From the Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland and ¶ INSERM U-338, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France
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ABSTRACT |
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ADP-ribosylation factors (ARFs) play important
roles in both constitutive and regulated membrane trafficking to the
plasma membrane in other cells. Here we have examined their role in
insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. These
cells express ARF5 and ARF6. ARF5 was identified in the soluble protein
and intracellular membranes; in response to insulin some ARF5 was observed to re-locate to the plasma membrane. In contrast, ARF6 was
predominantly localized to the plasma membrane and did not redistribute
in response to insulin. We employed myristoylated peptides
corresponding to the NH2 termini of ARF5 and ARF6 to investigate the function of these proteins. Myr-ARF6 peptide inhibited insulin-stimulated glucose transport and GLUT4 translocation by ~50%
in permeabilized adipocytes. In contrast, myr-ARF1 and myr-ARF5 peptides were without effect. Myr-ARF5 peptide also inhibited the
insulin stimulated increase in cell surface levels of GLUT1 and
transferrin receptors. Myr-ARF6 peptide significantly decreased cell
surface levels of these proteins in both basal and insulin-stimulated states, but did not inhibit the fold increase in response to insulin. These data suggest an important role for ARF6 in regulating cell surface levels of GLUT4 in adipocytes, and argue for a role for both
ARF5 and ARF6 in the regulation of membrane trafficking to the plasma membrane.
Insulin stimulates glucose disposal in peripheral tissues by
virtue of the expression of the GLUT4 glucose transporter isoform (1-3). In the absence of insulin, this transporter is intracellularly sequestered within the elements of the endosomal system, the
trans Golgi network and a specialized storage compartment
(4-7). Upon insulin stimulation or muscle contraction, GLUT4 is
re-distributed from these intracellular locations to the plasma
membrane, resulting in a dramatic increase in the rate of glucose entry
into these tissues (4-7). Several studies have suggested that the
insulin-stimulated translocation of GLUT4 to the plasma membrane is
mechanistically akin to the fusion of small synaptic vesicles with the
neuronal plasma membrane (reviewed in Ref. 2). This has been supported by the identification of a morphologically similar GLUT4 storage compartment within adipocytes, and by the identification of soluble N-ethylmaleimide-sensitive factor attachment protein
receptors (SNAREs)1 located
in GLUT4 vesicles (the v-SNAREs cellubrevin and vesicle-associated membrane protein 2) (8-11) which bind in a highly specific manner to
t-SNAREs located in the adipocyte plasma membrane (Syntaxin 4 and
Syndet) (8, 10, 12-14). Vesicle-associated membrane protein 2 has been
shown to be the predominant v-SNARE that targets small synaptic
vesicles to the pre-synaptic plasma membrane by interacting with the
cognate t-SNAREs, syntaxin1, and synaptosome-associated protein of 25 kDa (SNAP-25) found on the target membrane (15). Recent studies
implicating vesicle-associated membrane protein 2 in insulin-stimulated
GLUT4 translocation further strengthen the mechanistic parallels
between regulated exocytosis of small synaptic vesicles and the
insulin-stimulated movement of GLUT4-containing vesicles to the
adipocyte cell surface (9, 16).
ADP-ribosylation factors (ARFs) are a family of GTP-binding proteins
(17, 18). To date, 6 isoforms have been identified in mouse tissues
(19) which fall within three groups, ARFs1, -2 and -3 constitute group
I, ARFs4 and -5 group II and ARF6 is the sole member of group III
identified to date. ARF proteins have been proposed to play several
roles in the control of membrane traffic, including the formation of
secretory vesicles at the trans Golgi network, regulating
endosome-endosome fusion, and notably in regulating the fusion of
secretory vesicles with the plasma membrane in bovine adrenal medulla
cells (20-25). Given the importance of ARF proteins in regulated
membrane trafficking, we set out to identify which ARF isoforms were
expressed in murine 3T3-L1 adipocytes, how their distribution was
modulated by insulin, and whether they played a role in
insulin-stimulated GLUT4 translocation.
Here we show that adipocytes express ARF5 and ARF6, as determined by
immunoblotting with ARF-specific antibodies. ARF5 was observed to
exhibit modest re-distribution to the plasma membrane in response to
insulin. In contrast, ARF6 was predominantly located within plasma
membrane subcellular fractions, and its distribution was not altered by
insulin treatment. Using myristoylated ARF NH2-terminal
peptides to inhibit ARF action in permeabilized cells, we show that
myristoylated ARF6 peptide partially inhibits insulin-stimulated glucose transport and GLUT4 translocation, whereas ARF1- and
ARF5-myristoylated peptides were without effect. Insulin stimulates the
movement of other proteins to the cell surface, including the
transferrin receptor (TfR) and GLUT1 (26, 27). Strikingly, we observed marked inhibition of insulin-stimulated TfR and GLUT1 movement to the
cell surface in the presence of myristoylated ARF5 peptide, implying an
important role for ARF5 in the stimulated delivery of recycling
membrane proteins to the cell surface. Myristoylated ARF6 peptide
decreased the cell surface abundance of TfR and GLUT1 in both the basal
and insulin-stimulated states, but did not inhibit the ability of
insulin to increase cell surface levels of these proteins. These data
argue for an important role for ARF6 in regulating cell surface levels
of GLUT4 in adipocytes, and provide evidence for a role for both ARF5
and ARF6 in the regulation of membrane trafficking to the plasma membrane.
Materials--
Cell Culture--
3T3-L1 fibroblasts were grown and
differentiated into adipocytes exactly as described in Refs. 16 and 28.
Cells were used between passages 3 and 10 in all experiments, and
between days 8 and 13 after induction of differentiation. Prior to use,
cells were incubated in serum-free Dulbecco's modified Eagle's medium for 2 h.
Subcellular Fractionation of Adipocytes--
Adipocytes were
subjected to a differential centrifugation procedure as described
previously (28, 29). Briefly, cells were scraped and homogenized in
ice-cold HES (20 mM HEPES, 1 mM EDTA, 255 mM sucrose, pH 7.4, 5 ml/10-cm plate) containing protease inhibitors (1 µg/ml pepstatin A, 0.2 mM diisopropyl
fluorophosphate, 20 µM
L-transepoxysuccinyl-leucylamido-4-guanidiniobutane, and 50 µM aprotinin). The homogenate was centrifuged at
19,000 × g for 20 min at 4 °C. The pellet from this
spin was resuspended in 2 ml HES, layered onto 1 ml of 1.12 M sucrose in HES, and centrifuged at 100,000 × g for 1 h at 4 °C in a swing-out rotor. Plasma
membranes were collected from the interface by careful aspiration,
resuspended in HES, and collected by centrifugation at 41,000 × g for 20 min at 4 °C. The supernatant from the
19,000 × g spin was re-centrifuged at 41,000 × g to yield a high density microsomal pellet and the supernatant from this spin centrifuged at 180,000 × g
for 75 min at 4 °C to collect low density microsomes. All fractions
were resuspended in equal volumes of HES buffer (cell equivalents), snap frozen in liquid nitrogen, and stored at Permeabilization of 3T3-L1 Adipocytes--
3T3-L1 adipocytes
were washed twice with IC buffer (10 mM NaCl, 20 mM Hepes, 50 mM KCl, 2 mM
K2HPO4, 90 mM potassium glutamate, 1 mM MgCl2, 4 mM EGTA, 2 mM CaCl2, pH 7.4) at 37 °C, then incubated in 500 µl of ICR buffer (IC buffer plus 4 mM MgATP, 3 mM sodium pyruvate, 100 µg/ml bovine serum albumin, pH
7.4) containing Deoxyglucose Transport Measurements--
Plasma Membrane Lawn Assays for GLUT4 Translocation--
After
experimental manipulations, coverslips of adipocytes were rapidly
washed in ice-cold buffer for the preparation of plasma membrane lawns
exactly as described in Ref. 9. After fixation in paraformaldehyde,
plasma membrane lawns were incubated with anti-GLUT-4 (1:100 dilution)
antibodies for 1 h at room temperature (9). After washing, the
coverslips were then incubated with fluorescein
isothiocyanate-conjugated donkey anti-rabbit IgG, washed and mounted on
glass coverslips. Coversips were viewed using a × 40 objective
lens on a Zeiss Axiovert microscope operated in Laser Scanning Confocal
mode. Samples were illuminated at 488 nm and the signal at 510 nm
collected. Duplicate coverslips were prepared at each experimental
condition, and 10 random images of plasma membrane lawns collected from
each. These were quantified using MetaMorph (Universal Imaging, CA)
software on a DAN PC (Noran Instruments, Surrey, UK). Similar methods
were employed to assay GLUT1 levels in plasma membranes.
Cell Surface Transferrin Receptor Binding
Assays--
Transferrin receptors present at the cell surface were
quantified as outlined in Refs. 27 and 31. After experimental
manipulations, cells were rapidly chilled by three washes in ice-cold
KRP buffer (136 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO4, 1.25 mM
CaCl2, 5 mM NaH2PO4, pH
7.4) containing 1 mg/ml bovine serum albumin. Thereafter, cells were
incubated in the same buffer containing ~3 nM
125I-transferrin for 2 h on ice. After this time, the
media was aspirated and the monolayers washed three times with 1 ml of
KRP/bovine serum albumin for 1 min. Cells were then solubilized in 1 M NaOH and the radioactivity associated with each well
determined by Peptides and Antibodies--
The peptides employed in this study
were prepared either by Thistle Research (Glasgow, UK) or as outlined
in Ref. 22. The sequences of the peptides used is shown in Table
I. Antibodies to ARF5 were provided by
Dr. J. Moss (NHLBI, National Institutes of Health, Bethesda, MD) and
Dr. R. A. Kahn (Atlanta, GA) (17, 32). Antibodies to ARF6 were
provided by Dr. J. Donaldson (NHLBI, National Institutes of Health,
Bethesda, MD) (33). Anti-GLUT1 was generously provided by Professor
Geoff Holman (University of Bath).
Expression and Distribution of ARF5 and ARF6 in 3T3-L1
Adipocytes--
We have used a panel of ARF-specific antibodies to
examine the expression and subcellular distribution of ARFs 5 and 6 in 3T3-L1 adipocytes (Fig. 1). In the basal
(non-stimulated) state, ARF5 was predominantly localized to the soluble
protein fraction of adipocytes, with some association with
intracellular membranes. In response to insulin treatment, ARF5 levels
at the plasma membrane were observed to increase with a concomitant
decrease chiefly from the soluble protein fraction of the cells.
Similar results were obtained using two different anti-ARF5 antibodies.
In the same experiments, GLUT4 was observed to translocate to the
plasma membrane in response to insulin from intracellular membrane
fractions as has been extensively reported (1-3). In contrast, ARF6
was observed chiefly in the plasma membrane fraction of 3T3-L1
adipocytes (34), and did not exhibit appreciable alteration in
subcellular distribution in response to insulin (Fig. 1). The plasma
membrane localization of ARF6 is in agreement with studies in a range
of cell types (34, 35). Although insulin did not appear to modulate the
subcellular distribution of ARF6, it is possible that insulin may
modify the ARF6-GDP/ARF6-GTP ratio at the plasma membrane, indeed
several studies have suggested that unlike other members of the ARF
family, ARF6 remains membrane associated even in its GDP-bound state
(34, 35). Alternatively, insulin may modulate the rate of turnover from
membrane to cytosolic states of ARF6 without an apparent alteration in
distribution between these fractions. Hence the apparent lack of
altered subcellular distribution of ARF6 in response to insulin does
not preclude an important role for this protein in insulin action.
Functional Role of ARF Proteins in Insulin-stimulated Glucose
Transport and GLUT4 Translocation--
Myristoylated peptides
corresponding to the amino terminus of ARF proteins have been widely
used in many laboratories to probe the function of ARF proteins in
intracellular trafficking, including endoplasmic reticulum to Golgi
transport, intra-Golgi transport, and endocytic vesicle fusion
(36-38). Myristoylated peptides corresponding to residues 2 through 13 of the NH2 terminus of ARF6 have also been shown to inhibit
regulated exocytosis in permeabilized chromaffin cells (22), and to
inhibit stimulated phospholipase D activity in these cells in response
to agents which stimulate secretion (39). In contrast, a corresponding
peptide lacking the myristoyl group at Gly-2, or the cognate
myristoylated peptide from ARF1 were without effect (22).
We therefore chose to adopt similar methodology to examine the role of
ARF5 or ARF6 in insulin-stimulated glucose transport in
This data argues that ARF6 may play a role in the regulation of plasma
membrane GLUT4 levels in response to insulin, as GLUT4 is responsible
for the majority of insulin-stimulated glucose uptake in adipocytes. In
order to address this directly, we measured insulin-stimulated GLUT4
translocation to the cell surface using the plasma membrane lawn
technique. Permeabilized adipocytes were incubated with myristoylated
ARF1, ARF5, and ARF6 peptides prior to insulin stimulation and
assessment of plasma membrane GLUT4 levels. Data from a typical
experiment are presented in Fig. 2B, and the results of
three experiments of this type presented in Fig. 2C. We
observed marked inhibition of GLUT4 translocation in the presence of
100 µM myristoylated ARF6 peptide (42 ± 3%; n = 3). A modest inhibition of GLUT4 translocation was
also observed in the presence of 100 µM myristoylated
ARF5 peptide, but the effect was considerably less than that induced by
the ARF6 peptide and did not reach significance in every experiment.
100 µM Myristoylated ARF1 peptide was without effect in
this assay. A dose-response curve for these peptides on GLUT4
translocation is presented in Fig. 2D. No significant effect
on basal (unstimulated) plasma membrane levels of GLUT4 were observed
after incubation with the peptides (Fig. 2B), but low signal
precludes detailed quantification of the level of GLUT4 at the plasma
membrane in the absence of insulin.
ARF5 and ARF6 Regulate Plasma Membrane Transferrin Receptor and
GLUT1 Numbers in 3T3-L1 Adipocytes--
Insulin treatment of
adipocytes results in the movement of other proteins to the plasma
membrane, including the TfR, the IGF-II/cation-independent mannose-6-phosphate receptor (27, 40) and GLUT1 (26), albeit to a much
lesser extent than is observed for GLUT4 (typically ~2-fold compared
with 12-15-fold). This is probably due to movement of these proteins
from the recycling endosomal system to the plasma membrane. We wished
to determine whether the effect of the myristoylated ARF6 peptide to
inhibit insulin-stimulated GLUT4 translocation was specific for GLUT4,
or whether other proteins which traffic between intracellular membranes
and the cell surface in an insulin-regulated manner were also effected.
We therefore examined the effect of insulin on cell surface levels of
TfR and GLUT1 in permeabilized adipocytes incubated with myristoylated
ARF peptides (Fig. 3).
Insulin stimulation of permeabilized adipocytes results in a ~2-fold
increase in plasma membrane TfR levels, in agreement with published
studies (27, 40). Prior incubation with myristoylated ARF1 peptide did
not reduce the magnitude of this response (Fig. 3A). In
contrast, prior incubation of 3T3-L1 adipocytes with myristoylated ARF5
peptide significantly inhibited the ability of insulin to stimulate TfR
levels at the cell surface with no effect on the basal (unstimulated)
TfR levels. In contrast, myristoylated ARF6 peptide reduced cell
surface TfR levels in both basal and insulin-stimulated cells
significantly compared with control cells, without significantly reducing the fold increase in cell surface TfR levels observed in
response to insulin (Fig. 3A). Similar data were also
observed for GLUT1 (Fig. 3B), with the exception that the
reduction in plasma membrane GLUT1 levels in the presence of the ARF6
peptide were not as extensive as those observed for TfR (Fig.
3B). Consistent with this, we observed a diminution of basal
(unstimulated) deoxy-Glc uptake in cells incubated with the ARF6
peptide (~15% inhibition), but this effect did not reach statistical
significance, presumably because the rate of basal transport is low
(data not shown). Nevertheless, these data argue that both ARF5 and
ARF6 are intimately involved in the trafficking of membrane proteins
between the plasma membrane and the recycling endosomal system in this
cell type.
Here we have shown that insulin stimulation of 3T3-L1 adipocytes
results in the redistribution of ARF5 from the soluble protein (cytosolic) fraction to the plasma membrane. We hypothesize that ARF5
recycles between the plasma membrane and intracellular (cytosolic) fractions presumably as a consequence of GDP/GTP exchange, and that
insulin stimulates GTP loading of this protein. Despite this insulin-stimulated translocation of ARF5, our data is not consistent with a role for this protein in insulin-stimulated GLUT4 translocation. Rather, we suggest that ARF5 plays a role in the insulin-stimulated trafficking of membrane proteins between the recycling endosomal system
and the plasma membrane, as evidenced by the ability of myristoylated
ARF5 peptides to inhibit insulin-stimulated TfR and GLUT1 movement to
the cell surface (Fig. 3). To our knowledge, this is the first
demonstration of a functional role for ARF5 in membrane trafficking.
In contrast to the data relating to ARF5, we show that a myristoylated
peptide corresponding to the amino terminus of ARF6 partially inhibits
insulin-stimulated GLUT4 translocation and deoxy-Glc transport in
GLUT4 has been proposed to populate at least two distinct intracellular
compartments, one of which corresponds to the recycling endosomal
system, the other a specialized intracellular storage compartment
referred to as GLUT4 storage vesicles (reviewed in Refs. 1, 2, and 41).
Hence, the partial inhibition of insulin-stimulated GLUT4 translocation
by myristoylated ARF6 peptides may be explained by the selective
inhibition of translocation of one of the proposed multiple
intracellular GLUT4 pools. This is consistent with previous studies
which implicate ARF6 in the exocytosis of secretory vesicles in other
cell types (22).
A second model, however, should also be considered which may also
explain the experimental data presented here. Previous studies of ARF6
function in other cell types have shown that mutants of ARF6 disrupt
receptor-mediated endocytosis (42). Overexpression of ARF6
redistributed TfR to the cell surface, while a dominant negative mutant
of ARF6 was shown to redistribute TfR to intracellular membranes and
inhibit TfR recycling to the cell surface, suggesting that ARF6 is an
integral component of the endocytic apparatus and that its GTP
cycle/nucleotide status regulate progression through the endocytic
pathway (42). In agreement with these studies, we have shown that
myristoylated ARF6 peptide decreases the cell surface levels of TfR
both in the presence and absence of insulin without affecting the
magnitude of the insulin-dependent increase in TfR at the
cell surface (Fig. 3A), consistent with ARF6 regulating
progression of TfRs through the endocytic pathway. Hence, an
alternative explanation of the data presented here is that the
myristoylated ARF6 peptide functions in a fashion similar to a dominant
negative ARF6, resulting in a change in the steady-state distribution
of TfR such that the intracellular/plasma membrane ratio is increased.
It is possible that after insulin stimulation when cell surface GLUT4
levels are increased, ARF6 function regulates the
internalization/recycling of GLUT4 in a similar manner to that of the
TfR. When ARF6 function is disrupted by the myristoylated peptide,
intracellular levels of GLUT4 and TfR are increased, resulting in
decreased plasma membrane levels of both of these proteins. This effect
is only manifest for GLUT4 in the insulin-stimulated state as plasma
membrane levels of GLUT4 in the absence of insulin are already very
low. Hence, in this model, the role of ARF6 is not specific for any
particular GLUT4 compartment, but rather is manifest at the level of
GLUT4 recycling between the plasma membrane and intracellular
compartments, which is known to occur even in the presence of insulin
(1, 41, 43). Distinguishing between these (or other) models for ARF6
action represents an important goal.
These data also offer further insight into the ability of insulin to
regulate membrane trafficking in adipocytes. We have shown that
myristoylated ARF5 peptide inhibits insulin-stimulated GLUT1 and TfR
translocation to the plasma membrane without significantly inhibiting
GLUT4 translocation. This result implies that the pathways by which
these two proteins reach the plasma membrane after insulin stimulation
are distinct. Several studies have identified a GLUT4 compartment which
is relatively devoid of TfR in both adipocytes and muscle (4, 44-46).
This (GLUT4 storage vesicles) compartment has been suggested to be a
GLUT4 storage compartment which is rapidly mobilized in response to
insulin (2). Although there is ample data implicating some overlap
between GLUT4 and the TfR in endosomes, it is possible that this
overlap is mainly a consequence of these proteins sharing the same
components of the endocytic arm of the recycling pathway. Indeed
studies using mutant dynamin molecules has suggested that the slowly
recycling (GLUT1/TfR positive) GLUT4 compartment contributes minimally
to insulin-stimulated GLUT4 translocation (47). In response to insulin
when traffic through the recycling endosomal system is clearly
increased, some GLUT4 will move to the cell surface from this location.
We would suggest that this represents a modest proportion of the total insulin-stimulated GLUT4 translocation, as evidenced here by the lack
of significant inhibition of GLUT4 translocation under conditions when
GLUT1 and TfR translocation are significantly compromised (i.e. in the presence of ARF5 peptide). Hence we suggest
that the main effect of insulin is to recruit GLUT4 from the
post-endosomal storage compartment (GLUT4 storage vesicless), and that
unlike insulin-stimulated movement through the recycling endosomes,
this is independent of ARF5 function.
In conclusion, we suggest that both ARF5 and ARF6 regulate trafficking
of membrane proteins to and from the cell surface, and show that ARF6
plays an important regulatory role in the steady-state levels of GLUT4
at the adipocyte cell surface after insulin stimulation. We speculate
that the actions of ARF5 and ARF6 are chiefly mediated by effects on
the recycling endosomal system, at least in this insulin-responsive
cell type.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Toxin was from Calbiochem, United Kingdom,
wortmannin from Sigma, UK, and 125I-transferrin and
[14C]sucrose were from NEN Life Science Products Inc. and
Amersham International, respectively. All other reagents were as
described (16, 28).
80 °C prior to use.
-toxin at 250 hemolytic units/ml for 5 min to
permeabilize the plasma membrane. The medium was removed and the cells
covered with 500 µl of ICR buffer containing peptides, insulin, or
vehicle as described in the figure legends. This methodology has been
documented in Ref. 30.
-Toxin is a 34-kDa
protein which inserts into the plasma membrane and oligomerizes to form
a 3-nm aqueous pore that allows passage of molecules up to ~5 kDa
across the cell membrane. We therefore employed this experimental
system to determine the role of ARF proteins on
[3H]2-deoxy-D-glucose (deoxy-Glc) transport
as has been described previously (30). After permeabilization and
incubation with peptides and insulin as described in the figure
legends, 50 µl of radioisotope solution was added to each well of
adipocytes such that the final concentration of deoxy-Glc was 50 µM and 0.5 µCi/well. Also included in this 50-µl
aliquot was [14C]sucrose (final concentration 50 µM, 0.05 µCi per well) so as to allow estimation of the
nonspecific association of sugar with the cells. The transport rates
presented have been corrected for this calculation. Uptake was carried
out for 3 min, then the cells were rapidly washed three times in
ice-cold phosphate-buffered saline and air-dried. Cell associated
radioactivity was determined by solubilizing the cells in 1% Triton
X-100. Nonspecific association of radioactivity with the cells amounted
to less than 20% of the specific uptake under these conditions.
-counting. For each condition, duplicates plates were
incubated exactly as above, but in the presence of 1 µM
transferrin; the radioactivity associated with each well under these
conditions was the value of nonspecific binding at each condition, and
was found to vary between 5 and 10% of the total counts per well.
Sequence alignment of ARF proteins at the amino terminus
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunological analysis of GLUT4, ARF5, and
ARF6 in subcellular membranes of 3T3-L1 adipocytes in response to
insulin. 10-cm plates of 3T3-L1 adipocytes were incubated ± 1 µM insulin for 15 min then subjected to subcellular
fractionation as described under "Experimental Procedures." Samples
corresponding to 10% of the total yield of plasma membrane and low
density microsomal (LDM) fractions, and 5% of the soluble
protein were subjected to SDS-polyacrylamide gel electrophoresis and
immunoblot analysis using antibodies against ARF5, ARF6, and GLUT4 as
indicated. Data from a representative blot, repeated three times, is
shown in Panel A with the approximate position of molecular
weight markers indicated at right of the blots.
Quantification of three blots of this type is shown in Panel
B in which the relative distribution of the indicated protein is
expressed as a function of the protein level in basal plasma membranes
(note that in Panel A, the amount of soluble protein loaded
per lane is 50% of that of the plasma membrane and low density
microsomal fractions).
-toxin-permeabilized 3T3-L1 adipocytes. We synthesized peptides corresponding to residues 2 through 16 of murine ARF5 (with or without
a myristoyl group at position Gly-2), and employed myristoylated ARF1
and ARF6 peptides described by one of us previously (22). Permeabilized
adipocytes were incubated with the peptides for 10 min, then stimulated
with 1 µM insulin for a further 20 min. At the end of
this time, deoxy-Glc uptake was measured as described under
"Experimental Procedures." The results of a typical experiment are
presented in Fig. 2A. We
consistently observed a diminution in the rate of insulin-stimulated
deoxy-Glc uptake in cells incubated with myristoylated ARF6 peptide. In
three experiments of this type, the extent of inhibition of
insulin-stimulated deoxy-Glc transport was 47 ± 11%. In
contrast, neither myristoylated ARF1 nor myristoylated ARF5 peptides
inhibited insulin-stimulated deoxy-Glc uptake over the same
concentration range. In control experiments (not shown) the addition of
the peptide to intact 3T3-L1 adipocytes was without effect on either
basal or insulin-stimulated deoxy-Glc uptake.
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Fig. 2.
Myristoylated ARF6 amino-terminal
peptide inhibits insulin-stimulated deoxy-Glc transport and GLUT4
translocation in 3T3-L1 adipocytes. Panel A, 3T3-L1
adipocytes were permeabilized with -toxin as described. After
permeabilization, cells incubated with amino-terminal ARF peptides were
added (100 µM) in ICR buffer for 10 min, followed by
insulin addition (1 µM) and a further 20-min incubation
at 37 °C. Deoxy-Glc transport was determined as described under
"Experimental Procedures" and the results of a typical experiment
of this type is shown. Each point is the mean of three measurements,
corrected for nonspecific association of deoxy-Glc with cells. In three
experiments of this type, the myristoylated ARF6 peptide inhibited
deoxy-Glc uptake by 47 ± 11%, ARF1 and ARF5 peptides were
without effect. * indicates a statistically significant difference from
insulin alone, p < 0.05. Prior incubation with ARF1 or
ARF5 peptides did not diminish basal deoxy-Glc uptake; incubation with
ARF6 peptide reduced basal deoxy-Glc uptake ~15%, but this effect
did not reach statistical significance (data not shown). Panel
B, 3T3-L1 adipocytes were treated as described for Panel
A, then processed for plasma membrane lawn assay for GLUT4
translocation as described under "Experimental Procedures." Shown
is data from a typical experiment (Panel B) and the data
from three independent experiments of this type, quantified as
described under "Experimental Procedures" is shown in Panel
C. * indicates significant difference from insulin alone,
p < 0.05. A dose-response curve for the inhibition of
insulin-stimulated GLUT4 translocation by myristoylated ARF6 peptide is
shown in Panel D.
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Fig. 3.
Effects of ARF peptides on insulin-stimulated
cell surface transferrin receptor levels. 3T3-L1 adipocytes were
permeabilized, incubated with myristoylated ARF peptides, and
incubated ± 1 µM insulin as described in the legend
to Fig. 2. After this, cell surface transferrin receptor levels were
determined by radioactive Tf binding (Panel A) or cell
surface GLUT1 levels measured using plasma membrane lawns (Panel
B). Shown are the results of a typical experiment using
myristoylated ARF1, ARF5, and ARF6 peptides. In Panel A, *
indicates a statistically significant difference from insulin alone
(p ~ 0.01); ** indicates statistically significant
difference from basal cells (p < 0.05); *** indicates
a statistically significant difference from basal + myr-ARF6
peptide-loaded cells (p = 0.01). In Panel B,
* indicates a statistically significant difference from insulin alone
(p = 0.010); ** indicates statistically significant
difference from basal cells (p ~ 0.05); *** indicates
a statistically significant difference from basal + myr-ARF6
peptide-loaded cells (p = 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-toxin permeabilized 3T3-L1 adipocytes (Fig. 2), implicating ARF6 as
a key component of this response. Furthermore, we show that incubation
of 3T3-L1 adipocytes with myristoylated ARF6 peptide reduces TfR number
at the cell surface both in the basal state and after insulin
stimulation, without decreasing the fold increase in cell surface
levels in response to insulin (Fig. 3A); similar results
were observed for GLUT1, except the reduction in plasma membrane GLUT1
levels were not as great (Fig. 3B). Collectively, these data
argue that ARF6 plays a fundamental role in trafficking between
intracellular membranes and the cell surface, indeed ARF6 has been
proposed to mediate the targeting of recycling vesicles to the plasma
membrane, either from a perinuclear compartment (CHO cells (21)) or
from a unique tubular-vesicular compartment (HeLa cells (33)). With
this in mind, several models may be proposed to explain the inhibitory effect of myristoylated ARF6 peptides on insulin-stimulated GLUT4 translocation.
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ACKNOWLEDGEMENTS |
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We thank Drs. Donaldson, Holman, Kahn, and Moss for their generous provision of antibodies used in this study and Dr. Francis Barr for constructive comments on this manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from The Wellcome Trust and The British Diabetic Association (to G. W. G.).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.
Contributed equally to the results of this work.
§ Supported by a Ph.D. studentship from The British Diabetic Association.
To whom all correspondence should be addressed. Tel.:
44-141-330-5263; Fax: 44-141-330-4620; E-mail:
G.W.Gould{at}bio.gla.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; ARF, ADP-ribosylation factor; GLUT, glucose transporter; deoxy-Glc, 2-deoxy-D-glucose; TfR, transferrin receptor; Tf, transferrin.
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REFERENCES |
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