three distinct membrane bands; one band was
located above the 25% sucrose cushion representing membranes enriched
with plasma membranes (denoted as F25), and a second band separated on
top of the 30% sucrose layer (F30) contained membranes largely of
endosomal origin (27). A third band separated out on top of the 35%
sucrose layer (F35) which consisted of membranes largely of
intracellular origin that contain the insulin "recruitable" pool of GLUT4 glucose transporters (28). Protein content was determined
using the Bradford method (25).
SDS-PAGE and Immunoblotting--
Cell lysates (50 µg protein)
or membranes (20 µg protein) were subjected to SDS-PAGE and
immunoblotting as described previously (14). Separated proteins were
transferred onto nitrocellulose membranes and blocked with
Tris-buffered saline (pH 7.4) containing 5% milk protein and 0.05%
(v/v) Tween 20. Membranes were probed with polyclonal antibodies
against PKB, phospho-PKB Ser473, and phospho-GSK3 (used at
a final dilution of 1:1000), the SAT2 System A isoform (used at 1:3000
(7)), annexin II (used at 1:400, a gift from Prof. V. Gerke, University
of Munster, Munster, Germany), GLUT4 (final dilution 1:500), TfR
(1:1000), or a monoclonal antibody against the Measurement of System A Amino Acid Transport--
System A
activity was assayed by measuring the uptake of Me-AIB as described
previously (20). Briefly, L6 myotubes were incubated with 10 µM [14C]Me-AIB (1 µCi/ml) for 10 min.
Nonspecific tracer binding was determined using either
[3H]mannitol as an extracellular marker or by determining
cell-associated radioactivity in the presence of an excess saturating
dose of unlabeled Me-AIB (10 mM). In some experiments
glucose transport was measured simultaneously in the same population of
muscle cells used to assay System A activity by a dual label approach
(12). Uptake of 10 µM
2-deoxy-D-[3H]glucose (2DG; 1 µCi/ml, 26.2 Ci/mmol) was measured for 10 min. Carrier-mediated
glucose transport was determined by quantitating cell-associated
radioactivity in the presence of 10 µM cytochalasin B, an
inhibitor of facilitative glucose transporters (14). Uptake of both
Me-AIB and 2DG was determined by aspirating the radioactive medium,
followed by three successive washes in ice-cold isotonic saline
solution (0.9% NaCl, w/v). Cells were lysed in 0.05 M NaOH and the associated radioactivity determined by liquid scintillation counting. Total cell protein was determined by the method of Bradford (25).
Analysis of Cell Surface 125I-Transferrin
Binding--
The transferrin receptor is known to recycle
constitutively between the cell surface and an endosomal compartment.
To assess the effects of CQ and CD on endosomal recycling, cell surface transferrin (Tfn) binding in L6 cells was determined essentially as
described previously (29). Briefly, human Tfn (Sigma) was iodinated
(2.6 × 106 cpm/µg) in phosphate-buffered saline
(PBS) containing 1% bovine serum albumin (w/v), pH 7.4, with carrier
free Na125I (PerkinElmer Life Sciences) for 10 min.
Iodinated Tfn was isolated by passage through a Dowex-1 ion exchange column.
Muscle cells were incubated with CQ and CD for times and at
concentrations indicated in the figure legend (Fig. 2) prior to incubation for 30 min in MHB buffer (serum-free Statistical Analysis--
Statistical analysis for multiple
comparisons was performed using one-way analysis of variance (ANOVA)
followed by the Newman-Keuls test. Data analysis was performed
using GraphPad Prism software and considered statistically significant
at p values <0.05.
To investigate the effects of CQ on System A activity in L6
myotubes we first performed dose and time response studies with this
acidotropic agent. Muscle cells were preincubated with 100 µM CQ for up to 3 h, after which period basal and
insulin-stimulated Me-AIB uptake was assayed. In the absence of any
pretreatment with CQ, insulin increased Me-AIB uptake by ~65%, but
this stimulation was progressively lost upon pretreatment of cells with
CQ (Fig. 1A). Under these
conditions CQ also induced a modest reduction (~25%) in basal System
A transport activity (Fig. 1A). Insulin-stimulated System A
activity was virtually undetectable in cells pre-exposed to 100 µM CQ and was maximally suppressed following
preincubation with CQ for 2 h (Fig. 1, A-C). These
findings indicate that System A transporters may recycle constitutively
at the cell surface and that insulin stimulates System A by enhancing
its exocytosis from a CQ-sensitive endosomal compartment. If this
supposition is correct, then System A activity in the plasma membrane
will depend on the relative rates with which the carrier protein is exocytosed and endocytosed.
Studies in yeast and mammalian cells indicate strongly that actin
filaments and actin-based motor proteins play a key role in
receptor-mediated endocytosis (18-30). Thus, to examine further the
importance of exo- and endocytic events in the regulation of System A
activity, we investigated the effects of treating muscle cells with
cytochalasin D, a reagent that induces depolymerization of actin
filaments, in addition to CQ. To assess how each drug affects the
recycling of membrane proteins we monitored surface binding of
125I-transferrin as an index for plasma membrane
transferrin receptor (TfR) abundance, a paradigm marker protein that
recycles constitutively at the cell surface. Fig.
2 shows that surface Tfn binding was increased significantly (by 73%) following incubation of cells with
CD, whereas it fell by ~40% in cells that had been exposed to CQ
(Fig. 2). These findings are consistent with the view that CD and CQ
inhibit endocytosis and exocytosis of TfR, respectively. Previous work
using rat adipocytes has shown that the CQ-induced disruption in IGF-II
receptor recycling is reversible, suggesting that endosomal function
can be restored upon withdrawal of CQ from the incubation medium (31).
To test for whether this was also the case in L6 myotubes, muscle cells
were washed free of CQ, and Tfn binding was assayed during the
post-wash recovery period. Withdrawal of CQ from the incubation medium
for periods of up to 2 h led to a restoration in Tfn binding to a
level that was not significantly different from cells that had not been
exposed to CQ (Fig. 2).
Insulin Promotes the Cell Surface Recruitment of the SAT2/ATA2
System A Amino Acid Transporter from an Endosomal Compartment in
Skeletal Muscle Cells*
§,, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-minimal essential media,
-MEM), fetal calf serum, and antimycotic/antibiotic solution were
obtained from Invitrogen. Wortmannin, insulin, chloroquine, and
cytochalasin D were purchased from Sigma-Aldrich. Complete protease
inhibitor tablets were obtained from Roche Molecular Biochemicals.
Phospho-specific antibodies to PKB and GSK3 were obtained from New
England Biolabs (Hertfordshire, UK). Horseradish peroxidase conjugated
anti-rabbit IgG were obtained from Scottish Antibody Production Unit
(Carluke, Lanarkshire, UK). Hybond nitrocellulose membrane was obtained from Amersham Biosciences, and reagents for ECL were purchased from
Pierce & Warriner (Chester, UK).
-MEM
containing 2% (v/v) fetal calf serum and antimycotic/antibiotic
solution (100 units/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B) at 37 °C in an atmosphere of 5%
CO2, 95% air. Upon formation of myotubes, cells were
deprived of serum by incubating muscle cells in serum-free -MEM for
4 h followed by a 1 h incubation in amino acid-free
HEPES-buffered saline (HBS, 20 mM HEPES-Na (pH 7.4), 140 mM NaCl, 2.5 mM MgSO4, 5 mM KCl, 1 mM CaCl2). Additions (e.g. insulin, CQ, and cytochalasin D) to the cells were
made at times and at concentrations indicated in the figure legends.
1 subunit of the
Na,K-ATPase (Mck-1, used at 1:100, a gift from Dr K. Sweadner,
Laboratory of Membrane Biology, Massachusetts General Hospital,
Charlestown, MA). Following primary antibody incubation, membranes were
washed and then incubated with horseradish peroxidase conjugated
anti-rabbit IgG (1:1000). Immunoreactive protein bands were visualized
by enhanced chemiluminesence on Konica Medical film (Hohenbrunn, Germany).
-MEM medium, 20 mM HEPES (pH 7.4), 2 mg/ml bovine serum albumin) at
37 °C to deplete endogenous transferrin in the culture media. Cells
were subsequently incubated in MHB buffer containing 3 µg/ml
125I-Tfn (5 × 104 cpm/µg) for 60 min at
4 °C followed by three successive washes in ice-cold PBS and lysis
in 1% Triton X-100 prior to quantitating total cell-associated
radioactivity using a Beckman LS6000IC counter. To quantitate the
internalized radioactivity, the above protocol was repeated on a
duplicate set of cells, but this set was washed three times with PBS/25
mM glacial acetic acid (pH 4.2) following the initial three
washes with ice-cold PBS to strip surface-associated 125I-Tfn. The difference between the two samples gives an
indication of surface Tfn binding. Nonspecific surface binding was
corrected for by determining radioactivity associated with cells
incubated at 4 °C with MHB containing 3 µg/ml 125I-Tfn
with a 100-fold excess of unlabeled Tfn.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 1.
Effect of chloroquine on insulin-stimulated
System A amino acid transport in L6 myotubes. A, L6
myotubes were preincubated in the absence or presence of CQ (100 µM) for the times indicated prior to treatment with
(square) or without (circle) insulin (100 nM, 30 min). Uptake of Me-AIB (10 µM) was
then assayed as described under "Experimental Procedures."
B, muscle cells were preincubated with CQ for 2 h at
the concentrations indicated prior to treatment with
(square) or without (circle) insulin (100 nM, 30 min) and measurement of Me-AIB uptake. C,
depiction of net insulin-stimulated changes in Me-AIB uptake as a
function of CQ concentration. Results are the means ± S.E. of
three separate experiments, each performed in triplicate.
View larger version (20K):
[in a new window]
Fig. 2.
Effects of CD and CQ on cell surface Tfn
binding. L6 myotubes were preincubated in the absence or presence
of CD (2 µM, 2 h) or CQ (100 µM,
2 h) prior to analysis of cell surface 125I-Tfn
binding as described under "Experimental Procedures." In some
experiments, following pretreatment with CQ, cells were washed with PBS
and allowed to recover for the times indicated prior to quantitation of
surface Tfn binding. Results represent the mean ± S.E. for three
experiments, each performed in triplicate. The asterisks
represent a statistically significant change (p < 0.05) from the appropriate untreated control.
Having established that CD inhibits TfR internalization, we assessed
the effects of this drug on System A transport activity. Preincubation
of L6 myotubes with 2 µM CD alone induced a near 30%
stimulation in Me-AIB uptake (Fig. 3).
This increase most likely arises through a modest up-regulation in cell
surface System A carrier number that is associated with inhibition of
their internalization, whereas carrier exocytosis from the endosomal
compartment remains unaffected. This proposition is supported by the
finding that the CD-induced increase in System A activity did not take
place when muscle cells were treated with CQ just prior to incubation with CD (Fig. 3), under which conditions both endo- and exocytosis are
inhibited. Treatment of cells with insulin following incubation with CD
results in a further increase in System A activity over and above that
elicited by CD but to a level no greater than that observed in the
presence of insulin alone (Fig. 3) This finding provides further
support for the idea that exocytosis of carriers from an endosomal pool
is the mechanism by which insulin stimulates System A transport.
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PI3K has been implicated strongly in the control of both basal and insulin-stimulated exocytosis of recycling proteins such as TfR (32, 33). We hypothesized that if the stimulation of System A elicited by both CD and insulin relies upon carrier recruitment from an endosomal compartment, then it ought to be blocked by the PI3K inhibitor, wortmannin. Consistent with this idea, wortmannin prevented stimulation of Me-AIB uptake in response to CD and insulin (Fig. 3), and use of the structurally unrelated PI3K inhibitor, LY 294002, yielded similar results (data not shown).
The data presented in Fig. 2 indicate that endosomal function can be
restored slowly when muscle cells are washed and allowed to recover for
2 h following pretreatment with CQ. We postulated that if the
hormonal activation of System A was dependent on a functional endosomal
compartment, then the ability of insulin to stimulate System A activity
should also be recoverable during the post-wash period. Fig.
4 shows that CQ suppressed completely the
hormonal activation of System A but that this stimulation was restored
progressively in cells that had been washed "free" of the drug and
allowed to recover subsequently in media lacking CQ for up to 2 h.
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It is plausible that the inhibitory effects exerted by CQ on the
hormonal activation of System A might involve direct effects of the
drug on membrane transporters or through a blockade in insulin
signaling. To examine these possibilities we investigated the effects
of CQ on insulin-stimulated glucose transport and upon the
phosphorylation of two important insulin signaling intermediates, PKB
and glycogen synthase kinase-3 (GSK3) (34). Although CQ inhibited the
hormonal stimulation of System A transport, the drug had no apparent
effect on insulin-stimulated glucose transport when assayed in the same
cell population (Fig. 5A).
This observation is in agreement with previous work using adipocytes
showing that although CQ disrupts the recycling of IGF-II receptors, it
has little impact on insulin-stimulated glucose transport or upon transporter recruitment to the cell surface (31-35). These findings imply, first, that although a proportion of the insulin-regulated glucose transporter, GLUT4, localizes to recycling endosomes (36), the
effects of CQ on GLUT4 recycling are likely to be minimal. Second,
because CQ inhibits insulin-stimulated System A transport and
influences the recycling dynamics of TfR and the IGF-II receptor (31),
this indicates that such proteins are present in a distinct population
of endosomal vesicles from which GLUT4 is excluded. To assess the
possibility that CQ may have impaired insulin signaling, we monitored
the insulin-induced phosphorylation of PKB, an insulin signaling
intermediate implicated in the regulation of diverse end point
responses to the hormone such as glycogen synthesis (37), glucose
transport, and System A transport (14). Fig. 5B shows that
insulin promoted the phosphorylation of PKB and also that of its
downstream physiological target, GSK3. This finding is fully compatible
with the observed stimulation in glucose transport elicited by the
hormone (Fig. 5A) and indicates strongly that CQ is unlikely
to have inhibited activation of System A through suppression of early
insulin signaling events.
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Three SAT isoforms (SAT1-3, also known as ATA1-3) have
recently been cloned (3-6). Of these, SAT2 appears to be the most abundantly expressed isoform in skeletal muscle, whereas SAT3 expression occurs at a much lower level, and that of SAT1 is not detectable (6-7). Recent work from our laboratory has shown that SAT2
protein expression is enhanced in muscle and fat cells following a
period of amino acid deprivation, suggesting that changes in SAT2
expression are likely to underpin the adaptive increase in System A
activity (7). Moreover, the observation that SAT3 is a relatively poor
mediator of Me-AIB uptake compared with the other isoforms (38) implies
that SAT2 is the most likely isoform to mediate insulin-stimulated
Me-AIB uptake in muscle. To test this proposition, we investigated the
effects of insulin and chloroquine treatment on the subcellular
distribution of SAT2 in L6 myotubes. For these studies a 32, 40, and
50% (w/w) discontinuous sucrose gradient was utilized. The membranes
that float on top of the 32% sucrose cushion have been shown
previously to be enriched with plasma membrane (PM) markers such as the
1 subunit of the Na,K-ATPase (12-22). Membranes from the 32/40%
and 40/50% sucrose interfaces are depleted in PM markers but are
enriched with proteins of intracellular origin. Fig.
6A shows the relative
abundance of annexin II, a protein that is a major component of
fusogenic endosomal vesicles and thus a representative endosomal
marker, in the three membrane fractions. Annexin II was detected in all three membrane fractions, but its abundance was greatest in membranes recovered from the 32/40% sucrose gradient interface (Fig.
6A), indicating that this fraction was enriched with
endosomal membranes.
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Consistent with our recent work (7), SAT2 was detected in all three membrane fractions but was enriched in membranes recovered from the 32/40% sucrose interface (Fig. 6A). When SAT2 abundance was investigated in the three fractions following isolation of cells treated with insulin, we did not observe any changes in SAT2 content in membranes recovered from the 40/50% sucrose interface (data not shown). However, data from three separate experiments revealed that the hormone induced a greater than 2-fold increase in SAT2 in the PM-enriched fraction, Fig. 6B (this was associated with an increase in the Vmax of System A transport from 388 ± 10 pmol/min/mg to 584 ± 16 pmol/min/mg (p < 0.05), with no significant change in Km). The increase in cell surface SAT2 was associated with a concomitant reduction in SAT2 abundance in membranes recovered from the 32/40% sucrose interface (Fig. 6B). These findings imply that insulin promotes recruitment of SAT2 to the PM from the 32/40% fraction, which is enriched with endosomal membranes. Pretreatment of L6 cells with CQ for 2 h prior to subcellular fractionation reduced the amount of SAT2 recovered in the PM fraction, a finding consistent with the idea that this compound prevents the delivery of proteins to the cell surface from the endosomal compartment without affecting the internalization of PM proteins that are constitutively recycling at the cell surface. The loss in PM SAT2 was not recovered in any of the other membrane fractions isolated from the sucrose gradient, raising the possibility that, once internalized, SAT2 may have been routed for lysosomal degradation. Unlike SAT2, pretreatment of L6 cells with CQ had no detectable effect on the insulin-dependent recruitment of the GLUT4 glucose transporter to the PM (Fig. 6C). The increase in GLUT4 abundance elicited by insulin was, however, prevented by the PI3K inhibitor, wortmannin (Fig. 6C). These findings are consistent with the observation that CQ does not impair insulin-stimulated glucose transport (Fig. 5A) and with numerous reports showing that insulin-stimulated GLUT4 translocation requires PI3K activity (39).
Interestingly, analysis of SAT2 abundance in subcellular membrane fractions prepared from rat skeletal muscle indicate that, as in L6 cells, the carrier is present in membranes of endosomal origin (F30) based on enrichment of TfR (a marker of recycling endosomes). In contrast, SAT2 immunoreactivity was very low in membranes that harbor the insulin responsive GLUT4 pool (F35) (Fig. 6D). Indeed, in the present studies the carrier was only detected in this fraction upon prolonged exposure of autoradiographic film. Although SAT2 immunoreactivity was greatest in the PM (F25) fraction, it should be noted that the abundance of SAT2 in the F30 fraction is not insignificant, as the total protein recovery of this fraction is ~5-fold higher than that of PM enriched fraction (27). These findings thus raise the possibility that SAT2 may also be recruited to the sarcolemma of rat muscle from an endosomal pool as observed in our cell-based studies.
To further strengthen our assertion that SAT2 mediates
insulin-stimulated System A transport, we investigated (i) whether the
insulin-mediated increase in plasma membrane SAT2 was sensitive to
wortmannin, a PI3K inhibitor; and (ii) whether SAT2 translocation could
be rescued following CQ wash-out in a time-dependent
manner. Fig. 7A shows that the
increase in plasma membrane SAT2 elicited by insulin was suppressed by
prior treatment of cells with 100 nM wortmannin, a finding
consistent with the observation that wortmannin inhibits the hormonal
activation of System A transport (Fig. 3). In line with the data
presented in Fig. 4, we observed that CQ prevented the insulin-induced
increase in cell surface SAT2 but that cells "washed free" of CQ
displayed a time-dependent return of SAT2 recruitment (Fig.
7B). Taken together, the findings presented in Figs. 6 and 7
suggest strongly that SAT2 translocation underpins the functional
increase in System A transport that is observed in response to insulin
in L6 cells.
|
In summary, we have shown that treatment of skeletal muscle cells with
insulin increases System A amino acid transport due to the
translocation of SAT2 protein from an intracellular pool (which
contains a high abundance of the endosomal marker annexin II) to the
plasma membrane. CQ, a substance that disrupts the trafficking of
endosomal transferrin receptors to the plasma membrane, prevents both
the recruitment of SAT2 from this internal pool and the increase in
System A transport following insulin treatment. Furthermore, disrupting
actin microfilaments with CD moderately increases both System A
activity and transferrin binding, consistent with a role for the actin
network in endocytosis from the plasma membrane. Collectively these
results imply that insulin stimulates System A activity by increasing
the exocytosis of the SAT2 amino acid transporter from an intracellular
endosomal location to the plasma membrane. Our present working model
for the hormonal regulation of skeletal muscle System A transport is
shown in Fig. 8. Future experiments will
seek to determine the nature of the endosomal SAT2 vesicles in greater
detail and how the endosomal and cytoskeletal systems interact with
insulin signaling networks to regulate System A activity.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Gary Litherland and Graham Christie for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC), Medical Research Council, Diabetes UK, and The Wellcome Trust.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.
These authors contributed equally to the work reported.
§ Recipient of a BBSRC-SmithKline Beecham CASE (Co-Operative Awards in Science and Engineering) studentship.
¶ To whom correspondence should be addressed. Tel.: 44-1382-344969; Fax: 44-1382-345507; E-mail: h.s.hundal@dundee.ac.uk.
Published, JBC Papers in Press, February 7, 2002, DOI 10.1074/jbc.M108609200
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ABBREVIATIONS |
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The abbreviations used are:
Me-AIB, methylaminoisobutyric acid;
SAT, System A transporter;
PI3K, phosphoinositide 3-kinase;
PKB, protein kinase B;
GLUT, glucose
transporter;
CQ, chloroquine;
CD, cytochalasin D;
GSK3, glycogen
synthase kinase 3;
2DG, 2-deoxyglucose;
Tfn, transferrin;
TfR, transferrin receptor;
PBS, phosphate-buffered saline;
PM, plasma
membrane;
-MEM, -minimal essential media;
IGF, insulin-like
growth factor.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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|---|
| 1. |
Palacin, M.,
Estevez, R.,
Bertran, J.,
and Zorzano, A.
(1998)
Physiol. Rev.
78,
969-1054 |
| 2. |
Christie, G. R.,
Hyde, R.,
and Hundal, H. S.
(2001)
Curr. Opin. Clin. Nutr. Metab. Care
4,
425-431[CrossRef][Medline]
[Order article via Infotrieve] |
| 3. |
Varoqui, H.,
Zhu, H.,
Yao, D.,
Ming, H.,
and Erickson, J. D.
(2000)
J. Biol. Chem.
275,
4049-4054 |
| 4. |
Yao, D.,
Mackenzie, B.,
Ming, H.,
Varoqui, H.,
Zhu, H.,
Hediger, M. A.,
and Erickson, J. D.
(2000)
J. Biol. Chem.
275,
22790-22797 |
| 5. |
Sugawara, M.,
Nakanishi, T.,
Fei, Y. J.,
Huang, W.,
Ganapathy, M. E.,
Leibach, F. H.,
and Ganapathy, V.
(2000)
J. Biol. Chem.
275,
16473-16477 |
| 6. |
Sugawara, M.,
Nakanishi, T.,
Fei, Y. J.,
Martindale, R. G.,
Ganapathy, M. E.,
Leibach, F. H.,
and Ganapathy, V.
(2000)
Biochim. Biophys. Acta
1509,
7-13[Medline]
[Order article via Infotrieve] |
| 7. |
Hyde, R.,
Christie, G. R.,
Litherland, G. J.,
Hajduch, E.,
Taylor, P. M.,
and Hundal, H. S.
(2001)
Biochem. J.
355,
563-568[Medline]
[Order article via Infotrieve] |
| 8. |
Gazzola, R. F.,
Sala, R.,
Bussolati, O.,
Visigalli, R.,
Dall'Asta, V.,
Ganapathy, V.,
and Gazzola, G. C.
(2001)
FEBS Lett.
490,
11-14[CrossRef][Medline]
[Order article via Infotrieve] |
| 9. |
Guma, A.,
Testar, X.,
Palacin, M.,
and Zorzano, A.
(1988)
Biochem. J.
253,
625-629[Medline]
[Order article via Infotrieve] |
| 10. |
Tsakiridis, T.,
McDowell, H. E.,
Walker, T.,
Downes, C. P.,
Hundal, H. S.,
Vranic, M.,
and Klip, A.
(1995)
Endocrinology
136,
4315-4322[Abstract] |
| 11. |
McDowell, H. E.,
Eyers, P. A.,
and Hundal, H. S.
(1998)
FEBS Lett.
441,
15-19[CrossRef][Medline]
[Order article via Infotrieve] |
| 12. |
Hundal, H. S.,
Bilan, P. J.,
Tsakridis, T.,
Marette, A.,
and Klip, A.
(1994)
Biochem. J.
297,
289-295 |
| 13. |
Tramacere, M.,
Petronini, P. G.,
Severini, A.,
and Borghetti, A. F.
(1984)
Exp. Cell. Res.
151,
70-79[CrossRef][Medline]
[Order article via Infotrieve] |
| 14. |
Hajduch, E.,
Alessi, D. R.,
Hemmings, B. A.,
and Hundal, H. S.
(1998)
Diabetes
47,
1006-1013[Abstract] |
| 15. |
Holman, G. D.,
and Kasuga, M.
(1997)
Diabetologia
40,
991-1003[CrossRef][Medline]
[Order article via Infotrieve] |
| 16. |
Puertollano, R.,
and Alonso, M. A.
(1999)
Mol. Biol. Cell
10,
3435-3447 |
| 17. |
Sibille, J. C.,
Kondo, H.,
and Aisen, P.
(1989)
Biochim. Biophys. Acta
1010,
204-209[Medline]
[Order article via Infotrieve] |
| 18. |
Durrbach, A.,
Louvard, D.,
and Coudrier, E.
(1996)
J. Cell Sci.
109,
457-465[Abstract] |
| 19. |
Lamaze, C.,
Fujimoto, L. M.,
Yin, H. L.,
and Schmid, S. L.
(1997)
J. Biol. Chem.
272,
20332-20335 |
| 20. |
McDowell, H. E.,
Christie, G. R.,
Stenhouse, G.,
and Hundal, H. S.
(1995)
Am. J. Physiol.
269,
C1287-C1294 |
| 21. |
Mitsumoto, L.,
Burdett, E.,
Grant, A.,
and Klip, A.
(1991)
Biochem. Biophys. Res. Comm.
175,
652-659[CrossRef][Medline]
[Order article via Infotrieve] |
| 22. |
Bilan, P. J.,
Mitsumoto, Y.,
Ramlal, T.,
and Klip, A.
(1992)
FEBS Lett.
298,
285-290[CrossRef][Medline]
[Order article via Infotrieve] |
| 23. |
Hundal, H. S.,
Ramlal, T.,
Reyes, R.,
Leiter, L. A.,
and Klip, A.
(1992)
Endocrinology
131,
1165-1173[Abstract] |
| 24. |
McDowell, H. E.,
Walker, T.,
Hajduch, E.,
Christie, G.,
Batty, I. H.,
Downes, C. P.,
and Hundal, H. S.
(1997)
Eur. J. Biochem.
247,
306-313[Medline]
[Order article via Infotrieve] |
| 25. |
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve] |
| 26. |
Klip, A.,
Ramlal, T.,
Young, D. A.,
and Holloszy, J. O.
(1987)
FEBS Lett.
224,
224-230[CrossRef][Medline]
[Order article via Infotrieve] |
| 27. |
Aledo, J. C.,
Lavoie, L.,
Volchuk, A.,
Keller, S. R.,
Klip, A.,
and Hundal, H. S.
(1997)
Biochem. J.
325,
727-732 |
| 28. |
Douen, A.,
Burdett, E.,
Ramlal, T.,
Rastogi, S.,
Vranic, M.,
and Klip, A.
(1991)
Endocrinology
128,
611-616[Abstract] |
| 29. |
Zuk, P. A.,
and Elferink, L. A.
(1999)
J. Biol. Chem.
274,
22303-22312 |
| 30. |
Munn, A. L.,
Stevenson, B. J.,
Geli, M. I.,
and Riezman, H.
(1995)
Mol. Biol. Cell
6,
1721-1742[Abstract] |
| 31. |
Oka, Y.,
Kasuga, M.,
Kanazawa, Y.,
and Takaku, F.
(1987)
J. Biol. Chem.
262,
17480-17486 |
| 32. |
Shepherd, P. R.,
Soos, M. A.,
and Siddle, K.
(1995)
Biochem. Biophys. Res. Comm.
211,
535-539[CrossRef][Medline]
[Order article via Infotrieve] |
| 33. |
Jess, T. J.,
Belham, C. M.,
Thomson, F. J.,
Scott, P. H.,
Plevin, R. J.,
and Gould, G. W.
(1996)
Cell. Signal.
8,
297-304[CrossRef][Medline]
[Order article via Infotrieve] |
| 34. |
Cross, D. A. E.,
Alessi, D. R.,
Cohen, P.,
Andjelkovic, M.,
and Hemmings, B. A.
(1995)
Nature
378,
785-789[CrossRef][Medline]
[Order article via Infotrieve] |
| 35. |
Romanek, R.,
Sargeant, R.,
Paquet, M. R.,
Gluck, S.,
Klip, A.,
and Grinstein, S.
(1993)
Biochem. J.
296,
321-327 |
| 36. |
Livingstone, C.,
James, D. E.,
Rice, J. E.,
Hanpeter, D.,
and Gould, G. W.
(1996)
Biochem. J.
315,
487-495 |
| 37. |
Alessi, D. R.,
and Cohen, P.
(1998)
Curr. Opin. Genet. Dev.
8,
55-62[CrossRef][Medline]
[Order article via Infotrieve] |
| 38. |
Hatanaka, T.,
Huang, W.,
Ling, R.,
Prasad, P. D.,
Sugawara, M.,
Leibach, F. H.,
and Ganapathy, V.
(2001)
Biochim. Biophys. Acta
1510,
10-17[Medline]
[Order article via Infotrieve] |
| 39. |
Hajduch, E.,
Litherland, G. J.,
and Hundal, H. S.
(2001)
FEBS Lett.
492,
199-203[CrossRef][Medline]
[Order article via Infotrieve] |
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