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Molecular and Cellular Biology, July 2001, p. 4785-4806, Vol. 21, No. 14
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.14.4785-4806.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Insulin-Responsive Compartments Containing GLUT4 in 3T3-L1 and
CHO Cells: Regulation by Amino Acid Concentrations
Jonathan S.
Bogan,1,2
Adrienne E.
McKee,2 and
Harvey F.
Lodish2,3,*
Diabetes Unit, Department of Medicine,
Massachusetts General Hospital and Harvard Medical School, Boston,
Massachusetts 02114,1 and Whitehead
Institute for Biomedical Research2 and
Department of Biology, Massachusetts Institute of
Technology,3 Cambridge, Massachusetts 02142
Received 22 November 2000/Returned for modification 19 December
2000/Accepted 17 April 2001
 |
ABSTRACT |
In fat and muscle, insulin stimulates glucose uptake by rapidly
mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method
to quantify the relative proportion of GLUT4 at the plasma membrane,
using flow cytometry to measure a ratio of fluorescence intensities
corresponding to the cell surface and total amounts of a tagged GLUT4
reporter in individual living cells. Using this assay, we demonstrate
that both 3T3-L1 and CHO cells contain intracellular compartments from
which GLUT4 is rapidly mobilized by insulin and that the initial
magnitude and kinetics of redistribution to the plasma membrane are
similar in these two cell types when they are cultured identically.
Targeting of GLUT4 to a highly insulin-responsive compartment in CHO
cells is modulated by culture conditions. In particular, we find that
amino acids regulate distribution of GLUT4 to this kinetically defined
compartment through a rapamycin-sensitive pathway. Amino acids also
modulate the magnitude of insulin-stimulated translocation in
3T3-L1 adipocytes. Our results indicate a novel link between
glucose and amino acid metabolism.
| |
INTRODUCTION |
The GLUT4 glucose transporter is
expressed predominantly in adipose and muscle tissues, where it
accounts for the bulk of insulin-stimulated glucose uptake (12,
84, 95). In the presence of insulin, GLUT4 is redistributed from
an intracellular compartment to the plasma membrane, where it
facilitates the diffusion of glucose into the cell (15, 35, 73,
77, 102). Another glucose transporter isoform, GLUT1, is also
expressed in fat and muscle tissues and is present at high levels in
many other cell types and in cultured cell lines. A large proportion of
GLUT1 is present on the plasma membrane even in the absence of insulin. Thus, while both GLUT1 and GLUT4 recycle at the plasma membrane, only
GLUT4 recycling is characterized by significant intracellular sequestration, resulting from a slow rate of exocytosis, in the absence
of insulin. Insulin increases the rate of GLUT4 exocytosis, with little
or no decrease in its rate of endocytosis, so that in adipocytes the
proportion of GLUT4 at the cell surface increases from <10% in the
absence of insulin to 35 to 50% in its presence (41, 55, 85,
113, 114).
Characterization of the intracellular insulin-responsive
GLUT4-containing compartment is complicated by the fact that
GLUT4 resides in several morphologically distinct locations within the cell. Ultrastructural studies have shown that GLUT4 is present in
tubulovesicular structures distinct from lysosomes, as well as in a
perinuclear compartment that is in close vicinity to the trans-Golgi
network (39, 96-98). Recent work demonstrates that approximately 40 to 45% of intracellular GLUT4 localizes in a transferrin receptor (TfnR)-positive endosomal compartment, while 50 to
60% is in a second, TfnR-negative compartment; it is GLUT4 in this
TfnR-negative compartment that is rapidly mobilized upon insulin
addition (1, 32, 46, 55, 57, 65, 76). In primary
adipocytes, this TfnR-negative compartment can be subdivided into
separate storage and exocytic pools of GLUT4 (55, 56). Other data suggest that the TfnR-positive GLUT4 compartment is the
precursor of the TfnR-negative, insulin-responsive compartment (109). Moreover, targeting motifs within GLUT4 mediate its
distribution between TfnR-negative and TfnR-positive compartments
(66). Studies using the cation-dependent mannose
6-phosphate receptor (CD-M6PR) as an endosomal marker also find a
similar, highly insulin-responsive, M6PR-negative pool of intracellular
GLUT4 in 3T3-L1 adipocytes (64). Thus, it appears that
GLUT4 is sequestered out of endosomes and into a highly
insulin-responsive compartment. We have recently shown that 3T3-L1
adipocytes also possess an insulin-regulated secretory
compartment containing ACRP30, a tumor necrosis factor alpha-like
protein produced exclusively by adipocytes (7, 87, 92).
This regulated secretory compartment is distinct from the insulin-regulated compartment containing GLUT4 (7).
Kinetic studies are consistent with the notion that there are multiple
compartments through which GLUT4 traffics in adipocytes. GLUT4 recycles
between the plasma membrane and intracellular sites in both basal and
insulin-stimulated states, yet the initial externalization of GLUT4
after insulin addition is too rapid to be explained by the steady-state
rate constants for exocytosis and endocytosis in the presence of
insulin (14, 85, 113). It has therefore been argued that a
two-pool model, with one intracellular and one plasma membrane
compartment, does not explain the observed kinetics GLUT4
externalization after insulin addition and that GLUT4 must traffic
through three or more compartments (37, 85, 114). Among
these compartments is postulated to be a highly insulin-responsive intracellular compartment from which GLUT4 is rapidly mobilized by
insulin (37, 56, 114). The GLUT4 accumulated in this
highly insulin-responsive compartment in the basal state is depleted upon insulin addition, and the steady-state exocytosis rate for GLUT4
in the continued presence of insulin becomes limited at some other step
in the recycling pathway. This kinetically defined, highly
insulin-responsive compartment in adipocytes corresponds well with the
morphologically and biochemically defined, TfnR-negative compartments
containing GLUT4 in adipocytes and myocytes, which are also depleted of
GLUT4 after acute insulin stimulation (1, 32, 46, 55, 57, 65,
76).
The mechanisms controlling GLUT4 accumulation in this specialized
insulin-sensitive compartment are poorly understood. The compartment is
believed to be present only in muscle and fat and apparently develops
early during 3T3-L1 adipocyte differentiation in cell culture
(16, 19, 27, 34, 38, 39, 82, 89, 107). Some data indicate
that a similar compartment to which GLUT4 is targeted is present in CHO
cells, though this has been controversial (40, 44, 93, 105,
109). Kinetic studies suggest that even if some GLUT4 is
targeted to a highly insulin-responsive compartment in CHO cells,
sorting to this compartment is not efficient and does not constitute a
major pathway by which GLUT4 traffics, in contrast to the case in
adipocytes (3). Studies of the insulin-responsive aminopeptidase (IRAP), a protein of uncertain physiologic function that
is thought to cotraffic with GLUT4, show that this protein participates
in similar mechanisms for dynamic retention in the endosomal systems of
CHO cells and 3T3-L1 adipocytes (43, 100). The assertion
that the trafficking mechanisms employed by IRAP are identical those
used by GLUT4 rests upon evidence that these proteins colocalize and
cotraffic, with similar kinetics, under all conditions. Most data
support this hypothesis (20, 23, 45, 62, 65, 81, 101). Of
note, trafficking of IRAP, like that of GLUT4, is much more
insulin-responsive in 3T3-L1 adipocytes than in CHO cells
(43, 100). This is presumed to result from cell-type-specific differences, although, as with GLUT4, little is known about the mechanisms regulating IRAP accumulation and release from the highly insulin-responsive pool.
The present work began as a study of the cell type specificity of
insulin-regulated GLUT4 trafficking. Using a novel reporter molecule to
obtain detailed kinetic data, we find that GLUT4 participates in a
highly insulin-responsive compartment not only in the fully differentiated 3T3-L1 adipocytes that we employ, but in
undifferentiated 3T3-L1 preadipocytes as well. Such a compartment is
not present in all cell types, since NIH 3T3 cells do not exhibit
highly insulin-responsive trafficking. In CHO cells, we observe highly
insulin-responsive trafficking only when the cells are cultured
identically to 3T3-L1 adipocytes, in Dulbecco's modified Eagle's
medium (DMEM). In standard F12 culture medium, the cells are less
responsive. The highly insulin-responsive kinetics correlate with basal
state redistribution of intracellular GLUT4 from the perinuclear region
into punctate, peripheral structures. We pinpointed the amino acid
content of these media as the relevant difference causing these
trafficking characteristics: thus, given sufficient concentrations of
essential amino acids, GLUT4 accumulates in a highly insulin-responsive compartment in CHO cells. Our data show that amino acids regulate GLUT4
accumulation in this compartment through a rapamycin-sensitive pathway.
Finally, we demonstrate that amino acid sufficiency also modulates
highly insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes and
that this response is also rapamycin sensitive. Our data are consistent
with the notion that both 3T3-L1 cells and CHO cells contain
peripheral, highly insulin-responsive compartments through which GLUT4
traffics and that amino acid sufficiency modulates GLUT4 trafficking
through these compartments in both cell types.
| |
MATERIALS AND METHODS |
Antibodies and reagents.
Cell culture media and
supplements were purchased from Life Technologies (Grand Island, N.Y.)
and JRH Biosciences (Lenexa, Kans.). Anti-c-Myc monoclonal antibody
(clone 9E10) was from Babco/Covance (Richmond, Calif.) and from Roche.
An anti-insulin receptor 
-chain antibody was purchased from BD
Transduction Laboratories. Normal donkey serum and R-phycoerythrin
(PE)-conjugated donkey F(ab')2 anti-mouse
immunoglobulin G (IgG) secondary antibody were purchased from
Jackson Immunoresearch (West Grove, Pa.). Restriction enzymes were from
New England Biolabs (Beverly, Mass.) and Pfu
and Taq DNA polymerases were from Stratagene (La Jolla,
Calif.). Wortmannin, LY294002, and rapamycin were from Calbiochem
(La Jolla, Calif.). Oil red O and other chemicals were from Sigma
(St. Louis, Mo.).
Cell culture.
Murine 3T3-L1 fibroblasts were cultured in
DMEM containing 10% fetal bovine serum (or 10% calf serum, where
noted), and differentiation was induced according to established
protocols (7, 22). Briefly, cells were allowed to reach
confluence at least 2 days prior to the induction of differentiation.
Differentiation was induced (on day 0) with medium containing 0.25 µM
dexamethasone, 160 nM insulin, and 500 µM methylisobutylxanthine.
After 48 h (day 2), the cells were fed with medium containing 160 nM insulin. After an additional 48 h (day 4), the cells were refed
every 2 days with DMEM-10% fetal bovine serum. All media were
supplemented with 2 mM glutamine, 100 U of penicillin per ml, and 0.1 mg of streptomycin per ml. Differentiation was monitored by noting the accumulation of lipid droplets, which typically began by day 4 of
differentiation. Cells were considered fully differentiated between
days 8 and 12. Throughout this report, the terms day 0 3T3-L1 cells and
confluent 3T3-L1 preadipocytes are used interchangeably.
CHO-K1 cells stably expressing the murine ecotropic retrovirus receptor
were kindly provided by David Hirsch, Roger Lawrence, and Monty Kreiger
(Massachusetts Institute of Technology, Cambridge, Mass.) and
maintained in Ham's F12 medium (F12) with 10% fetal bovine serum and
2 mM glutamine plus 100 U of penicillin and 0.1 mg of streptomycin per
ml (5). NIH 3T3 cells were cultured in DMEM containing
10% calf serum, glutamine, penicillin, and streptomycin as above.
Phoenix ecotropic retrovirus packaging cells were a gift from Garry
Nolan (Stanford University Medical Center), and VE23 ecotropic
retrovirus packaging cells were a gift from Merav Socolovsky (Whitehead
Institute, Cambridge, Mass.) (51, 99, 103). Both
retrovirus packaging cell lines were cultured in DMEM-10% fetal
bovine serum with glutamine, penicillin, and streptomycin as above.
For experiments involving culture in minimal essential medium (MEM)
with various amino acid concentrations, we used the MEM
Select-Amine
kit (Life Technologies). The 1× concentration of
each amino acid was
as follows (free base, in milligrams per liter):
L-Arg,
102;
L-Cys, 36;
L-His, 30;
L-Ile,
52;
L-Leu, 52;
L-Lys,
57;
L-Met,
15;
L-Phe, 32;
L-Thr, 48;
L-Trp,
10;
L-Tyr, 35; and
L-Val, 46. Relative to these
concentrations, DMEM contains 2×
Cys, Ile, Leu, Lys, Met, Phe, Thr,
Tyr, and Val; 1.6× Trp; 1×
His; and 0.67× Arg. F12 contains 1.67×
Arg; 1.25× Cys; 0.5× His
and Lys; 0.3× Met; 0.25× Leu, Thr, and
Val; 0.2× Trp; 0.15× Phe
and Tyr; and 0.08× Ile. All media contained
2 mM glutamine as
well as penicillin and streptomycin as
above.
Construction of a GLUT4 reporter.
A human GLUT4 cDNA
containing a c-Myc epitope tag in the first exofacial loop was kindly
provided by Zhijun Luo and Joseph Avruch (Massachusetts General
Hospital, Boston). This clone had been constructed as described by
Kanai et al. (44). We fused the green fluorescent protein
(GFP) coding sequence in frame to the carboxy terminus of this GLUT4
clone based on the results of Dobson et al. (18) that
GLUT4-GFP appears to localize and traffic similarly to wild-type GLUT4.
The GFP coding sequence from pEGFP-N1 (Stratagene) was first cloned
into the pMX retroviral vector using EcoRI and
NotI to generate plasmid pMX-GFP (68). PCR was
done using primers 5'-GACATTTGACCAGATCTCGG-3' and
5'-GGCCCGCGGGTCATTCTCATCTGGCCC-3' to generate a
~110-bp BglII-SacII fragment from the 3' end of the rat GLUT4 cDNA (11). This PCR product and an
EcoRI-BglII fragment containing most of the
GLUT4myc cDNA were used in a three-way ligation with
EcoRI- and SacII-digested pMX-GFP to generate
pMX-GLUT4myc-GFP. Next, six additional myc
epitope tags were added in tandem with the existing
myc epitope tag, for a total of seven myc epitope tags. We first used PCR to amplify a ~240-bp
EcoRI-HindIII fragment including the 5' end
of the rat GLUT4 cDNA and part of a myc tag, using primers
5'-CCGGCCGAATTCATGCCGTCGGGTTTCCAGCAGATC-3' and
5'-CTTCAGAAATAAGCTTTTGCTCCTCTGCAGGACCCTGCCTACCCAGCCAAGTTGC-3'. This fragment was used to replace a corresponding fragment in pMX-GLUT4myc-GFP, creating a unique HindIII
site within the myc epitope tag. A HindIII
fragment containing six tandem myc epitope tags was
amplified from plasmid pCS2+MT, a gift from Bill Schiemann (Whitehead
Institute), using primers
5'-CCATCGATTTAAAGCTATGGAGCAAAAGCTTATTTCTGAAGAGG-3' and
5'-CAGAAATAAGCTTTTGCTCCTCTGCAGGCTCAAGAGGTCTTGAGTTCAAGTCCTCTTC-3'. This fragment was inserted into the HindIII site
of pMX-GLUT4myc-GFP, creating pMX-GLUT4myc7-GFP.
The entire coding regions of the pMX-GLUT4myc-GFP and
pMX-GLUT4myc7-GFP plasmids were verified by sequencing. The GLUT4myc7-GFP coding sequence was also placed in the pB
retrovirus vector in order to optimize the potential translation
efficiency; pB is identical to pMX except that two point mutations were
introduced to eliminate potential start codons 5' of the cloning site
(J. S. Bogan, X. Liu, A. E. McKee, and H. F. Lodish,
unpublished data). In this report, the GLUT4 reporter refers to that
encoded by the GLUT4myc7-GFP sequence.
Production of retroviral supernatant and isolation of
infected cell populations.
Phoenix or VE23 ecotropic packaging
cells were transfected with the pMX-GLUT4myc-GFP,
pMX-GLUT4myc7-GFP, or pB-GLUT4myc7-GFP plasmid using calcium phosphate as described (51, 99,
103). In some instances, Phoenix cells were transfected using
Fugene 6 (Roche) per the manufacturer's protocol. Media containing
recombinant retroviruses were harvested 48 or 72 h after
transfection and used to infect dividing 3T3-L1 preadipocytes, NIH 3T3
cells, or CHO cells expressing the murine ecotropic receptor. For
3T3-L1 preadipocytes infected with
pMX-GLUT4myc7-GFP, flow cytometry demonstrated the presence
of GFP in >90% of cells after infection. We isolated stable
populations of infected cells expressing large, medium, and small
amounts of the reporter by flow sorting cells falling within narrow
ranges of GFP fluorescence. The sorted cells were expanded and
differentiated into adipocytes, and insulin-stimulated GLUT4
trafficking (stimulated/basal) was measured by flow cytometry in all
cases. We reasoned that there might be a trade-off between signal/noise
(with small amounts of the reporter) and potential saturation of a
trafficking mechanism (at large amounts of the reporter). In general,
we chose medium- or high-expressing cells for use in subsequent
experiments, since these had the greatest fold increase in cell surface
GLUT4 after insulin treatment. These cells generally contained 5 to 10 times as much reporter protein as native GLUT4 in the mature 3T3-L1
adipocytes, as judged by immunoblotting with antibodies directed
against the N and C termini of GLUT4 (kindly provided by Maureen
Charron, Albert Einstein College of Medicine, Bronx, N.Y.). Similar
optimization of reporter protein expression levels was carried out in
NIH 3T3 and CHO cells.
Measurement of plasma membrane GLUT4 trafficking by flow
cytometry.
Confluent cells were reseeded on the indicated day of
differentiation to six-well plates (Corning; Costar no. 3506) 1 day before use in experiments and starved in DMEM without fetal bovine serum for at least 3 h before insulin stimulation. Insulin was generally used at 160 nM; we occasionally used 80 or 200 nM and noted
no difference between these concentrations in either 3T3-L1 or CHO
cells. Insulin was added directly to the wells from a 100× stock.
After treatment in the presence or absence of insulin for the times
indicated in each figure, cells were quickly transferred to 4°C and
washed with cold phosphate-buffered saline (PBS) containing 0.9 mM
Ca2+ and 0.5 mM Mg2+ (PBS++). All
subsequent steps were carried out at 4°C, and staining of
externalized myc epitope was done on adherent cells. Cells were incubated with a 1:200 dilution of anti-Myc (9E10) ascites or with
purified 9E10 (25 µg/ml) in PBS++-2% bovine serum
albumin (BSA)-4% donkey serum for 1.5 h. Cells were then washed
twice in PBS++ for 5 min each time. They were then
incubated for 45 min in 12.5 to 25 µg of PE-conjugated donkey
F(ab')2 anti-mouse IgG secondary antibody per ml, diluted
in PBS++-2% BSA-4% donkey serum. Cells were rinsed
twice in PBS++, then washed three times in
PBS++ for 10 min each, and resuspended by gentle scraping
in PBS++-2% BSA or PBS++-5% fetal bovine
serum for flow cytometry. For experiments involving insulin removal,
cells were chilled as above after insulin stimulation and then washed
twice with 5 mM sodium acetate-150 mM NaCl (pH 4.0) (48,
112). Cells were rewarmed in 37°C DMEM for the times indicated, restimulated with insulin or not restimulated, then returned
to 4°C, and chilled with cold PBS++ before staining as above.
Flow cytometry was done on a FACScan or FACSCalibur cytometer (Becton
Dickinson). Appropriate compensation between the FL1
and FL2 channels
was set using uninfected (GFP negative) cells
or cells stained with PE
only (e.g., using a PE-conjugated anti-TfnR
antibody [Pharmingen]).
Pilot experiments demonstrated minimal
loss of viability; only 2 to 3%
of the cells typically stained
with propidium iodide using the protocol
described above, so propidium
iodide was not used in experiments when
accurate compensation
and quantitation of fluorescence intensities was
essential. For
each sample, data from
10,000 cells were collected. We
used median
fluorescence intensities for quantification, since this
measure
of central tendency is least sensitive to outliers. For each
sample,
the PE and GFP fluorescences specifically attributable to the
presence of the GLUT4 reporter were determined by subtracting
background fluorescences, measured using control unstained cells
and
cells not expressing the reporter, respectively. These control
cells
were treated with the same conditions (e.g., type of serum
and medium
and amino acid concentrations) used for the experimental
cells. The
ratio of fluorescence intensities plotted on the vertical
axes of many
figures is a relative, not absolute, measure of the
proportion of GLUT4
at the cell surface. The scales are numbered
arbitrarily and are not
intended to permit comparison in absolute
terms of data obtained in
different experiments. In some of the
kinetic studies (Fig.
7 to
10),
the data were subjected to a simple
smoothing operation. This consisted
of calculating an equation,
0.25
rt 
1 + 0.5
rt + 0.25
rt + 1, where
r is the ratio of specific
fluorescences for time point
t. This calculated value was used
for time points
t = 2 to
t =
n 
1, where
n is the total number
of time points. For the last time
point,
t =
n, 0.33
rt 1 + 0.67
rt was calculated. Basal
values of
r (
r1) were
unchanged.
Subcellular fractionation.
Four 10-cm plates of 3T3-L1
adipocytes were used per condition to isolate low-density microsomal
and plasma membrane fractions (Fig. 1b). Eight and ten plates per
condition were used to isolate low-density microsomes for analysis by
sucrose density gradient centrifugation (Fig. 1c) and in vesicle
immunopurification experiments (Fig. 1d), respectively. Five plates of
adipocytes and 10 plates of preadipocytes were used per condition in
the experiments presented in Fig. 3b and 4b, respectively. For
experiments presented in Fig. 1b and 1c, cells were serum starved
overnight, treated in the presence or absence of insulin (160 nM, 10 min), then transferred to 4°C, and washed with cold
PBS++. For the vesicle immunopurification (Fig. 1d), cells
were starved overnight, then transferred to 4°C, and washed with cold
PBS++ without insulin stimulation. For kinetic studies
(Fig. 3b and 4b), 480 nM insulin was used (added from a prewarmed 3×
stock) in order to maximally and simultaneously stimulate all of
the cells on each 10-cm dish. Cell were transferred to 4°C and washed with cold PBS++ at the indicated times. In all cases, cells
were next washed once with cold 250 mM sucrose-10 mM Tris (pH
7.4)-0.5 mM EDTA (buffer A). Cells were resuspended by scraping in
cold buffer A with Complete protease inhibitors (Roche) and then
homogenized using 16 strokes (four plates) or 25 strokes (eight plates)
in a Dounce-type Teflon tissue grinder (Kontes no. 22; VWR). All subsequent steps were performed at 4°C. The homogenate was
centrifuged at 11,500 rpm in an SS-34 rotor (16,000 × g) for 20 min. The pellet was resuspended in buffer A and then
layered on top of 1.12 M sucrose-10 mM Tris (pH 7.4)-0.5 mM EDTA in a
~2-ml centrifuge tube. The samples were centrifuged in a TLS-55 rotor
at 36,000 rpm (158,000 × g) for 20 min. The interface
was removed using a syringe, diluted in buffer A, and centrifuged in a
TLA-100.2 rotor at 37,000 rpm (74,000 × g) for 9 min.
The pellet was resuspended in buffer A and centrifuged again under
identical conditions. The pellet from this centrifugation, designated
PM, was resuspended in TNET (1% Triton X-100, 150 mM NaCl, 20 mM Tris
[pH 8.0], 2 mM EDTA) or in radioimmunoprecipitation assay (RIPA)
buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate [SDS]) and stored at 20°C until needed.
The supernatant from the initial centrifugation was recentrifuged at
19,000 rpm in an SS-34 rotor (43,000 ×
g) for 30 min.
The pellet (designated HDM) was resuspended in TNET or RIPA buffer
and
stored at
20°C. The supernatant was centrifuged at 65,000
rpm in a
Ti70.1 rotor (39,000 ×
g) for 75 min. The pellets from
this centrifugation, designated LDM, were resuspended in either
TNET or
RIPA buffer and stored at
20°C until needed or resuspended
in 500 µl of 50 mM sucrose-10 mM Tris (pH 7.4)-0.5 mM EDTA and
loaded on
top of a 4.9-ml 10 to 30% (wt/vol) linear sucrose gradient
prepared in
10 mM Tris (pH 7.4)-0.5 mM EDTA. Separation of low-density
microsomes
by sedimentation was done essentially as described
with minor
modifications (
19). The samples were centrifuged
in an
SW50.1 rotor at 48,000 rpm (280,000 ×
g) for 55 min.
Fractions
were collected from the tops of the gradients. Equal volumes
of
each fraction were analyzed by SDS-polyacrylamide gel
electrophoresis
(PAGE) and immunoblotting, and the total protein
content in each
fraction was determined using the Micro-BCA kit
(Pierce). In experiments
where LDM and PM fractions were analyzed
directly (without sedimentation
of the LDM), total protein content was
first determined and then
equal amounts of protein were analyzed by
SDS-PAGE and
immunoblotting.
For vesicle immunopurification experiments, LDM fractions were
resuspended in PBS
++-2% BSA and then incubated overnight
at 4°C in the presence of
25 µl (10 µg) of anti-GFP monoclonal
antibodies (Roche). Protein
G-Sepharose beads were preblocked with
PBS
++-2% BSA, then added to each sample, and incubated
for 1 h at 4°C.
The beads were pelleted, and the supernatant was
transferred to
new tubes and frozen until needed. The beads were washed
five
times in PBS
++-2% BSA and then three times more in
PBS
++ without BSA. Material was eluted from the beads at
65°C for 30
min in SDS-PAGE sample buffer, and equal volumes were
loaded for
electrophoresis and subsequent
immunoblotting.
Other methods.
Immunofluorescence microscopy of permeablized
cells was done essentially as described (7). Cells were
fixed with 4% paraformaldehyde in PBS for 40 min, permeablized with
0.2% Triton X-100 for 5 min, and then washed extensively with
PBS++. Staining of CHO cells expressing the GLUT4 reporter
was done in order to increase the fluorescent signal from that due to
GFP alone and employed a monoclonal anti-Myc antibody and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody.
For immunofluorescence microscopy of unpermeablized cells (Fig.
6a),
cells on coverslips were stimulated with insulin, then
transferred to
4°C, and washed with cold PBS
++. Living cells were
stained at 4°C using anti-Myc (9E10) monoclonal
antibody (25 µg/ml)
in PBS
++-2% BSA-4% goat serum. After 1 h at 4°C,
cells were washed and
incubated with Alexa594-conjugated goat
anti-mouse IgG secondary
antibody (10 µg/ml) (Molecular Probes).
Cells were then fixed
with parafomaldehyde as described above, and
coverslips were mounted
using Prolong antifade reagent (Molecular
Probes). Microscopy
was done using a Zeiss Axiophot microscope, and
images were acquired
on film. In order to compare fluorescence
intensity due to externalized
Myc epitope, GFP images were acquired
first using an exposure
time calculated by the camera, and the exposure
time used for
the corresponding Alexa594 images was set as a constant
fraction
of the GFP exposure time. In this way, we attempted to
normalize
the images of externalized Myc epitope tag for variations in
the
total amount of the reporter protein and cell
density.
Oil red O staining was done on cells grown in 10-cm dishes. Cells were
fixed with 4% paraformaldehyde for 45 min at room temperature,
permeablized with 0.2% Triton X-100 for 5 min at 4°C., and stained
using a 2-mg/ml solution of Oil red O in ethanol (
24).
Phase-contrast
and bright-field microscopy was done using an Olympus
inverted
microscope.
| |
RESULTS |
Novel assay for GLUT4 trafficking at the plasma
membrane.
To assay changes in the proportion of GLUT4 that is
present at the plasma membrane, we constructed a cDNA encoding a GLUT4 reporter protein. This protein contains seven c-Myc epitope tags in the
first exofacial loop of GLUT4 and GFP fused in-frame at the carboxy
terminus. As shown in Fig. 1a, expression
of this protein in cells enables us to measure changes
in the proportion of GLUT4 at the cell surface as changes in a ratio of
fluorescence intensities. We use an anti-Myc monoclonal antibody
followed by a PE-conjugated secondary antibody to detect reporter
protein present at the surface of living cells. Green (GFP)
fluorescence indicates the total amount of the reporter present in each
cell. Thus, the ratio of PE to GFP fluorescence intensities
corresponds to the proportion of total GLUT4 that is present at the
plasma membrane. We employ flow cytometry to measure these fluorescence intensities simultaneously and on a cell-by-cell basis.
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FIG. 1.
Assay for changes in proportion of GLUT4 at the plasma
membrane. A GLUT4 reporter containing Myc epitope tags in the first
exofacial loop as well as GFP fused in frame at the carboxy terminus
was constructed as described in Materials and Methods. As shown in
panel a, this reporter enables measurement of changes in the proportion
of GLUT4 at the plasma membrane as changes in the ratio of fluorescence
intensities corresponding to cell surface and total amounts of the
reporter. Cell surface GLUT4 reporter is detected using an anti-Myc
primary antibody and PE-conjugated secondary antibody. Total GLUT4
reporter is proportional to GFP fluorescence. (b) Low-density microsome
(LDM) and plasma membrane (PM) fractions were isolated from 3T3-L1
adipocytes expressing the reporter and analyzed by SDS-PAGE and
immunoblotting. Equal amounts of protein were loaded in each lane.
Immunoblotting was done using an antibody directed against the carboxyl
terminus of GLUT4 and demonstrates that both the reporter (95 kDa) and
native GLUT4 (50 kDa) are redistributed from the LDM fraction to the PM
after acute insulin treatment. The amount of translocation is
quantitatively similar. (c) The LDM fractions from basal and
insulin-stimulated 3T3-L1 adipocytes expressing the reporter and from control
cells were further separated by sedimentation on a 10 to 30% linear
sucrose gradient, as described in Materials and Methods. Equal volumes
of each gradient fraction were analyzed by to determine total protein
and by SDS-PAGE and immunoblotting to detect native GLUT4 (in control
cells, using an anti-GLUT4 antibody) and the GLUT4 reporter (using an
anti-Myc antibody). As shown in the top panels, the reporter and
endogenous GLUT4 cosediment in both basal (left) and insulin-stimulated
(right) cells. Densitometry was used to quantify the bands (middle
panels), and data are plotted as the percentage of the total reporter
or native GLUT4 present in each gradient fraction; these profiles are
quite similar. As a control, the percentage of total protein present in
each gradient fraction is plotted (lower panels); these profiles are
similar to each other and distinct from those of the GLUT4 reporter and
endogenous GLUT4. (d) LDM fractions from unstimulated 3T3-L1 adipocytes
expressing the reporter and from control cells not expressing the
reporter were used in vesicle immunopurification experiments. LDM
fractions were incubated with two pooled anti-GFP monoclonal
antibodies, followed by protein G-Sepharose beads. After pelleting and
washing of the beads, the immunopurified material was eluted in sample
buffer and analyzed by SDS-PAGE and immunoblotting with an anti-GLUT4
rabbit polyclonal antibody. As shown in the upper two panels, native
GLUT4 is detected in the immunopurified material. The lower panels
present a similar immunoblot of the supernatants and demonstrate that
even though the immunopurification did not quantitatively remove all of
the GLUT4 reporter, the endogenous GLUT4 is depleted, as expected. (e)
Flow cytometry is used to quantify the insulin-stimulated change in the
proportion of GLUT4 at the plasma membrane of 3T3-L1 adipocytes
expressing the reporter protein. Serum-starved cells were treated or
not with insulin, chilled, stained for externalized Myc epitope tag,
and analyzed by FACS as described in Materials and Methods. PE and GFP
fluorescence intensities are plotted on the vertical and horizontal
axes, respectively, of the dotplots presented. Note that both scales
are logarithmic. Compared to the background fluorescence of cells not
expressing the reporter (yellow), cells expressing the reporter (blue)
have increased GFP fluorescence (leftmost panel). Among cells
expressing the reporter, unstained cells (blue) and basal (stained for
cell surface Myc, shown in red) and insulin-stimulated (stained for
cell surface Myc, shown in green) populations have progressively
increasing PE fluorescence with no change in GFP fluorescence. Control
experiments show that the background staining is negligible (not
shown). The four panels allow direct comparison of pairs of samples. In
this experiment, insulin caused a fourfold increase in the ratio of
median fluorescence intensities attributable to externalized Myc
epitope and to GFP expression, corresponding to a fourfold increase in
the proportion of total GLUT4 present at the cell surface. (f) Flow
cytometry was used to measure insulin-stimulated GLUT4 translocation in
confluent CHO cells. As in panel e, PE fluorescence (proportional to
cell surface GLUT4 reporter) is plotted on the vertical axis and GFP
fluorescence (proportional to total GLUT4 reporter) is plotted on the
horizontal axis; both scales are logarithmic. Background (unstained)
cells expressing the reporter are shown in blue, and basal and
insulin-stimulated populations are shown in red and green,
respectively. The three panels allow direct comparison between each
pair of samples. There is a minor population of unstained cells (blue)
within the first decade of each scale; these cells do not express the
GLUT4 reporter and conveniently demonstrate that the flow cytometer is
properly adjusted to compensate for fluorophore bleedthrough. Compared
to 3T3-L1 adipocytes, the background fluorescences (both PE and GFP)
account for much less of the total fluorescence intensities in CHO
cells, and the signal-noise ratio is correspondingly increased (compare
panels e and f). In this experiment, insulin stimulated a 3.5-fold
increase in the proportion of total GLUT4 present at the cell
surface.
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We placed the GLUT4 reporter in a murine retrovirus vector and infected
3T3-L1 preadipocytes. Using fluorescence-activated
cell sorting (FACS),
we isolated a population of cells falling
within a narrow range
of GFP fluorescence intensities; individual
cells in this population
express similar amounts of the reporter
protein. These 3T3-L1 cells
underwent normal adipose cell differentiation
(see below), and we
took several approaches to confirm that the
GLUT4 reporter traffics
appropriately. We first used differential
centrifugation to isolate
low-density microsomal (LDM) and plasma
membrane (PM) fractions from
cells expressing the GLUT4
myc7-GFP
reporter and from
control cells. As shown in Fig.
1b, acute insulin
treatment causes
similar decreases in the amounts of both native
GLUT4 (~50 kDa) and
the reporter protein (~95 kDa) in the LDM
fraction.
Correspondingly, insulin treatment causes similar increases
in
both proteins in the PM fraction. Of note, the increases in
the plasma
membrane fraction are in the 5- to 10-fold range
(insulin/basal),
as judged by densitometry of several
experiments. Thus, both the
reporter and endogenous GLUT4 proteins
redistribute to the plasma
membrane in a quantitatively similar
manner.
Figure
1c presents data further substantiating that the
GLUT4 reporter codistributes with endogenous GLUT4 in low-density
microsomes prepared from 3T3-L1 adipocytes. In this experiment,
we
first isolated LDM fractions from basal and insulin-stimulated
cells
containing the reporter and from control cells not expressing
the
reporter. We layered these fractions on top of 10 to 30% linear
sucrose density gradients and sedimented the microsomes by
centrifugation.
We then collected fractions from each sample and
immunoblotted
to detect endogenous GLUT4 or the reporter. As shown in
the upper
left panels of Fig.
1c, both native GLUT4 and the reporter
sediment
similarly in unstimulated cells, with the bulk of both
proteins
present in fractions 8 to 13 and in the pellet (middle left
panel).
In insulin-stimulated cells, the distributions of both
endogenous
GLUT4 and the reporter shift to the pellet; fractions 8 to
13
contain most of the remaining protein, as in the unstimulated
cells
(top and middle right panels). As a control, total protein
was measured
in each fraction and is plotted at the bottom of
the figure for
unstimulated cells (left) and for insulin-stimulated
cells (right).
These profiles are similar for cells containing
the GLUT4 reporter and
for control cells and are distinct from
the distributions of
endogenous GLUT4 and the GLUT4 reporter.
The distribution of the GLUT4
reporter on these gradients is broader
than that of native GLUT4,
perhaps because the reporter is expressed
at roughly fivefold-higher
levels (not shown) and may be present
in a more heterogeneous
population of vesicles. Nonetheless, the
bulk of GLUT4 reporter in
low-density microsomes is present in
vesicles that sediment similarly
to those containing native GLUT4
in both basal and insulin-stimulated
cells.
To further determine if endogenous GLUT4 and the GLUT4 reporter are
present in the same vesicles, we used an anti-GFP antibody
and protein
G-Sepharose beads to immunopurify vesicles from the
LDM fraction of
unstimulated 3T3-L1 adipocytes expressing the
reporter. As shown in
Fig.
1d, immunoblotting demonstrates the
presence of endogenous GLUT4
in these vesicles, as well as confirming
the presence of the reporter.
As a control, 3T3-L1 adipocytes
not expressing the GLUT4 reporter were
treated in parallel; in
this case, neither the GLUT4 reporter nor
endogenous GLUT4 is
detected in the material eluted from the beads.
Endogenous GLUT4
is detected in the supernatants from both samples and,
as expected,
is depleted from that of the cells expressing the
reporter. Some
GLUT4 reporter also remains in the supernatant, and we
estimate
that the immunopurification removed only 50 to 75% of
vesicles
containing the reporter protein from the starting microsomes.
The results indicate that endogenous GLUT4 and the GLUT4 reporter
are
present together in a population of vesicles within the low-density
microsomal
fraction.
To measure insulin-stimulated GLUT4 externalization in differentiated
3T3-L1 adipocytes expressing the reporter, we used flow
cytometry as
shown in Fig.
1e. This technique allows simultaneous
measurement of PE
fluorescence (corresponding to cell surface
GLUT4 reporter and shown on
the vertical scale) and GFP fluorescence
(corresponding to total GLUT4
reporter and shown on the horizontal
scale). Control 3T3-L1 adipocytes
not expressing the reporter
(shown in yellow) have background
fluorescences that are highly
correlated across the relevant
wavelengths and appear as a diagonal
population. For cells expressing
the reporter but not stained
for cell surface Myc epitope (shown in
blue), fluorescence along
the PE axis is due entirely to this
background autofluorescence.
Control experiments using secondary
antibody without primary (anti-Myc)
antibody, as well as control
experiments using both primary and
secondary antibodies on cells not
expressing the reporter, demonstrate
that background staining is
negligible (data not shown); thus,
essentially all of the increase in
PE fluorescence observed in
the basal (red) and insulin-stimulated
(green) populations is
due to detection of Myc on the cell surface.
Similarly, the GFP
fluorescence attributable to the GLUT4 reporter can
be determined
by subtracting the background autofluorescence (yellow).
Within
each population of stained cells (basal and insulin stimulated),
the amount of staining for cell surface Myc correlates with the
amount
of the reporter present and therefore with GFP fluorescence.
The
populations therefore lie along a diagonal, and GLUT4 exocytosis
results in a net translocation of the entire population upwards,
along the PE axis, with no change in the slope of the diagonal.
Within
this defined population of cells we observe no saturation
of the
recycling mechanism: changes in the proportion of GLUT4
at the cell
surface are equivalent, even among cells expressing
~50-fold-different amounts of the
reporter.
To measure GLUT4 trafficking at the surface of CHO cells, we infected
CHO cells expressing the murine ecotropic retrovirus
receptor with a
retrovirus carrying the GLUT4 reporter and used
FACS to isolate cells
falling within a narrow range of GFP fluorescence
intensities. Upon
insulin stimulation of these cells, we again
noted externalization of
the GLUT4 reporter, as detected by flow
cytometry. As shown in Fig.
1f,
autofluorescence accounts for
much less of the total fluorescent
signals in CHO compared to
3T3-L1. Thus, the unstained cells expressing
the reporter do not
fall along a diagonal because autofluorescence
contributes minimally.
Similarly, whereas autofluorescence
contributes perhaps one-quarter
of the total PE fluorescence in 3T3-L1
adipocytes, in CHO cells
this figure is reduced to 1 to 2%. As with
3T3-L1 adipocytes,
the distribution of each of the stained
populations (basal and
insulin stimulated, shown in red and green,
respectively) falls
along a diagonal because the amount of Myc epitope
at the surface
of each cell is proportional to the amount of the
reporter present
within that cell. Insulin stimulates the entire
population to
shift upward (~4-fold) along the PE (Myc) axis,
with no change
in the slope of the diagonal, consistent with exocytosis
of the
GLUT4 reporter equally and with no saturation of the recycling
mechanism among the infected
cells.
Insulin stimulates GLUT4 translocation similarly in
undifferentiated 3T3-L1 cells and throughout 3T3-L1 adipose
differentiation.
One difficulty in working with 3T3-L1 cells is
that if the undifferentiated fibroblasts are allowed to become
confluent, they must be induced to undergo adipose differentiation or
they will lose that capacity. This characteristic makes the
introduction of exogenous proteins by stable transfection technically
difficult, since the cells invariably become confluent during clonal
selection. We circumvented this difficulty by isolating a pool of cells
infected with a replication-deficient retrovirus encoding the reporter protein. Over 90% of the target 3T3-L1 fibroblasts were infected, and
those falling within a narrow window of GFP fluorescence were isolated
by FACS; individual cells in this sorted population express similar
amounts of the reporter. Figure 2a
demonstrates that these sorted cells undergo normal 3T3-L1 adipose
differentiation, as assessed by Oil red O staining to highlight the
development of intracellular lipid droplets.
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FIG. 2.
Adipose differentiation and GLUT4 translocation in
3T3-L1 cells. 3T3-L1 preadipocytes were infected with a retrovirus
containing the GLUT4 reporter, and flow sorting was used to
isolate a population of cells falling within a narrow range of GFP
fluorescence intensities. These cells were expanded and used
in experiments; because the retrovirus integrates into the genome, the
population is stable. These cells undergo normal 3T3-L1 adipose
differentiation, as demonstrated by staining lipid with Oil red O. (a)
Phase-contrast (upper left) and bright-field (upper right and lower
left and right) microscopy of cells at the indicated days of
differentiation is shown. Scale bar, 50 µm. (b) Confluent 3T3-L1
preadipocytes (day 0) or 3T3-L1 cells that had undergone
differentiation for various lengths of time were stimulated or not with
insulin (160 nM, 10 min), and changes in the proportion of GLUT4
reporter present at the cell surface were measured using flow cytometry
as described in the text. Some samples were treated with either 100 nM
wortmannin (42) or 50 µM LY294002 (8, 13)
for 40 min prior to insulin addition, as noted. The amount of
the reporter within each cell varies during 3T3-L1 differentiation and
is increased approximately threefold on days 2 and 4 (not shown).
We attribute this to increased activity of the retroviral promoter as
the cells undergo clonal expansion at the onset of adipocyte
differentiation, especially since we also observed increased expression
of the reporter in preconfluent, dividing cells (91).
Because the assay measures changes in the ratio of cell surface to
total GLUT4 rather than in the absolute amount of cell surface GLUT4,
the data presented are internally controlled for this variation, and
data from different days of differentiation can be meaningfully
compared. The numbering on the vertical scale indicates a relative
measure of GLUT4 at the cell surface, and these arbitrary units cannot
be compared in absolute terms to those in other figures. In all
instances, insulin stimulates GLUT4 exocytosis, and this effect is
blocked by either of the two phosphatidylinositol-3-kinase
inhibitors.
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We next sought to determine when during the course of 3T3-L1 adipocyte
differentiation the cells become competent to translocate
GLUT4 to the
plasma membrane after insulin addition. Cells expressing
the reporter
were differentiated to different days, and the ability
of insulin to
stimulate GLUT4 exocytosis was tested as described
above. Unexpectedly,
and as shown in Fig.
2b (and subsequently
in Fig.
3a), we found that
insulin stimulates GLUT4 exocytosis
at all times during 3T3-L1
differentiation, even in undifferentiated,
confluent fibroblasts.
Moreover, as shown in Fig.
2b, insulin-triggered
GLUT4 exocytosis is
invariably blocked by treatment of the cells
with
phosphatidylinositol-3-kinase inhibitors, either wortmannin
or
LY294002. In several experiments we noted that the overall
fold
increase in cell surface GLUT4 was greater at day 2 of differentiation
than at day 0, primarily due to more pronounced intracellular
sequestration in unstimulated cells; nonetheless, the effect at
day 0 is robust and consistently observed (Fig.
2b and see
below).
To determine if the kinetics of insulin-stimulated GLUT4
externalization vary during the course of 3T3-L1 differentiation,
we
stimulated cells at different days of differentiation and assayed
changes in the proportion of GLUT4 at the cell surface as a function
of
time. As shown in Fig.
3a, insulin
triggers a rapid redistribution
of GLUT4 to the cell surface, with
identical kinetics at all days
of 3T3-L1 differentiation. In all cases
there was a biphasic response
to insulin addition, with an initial
rapid externalization of
GLUT4 such that the greatest proportion
was present on the plasma
membrane 4 to 5 min after insulin addition.
Subsequently, in all
cases, the fraction of GLUT4 on the plasma
membrane fell by 20
to 40% and reached a steady state by 15 to 20 min
after insulin
addition. This overshoot of the steady-state proportion
of GLUT4
at the cell surface in the presence of insulin presumably
corresponds
to rapid mobilization (and depletion) of GLUT4 in a
highly insulin-sensitive
compartment, as noted in the introduction.
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FIG. 3.
Kinetics of GLUT4 trafficking in 3T3-L1 cells. (a)
Confluent 3T3-L1 preadipocytes (day 0) or 3T3-L1 cells at various
stages of adipocyte differentiation (as indicated) were treated with
insulin for various lengths of time, and changes in the proportion of
GLUT4 reporter present at the cell surface were analyzed. Data are
plotted for basal cells and for cells treated with 80 nM insulin for
0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, or 30 min. Membrane
trafficking was stopped by washing with cold PBS, cells were stained at
4°C for externalized Myc epitope tag using a PE-conjugated secondary
antibody, and PE and GFP fluorescence intensities were measured using
flow cytometry as described in the text. Regardless of the state of
differentiation, insulin causes a rapid externalization of GLUT4 that
peaks 4 to 5 min after insulin addition. Subsequently, there is a net
internalization, so that a steady state in the presence of insulin is
reached 20 min after insulin addition. The numbering on the vertical
scale indicates a relative measure of GLUT4 at the cell surface, and
these arbitrary units cannot be compared in absolute terms to those in
other figures. (b) Insulin-stimulated translocation of endogenous GLUT4
to the plasma membrane of 3T3-L1 adipocytes was analyzed by subcellular
fractionation. Cells were starved overnight and then stimulated with
480 nM insulin (added from a prewarmed, 3× stock) for various amounts
of time. Cells were transferred to 4°C, washed with cold PBS, and
fractionated as described in Materials and Methods. Plasma membrane
(PM) and low-density microsomal (LDM) fractions were analyzed by
immunoblotting. Equal amounts of protein were loaded in each lane.
GLUT4 translocates to the plasma membrane in a biphasic pattern, with a
peak at 7 min after insulin addition, followed by a subsequent
decrease. A reciprocal pattern is observed in the LDM fraction. As a
control, the PM immunoblot was reprobed with an anti-insulin receptor
(-subunit) antibody, which demonstrates similar amounts of insulin
receptor at the plasma membrane at all time points. The experiment was
performed twice, with similar results each time. (c) 3T3-L1
preadipocytes (day 0) or cells at various times during adipocyte
differentiation were stimulated with 80 nM insulin for 20 min, then
placed at 4°C, and washed with an acidic buffer to remove insulin.
Cells were rewarmed in serum-free medium to allow GLUT4
reinternalization for 6, 12, 18, 24, 30, 40, 60, 90, or 120 min; some
cells that had been rewarmed for 120 min were restimulated with 80 nM
insulin for 5, 10, or 15 min. Cells were stained for cell surface Myc
epitope and analyzed by flow cytometry as described in the text. In all
instances, the GLUT4 reporter was reinternalized after removal of
insulin and recycles upon readdition of insulin.
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Because of the relative ease with which we are able to measure GLUT4 at
the cell surface, all of the data points presented
in all six panels of
Fig.
3a were acquired in the same experiment.
As noted above, our assay
is internally controlled for the amount
of reporter present within each
cell. Thus, any data point in
any of the six panels can be directly
compared to any other data
point in the figure. Clearly, unstimulated
day 2 cells have a
lower proportion of GLUT4 on the cell surface than
unstimulated
confluent fibroblasts (day 0 cells), in agreement with the
data
presented in Fig.
2b. While the slightly higher basal level of
GLUT4 at the surface of confluent fibroblasts lessens the overall
fold
increase in cell surface GLUT4 after insulin addition, the
overall
picture is similar in undifferentiated 3T3-L1 cells and
in cells that
have undergone any degree of adipose differentiation.
Importantly, the
overshoot of the final, steady-state response
in the presence of
insulin is present in all
cases.
To determine if insulin stimulates externalization of native GLUT4 with
a similar overshoot of the final steady-state response,
we performed
subcellular fractionation of 3T3-L1 adipocytes. These
cells do not
express the GLUT4 reporter protein, and we detected
endogenous GLUT4 by
immunoblotting equal amounts of total protein
from each time point. As
shown in Fig.
3b, we observed a rapid
accumulation of GLUT4 in the
plasma membrane fraction (PM GLUT4)
and a corresponding decrease in
GLUT4 present in the low-density
microsomal fraction (LDM GLUT4). The
presence of GLUT4 in the
PM fraction peaks at 7 min after insulin
addition; subsequently,
there is a decrease in the amount present.
Similarly, GLUT4 in
the LDM fraction is first depleted and subsequently
reaccumulates
slightly. As a control, we reprobed the blot of the
plasma membrane
fractions with an anti-insulin receptor
-chain
antibody. As shown
in the lower panel of the figure, this detected
similar amounts
of insulin receptor at the plasma membrane at most time
points.
There is a slight decrease immediately (3 min) after insulin
addition,
perhaps due to internalization of the receptor, and the
amount
normalizes at subsequent times. Thus, immunoblotting of
subcellular
fractions demonstrates that native GLUT4 traffics with
kinetics
similar to those we observed using the tagged GLUT4 reporter
and
the FACS-based
assay.
We next examined reinternalization and recycling of the GLUT4 reporter
after insulin removal. Based on the results shown in
Fig.
3a, cells
were stimulated with insulin for 20 min so that
the redistribution of
GLUT4 to the plasma membrane would be at
steady state, then chilled,
washed with a low-pH buffer to remove
insulin, and rewarmed in
serum-free medium for various amounts
of time. Cells were allowed to
reinternalize GLUT4 for up to 2
h, at which time they were
restimulated with insulin for 5, 10,
or 15 min. As shown in Fig.
3c,
the reporter protein is reinternalized
in undifferentiated 3T3-L1 cells
and at all times of 3T3-L1 adipocyte
differentiation and is
recycled upon restimulation with insulin
in all cases. As in Fig.
3a,
all of the data points in Fig.
3c
were collected in parallel and
can be compared, even if presented
in different panels of the figure.
The rate of reinternalization
is slightly prolonged in more
differentiated cells compared to
less differentiated cells.
Finally, restimulation with insulin
causes reexternalization of the
reporter; the magnitude and kinetics
of this effect are similar to the
initial response, and the biphasic
pattern described above is likely
present. We conclude that the
addition of Myc epitope tags and fusion
of GFP to the carboxy
terminus of GLUT4 do not impair its ability
to undergo endocytosis
or insulin-stimulated recycling at the plasma
membrane.
As noted above, we were surprised to observe insulin-stimulated GLUT4
translocation to the plasma membrane of
undifferentiated
3T3-L1 preadipocytes. To determine
if this response is general
to other cell types, we expressed the GLUT4
reporter protein in
NIH 3T3 cells by retrovirus infection, isolated a
stable pool
of cells by flow sorting, and compared insulin-stimulated
externalization
in these cells and in 3T3-L1 preadipocytes. As shown in
Fig.
4a,
the NIH 3T3 cells respond poorly
to insulin stimulation, with
a less than twofold increase in the
proportion of GLUT4 at the
cell surface. In contrast, the 3T3-L1
preadipocytes demonstrate
a rapid externalization, such that the
increase in cell surface
GLUT4 reached almost fourfold at 5 min after
insulin addition.
Subsequently, the proportion of GLUT4 at the cell
surface decreases
markedly, so that the first phase of the response
overshoots the
final steady state. The data are consistent with the
presence
of a highly insulin-responsive pool of GLUT4 in the basal
state
in 3T3-L1 preadipocytes but not in NIH 3T3 cells. After the
initial,
rapid mobilization (and depletion) of this pool, the 3T3-L1
preadipocytes
may be only marginally able to recycle GLUT4 faster than
NIH 3T3
cells, so that the difference in steady-state presence of
insulin
is minimal. We also expressed the GLUT4 reporter in a cultured,
nontransformed hepatocyte cell line (AML12 [
110]) and
found that
in these cells as well, insulin stimulated minimal
translocation
and there was no overshoot (not shown). We conclude that
insulin
regulates GLUT4 recycling through a highly insulin-responsive
mechanism present in 3T3-L1 adipocytes and, at least to some degree,
in
3T3-L1 preadipocytes, but not characteristic of all cell types.
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FIG. 4.
Kinetics of GLUT4 trafficking in 3T3-L1 preadipocytes
and NIH 3T3 cells. (a) Confluent 3T3-L1 preadipocytes and NIH 3T3 cells
expressing similar amounts of the reporter were treated with 160 nM
insulin for various lengths of time, chilled, and analyzed by FACS to
measure changes in the proportion of GLUT4 reporter present at the cell
surface. Insulin caused a much more marked redistribution of GLUT4 to
the plasma membrane of 3T3-L1 preadipocytes than of NIH 3T3 cells. (b)
Translocation of the GLUT4 reporter in 3T3-L1 preadipocytes was assayed
by subcellular fractionation and immunoblotting. After serum
starvation, cells were stimulated with 480 nM insulin (added from a
prewarmed 3× stock) for various amounts of time, then washed with cold
PBS++, and fractionated as described in Materials and
Methods. Equal amounts of protein were loaded in each lane. Insulin
caused an increase in the amount of GLUT4 reporter in the plasma
membrane (PM) fraction, with a peak response at 8 min after insulin
addition and a subsequent decrease at 20 min. A reciprocal pattern is
apparent in the low-density microsomal (LDM) fraction. The experiment
was performed twice, with similar results each time. (c) Confluent
3T3-L1 preadipocytes were cultured for 3 days in either 10% fetal
bovine serum or 10% calf serum. Cells were then starved, stimulated
with 160 nM insulin for various amounts of time, and analyzed by flow
cytometry. Samples were measured in duplicate (control samples were in
triplicate or quadruplicate). Insulin caused similar increases in the
fraction of GLUT4 reporter at the plasma membrane under both
conditions. The experiment was done twice, with similar results each
time.
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To further demonstrate that insulin mobilizes GLUT4 to the plasma
membrane of 3T3-L1 preadipocytes, we performed subcellular
fractionation and immunoblotting. Since native GLUT4 is not expressed
in undifferentiated 3T3-L1 cells, we used cells expressing the
GLUT4 reporter. As shown in Fig.
4b, insulin stimulates movement
of the
GLUT4 reporter out of the LDM fraction and into the PM
fraction. The
peak response is at 8 min after insulin addition.
By 20 min there is a
decrease from this peak in the PM fraction
as well as a slight
increase in the amount present in the LDM
fraction. These
subcellular fractionation data not only indicate
that the GLUT4
reporter is translocated in 3T3-L1 preadipocytes,
but also suggest that
there is an early overshoot before the final
steady-state response.
Thus, the subcellular fractionation data
are in accord with our flow
cytometry
data.
One possible explanation for why we observe GLUT4 translocation in
3T3-L1 preadipocytes where others have not is that we routinely
culture
these cells in 10% fetal bovine serum rather than the
more usual 10%
calf serum. We therefore cultured confluent 3T3-L1
preadipocytes in
each of these sera for 3 days prior to assaying
the effect of insulin
on GLUT4 trafficking. As shown in Fig.
4c,
cells cultured in 10% calf
serum and in 10% fetal bovine serum
responded indistinguishably. In
both cases, insulin causes a fourfold
increase in the fraction of GLUT4
present at the plasma membrane
at early (5 min) time points, with a
subsequent decrease, as previously
noted. These data are consistent
with the those presented in Fig.
3a and
4a and indicate that the effect
that we observed does not
result from culture of 3T3-L1 preadipocytes
in fetal bovine
serum.
Kinetics of insulin-stimulated GLUT4 translocation in CHO cells are
medium dependent.
To determine if the kinetics of
insulin-stimulated GLUT4 externalization are similar in CHO cells and
in 3T3-L1 cells, we assayed changes in the proportion of GLUT4 at the
cell surface of CHO cells stimulated for various amounts of time. To
parallel the conditions used for 3T3-L1 cells, some CHO cells were
placed in DMEM 2 days before the experiment; others were left in
standard CHO medium (F12). As shown in Fig.
5, CHO cells cultured in DMEM respond to
insulin with a dramatic, biphasic redistribution of GLUT4 to the cell
surface. In contrast, CHO cells cultured in F12 medium redistributed
GLUT4 less dramatically and with no overshoot of the final steady-state
proportion of GLUT4 at the cell surface in the presence of insulin. As
with 3T3-L1 cells, GLUT4 externalization in DMEM-cultured CHO cells
peaks at 4 to 5 min after insulin addition and then decreases to reach
a steady state by 20 min after insulin addition. The peak fraction of
GLUT4 at the cell surface is 5.5-fold more than that in unstimulated
cells, and in several experiments 50 to 60% of this increase is
eliminated in the subsequent decrease. For comparison, the peak
response of 3T3-L1 adipocytes in Fig. 3a was 5.4-fold over basal
(average of days 8 and 10), though the subsequent decrease to steady
state was only ~20% of this peak response.
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FIG. 5.
Culture conditions modulate the kinetics of
insulin-stimulated GLUT4 translocation in CHO cells. Two days before
the experiment, confluent CHO cells were placed in DMEM identical to
that used for 3T3-L1 adipocytes or left in F12 culture medium. Cells
were starved overnight before the experiment. On the day of the
experiment, cells were stimulated with 80 nM insulin for 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 30 min,
then transferred to 4°C, and washed with cold PBS. Staining and flow
cytometry were done as described in the text. For cells cultured in
DMEM, insulin stimulated a rapid increase in GLUT4 reporter present at
the cell surface, peaking at 4 to 5 min after insulin addition.
Subsequently, the proportion of GLUT4 on the plasma membrane decreases,
and a steady state in the presence of insulin is reached 20 min after
insulin addition. In contrast, cells cultured in F12 medium
externalized GLUT4 with monophasic kinetics, characterized by no
overshoot of the final steady-state response in the presence of
insulin. Both the initial and final proportions of GLUT4 at the cell
surface are essentially unaffected by the culture conditions, despite
the marked effect on intermediate time points. For cells cultured in
DMEM, the peak response is a 5.5-fold increase in the proportion of
GLUT4 at the cell surface compared to the basal state. The kinetics and
the amplitude of the peak response are similar in CHO cells cultured in
DMEM and in 3T3-L1 cells (compare to Fig. 3a, 4a, and 4c).
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To examine the subcellular distribution of the GLUT4 reporter in CHO
cells cultured in these two distinct media, we performed
immunofluorescence microscopy. Cells were cultured in F12 or DMEM
for 2 days, stimulated with insulin for 0, 5, or 20 min, chilled,
and stained
without permeablization to detect the externalized
Myc epitope tag. As
shown in Fig.
6a, cells cultured in F12
have
a small increase in the amount of externalized Myc epitope at
5 and significantly more at 20 min after insulin addition (top
row). In
contrast, cells cultured in DMEM have greater cell surface
staining at 5 min than at 20 min after insulin addition (third
row);
after 5 min of insulin treatment, the amount of surface
Myc-GLUT4
fluorescence is much greater in cells cultured in DMEM
than in F12. We
also examined the distribution of GLUT4 reporter
within the cells using
GFP fluorescence. In this case, we noted
that cells cultured in F12
have an abundance of the reporter protein
in the perinuclear region,
both in the absence of insulin and
after 5 or 20 min of insulin
treatment (second row). In cells
cultured in DMEM, this perinuclear
accumulation is less marked
(fourth row). Though these microscopy data
are more difficult
to quantify accurately than the flow cytometry
data (Fig.
5),
it is clear that the kinetics that we observe by
microscopy are
similar to those observe using flow cytometry.
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FIG. 6.
Culture conditions modulate the subcellular
distribution of GLUT4 in CHO cells. CHO cells expressing the GLUT4
reporter were plated on coverslips, allowed to reach confluence, and
then treated as described in the legend to Fig. 5. Two days before
microscopy, cells were changed to DMEM or continued in F12 culture
medium. On the day of microscopy, cells were serum starved for 3 h
and then stimulated with 160 nM insulin for the times indicated. (a)
Cells were chilled and stained without fixation or permeablization to
detect externalized Myc epitope tag. A red (Alexa594-conjugated)
secondary antibody was used, and the images are shown in the first
(F12) and third (DMEM) rows. GFP was used to detect the total cellular
GLUT4 reporter, and images are shown in green in the second (F12) and
fourth (DMEM) rows. Cells cultured in F12 medium have the greatest
amount of GLUT4 at the cell surface at 20 min, whereas those cultured
in DMEM have more at the cell surface at 5 min. Scale bar, 10 µm. (b)
The subcellular distribution of the GLUT4 reporter is more closely
examined. These cells were fixed, permeablized, and stained with
anti-Myc antibody and a FITC-conjugated secondary antibody in order to
increase the total green fluorescent signal, which is then due to the
combination of GFP and FITC. In cells cultured in F12, there is
prominent staining of the GLUT4 reporter in the perinuclear region;
this does not change significantly with short-term insulin treatment
(bottom panels, arrowheads). In contrast, the GLUT4 reporter is absent
from the perinuclear region in unstimulated cells cultured in DMEM and
is present more prominently in punctate, peripheral structures (top
left panel). Insulin treatment for 5 min results in a dramatic
accumulation of GLUT4 at the plasma membrane in the cells cultured in
DMEM (top center panel, arrows). In contrast, cells cultured in F12
medium have much less plasma membrane GLUT4 after 5 min of insulin
treatment (bottom center panel). By 20 min after insulin addition,
plasma membrane GLUT4 is similar in cells cultured in the two culture
media (top right and bottom right panels) and is less prominent than at
5 min in the cells cultured in DMEM (compare to top center panel). The
changes observed at the plasma membrane by microscopy correlate well
with those quantified by flow cytometry (Fig. 5). Additionally,
microscopy demonstrates that when the cells are cultured in DMEM rather
than F12 medium, GLUT4 is distributed away from the perinuclear region
and into punctate structures in the periphery. Scale bar, 10 µm.
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Figure
6b presents a higher magnification demonstrating the
intracellular distribution of the GLUT4 reporter in CHO cells
cultured
in these two distinct media. In the basal state, GLUT4
is prominent in
the perinuclear region in cells cultured in F12
medium (Fig.
6b, lower
left panel, arrowheads). In contrast, cells
cultured for 2 days in DMEM
have less GLUT4 in the perinuclear
region and more that is present in
punctate structures in the
periphery (upper left panel). Insulin
treatment for 5 min causes
a dramatic increase in plasma membrane GLUT4
in cells cultured
in DMEM (Fig.
6b, arrows, upper center panel). Cells
cultured
in F12 have a less marked accumulation of GLUT4 at the plasma
membrane after 5 min of insulin treatment (Fig.
6b, lower center
panel). By 20 min after insulin addition, the amount of GLUT4
at the
plasma membrane is similar in cells cultured in both media
and is less
than the peak response at 5 min of insulin treatment
for cells cultured
in DMEM (right panels of Fig.
6b). Of note,
cells cultured in F12
medium have continued prominent perinuclear
GLUT4 accumulation even
after 5 or 20 min of insulin treatment
(Fig.
6b, arrowheads, lower
center and right panels). These data
are consistent with the flow
cytometry data presented in Fig.
5. Thus, correlation of kinetic and
microscopy data suggests that
GLUT4 accumulates in a peripheral, highly
insulin-responsive compartment
in the basal state when the cells are
cultured in DMEM. The perinuclear
GLUT4 accumulation seen in the
cells cultured in F12 may represent
a longer-term reservoir.
Finally, the overshoot of the steady-state
proportion of GLUT4 at
the cell surface in the presence of insulin
corresponds, to a
first approximation, to the amount of GLUT4
that has accumulated
in the peripheral compartment in CHO
cells.
Amino acid sufficiency modulates highly insulin-responsive GLUT4
trafficking in CHO cells and in 3T3-L1 adipocytes.
DMEM and F12
medium differ in several respects. Though DMEM has greater glucose and
calcium concentrations, neither of these components alone or in
combination proved necessary or sufficient to cause highly
insulin-responsive (i.e., biphasic) kinetics (data not shown). We noted
that many essential amino acids are present at markedly higher
concentrations in DMEM than in F12 (see above) and tested the
possibility that these are required for highly insulin-responsive
GLUT4 trafficking. After culture for 24 to 36 h in various
media, we examined the kinetics of insulin-stimulated GLUT4
translocation in CHO cells. As shown in Fig.
7a, the degree of overshoot of the final
steady-state response in the presence of insulin correlates quite well
with the concentration of most essential amino acids in different
media. DMEM has twofold the concentrations of most amino acids present
in MEM, which in turn has 2- to 12-fold greater concentrations of most
amino acids than F12. In MEM made without any amino acids, we observed
no overshoot of the final, steady-state proportion of GLUT4 at the
plasma membrane in the presence of insulin. We also tested the kinetics
of GLUT4 externalization in CHO cells cultured in MEM with 2×, 1×,
0.2×, or no amino acids, as shown in Fig. 7b. Higher concentrations of
amino acids result in a greater overshoot of the final
steady-state response in the presence of insulin. Since glutamine was
held constant, we surmise that flux through the glucosamine pathway is
not likely to be responsible for this effect.
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FIG. 7.
Amino acid concentrations regulate the amount of rapidly
insulin-mobilized GLUT4 in CHO cells. (a) CHO cells expressing the
reporter were cultured in the indicated media for 36 h and serum
starved during the last 12 h of this period. Cells were stimulated
with 160 nM insulin for various amounts of time, then chilled, stained
for externalized Myc epitope tag, and analyzed by FACS as described in
the text to determine the relative proportion of GLUT4 at the cell
surface in each sample. Compared to the amino acid concentrations in
standard MEM (defined as 1× amino acids), concentrations of most amino
acids in DMEM are twofold higher, and concentrations of most amino
acids in F12 are only 0.08 to 0.5 times as high (depending on the
particular amino acid; see Materials and Methods). All media contained
2 mM glutamine. The degree to which insulin stimulates a transient
overshoot of the final, steady-state proportion of GLUT4 at the plasma
membrane correlates well with the concentrations of essential amino
acids in the various media. The data shown are from two separate
experiments (mean ± standard deviation) and are normalized to the
steady-state response in the presence of insulin (30-min time point).
(b) A similar experiment was performed with cells cultured in MEM
containing various concentrations of essential amino acids except for
glutamine, which was held constant (see text). Higher amino acid
concentrations cause a greater overshoot of the final, steady-state
fraction of GLUT4 at the plasma membrane after insulin addition.
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Another insulin signaling output that is sensitive to amino
acid availability is the phosphorylation of p70 S6-kinase
(
28).
In this case, withdrawal of most individual amino
acids inhibits
the ability of insulin to stimulate p70
phosphorylation to various
degrees; the most potent were leucine
and arginine, and the effect
was mimicked by rapamycin. We also
observed a modest decrease
in the proportion of GLUT4
rapidly mobilized by insulin in CHO
cells cultured without
leucine and arginine (not shown). More
striking is the
ability of rapamycin to alter the kinetics of
insulin-stimulated
GLUT4 externalization. As shown in Fig.
8,
rapamycin used over a range of
concentrations progressively eliminated
the rapid first phase of
insulin-stimulated GLUT4 externalization,
so that at the highest
concentration there was no overshoot of
the final steady-state
response. Of note, the amount of GLUT4
reporter protein, as
assessed by GFP fluorescence, did not change
by more than ~10% after
amino acid deprivation or rapamycin treatment
(not shown). These
data parallel those presented in Fig.
7 and
indicate that amino acid
sufficiency modulates GLUT4 targeting
to a highly
insulin-responsive compartment through a rapamycin-sensitive
mechanism.
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FIG. 8.
Rapamycin treatment diminishes the amount of rapidly
insulin-mobilized GLUT4 in CHO cells. CHO cells expressing the GLUT4
reporter were cultured in DMEM, treated with the indicated
concentrations of rapamycin for 36 h, and serum starved during the
last 12 h of this period. Cells were stimulated with 160 nM insulin for
the indicated times, then chilled, stained for externalized Myc
epitope, and subjected to flow cytometry to determine the relative
proportion of GLUT4 at the cell surface in each sample. Increasing
concentrations of rapamycin caused a progressive diminution of the
first phase of GLUT4 externalization (i.e., the overshoot before the
steady-state response). Simultaneously, the proportion of GLUT4 at the
cell surface in the basal state increased slightly but progressively
with increasing rapamycin concentration. Together with the data
presented in Fig. 7, these data show that amino acid abundance
regulates the amount of rapidly insulin-translocated GLUT4 in CHO cells
through a rapamycin-sensitive mechanism.
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To learn whether our observations in CHO cells are relevant to GLUT4
trafficking in 3T3-L1 adipocytes, we varied the amino
acid
concentrations in which fully differentiated 3T3-L1 adipocytes
expressing the reporter were cultured. After 36 h, we examined
the
externalization of GLUT4 after insulin addition. As shown
in Fig.
9, 3T3-L1 adipocytes cultured in MEM
with 2× amino acids
(relative to standard MEM and similar to
DMEM) mobilized GLUT4
similarly to cells cultured in DMEM (e.g., Fig.
3a). Strikingly,
culture in progressively lower concentrations of amino
acids resulted
in decreased magnitude of GLUT4 translocation (Fig.
9).
In all
cases, the overshoot of the steady-state response remains
intact,
even in the absence of amino acids (except glutamine). Indeed,
for 3T3-L1 adipocytes cultured in the absence of amino acids,
the
response demonstrates a marked overshoot followed by only
a modest fold
increase in GLUT4 at the cell surface in the steady
state; this is
somewhat reminiscent of the response we observed
using CHO cells
cultured in DMEM (Fig.
5). Culture of 3T3-L1 cells
in the presence of
rapamycin has a similar effect, as shown in
Fig.
10. Here, a progressive increase
in the concentration of rapamycin
caused a progressive decrease in the
magnitude of GLUT4 translocation
by insulin. As with amino acid
insufficiency, rapamycin treatment
of 3T3-L1 adipocytes does not alter
the presence of biphasic kinetics.
Finally, the amount of GLUT4
reporter present in each cell, as
assessed by GFP fluorescence,
did not decrease by more than 10%
after amino acid
starvation or rapamycin treatment (not shown).
Overall, the data are
consistent with the notion that amino acid
sufficiency
modulates GLUT4 trafficking through a kinetically
defined, highly
insulin-responsive compartment in 3T3-L1 adipocytes
and that this
effect is rapamycin sensitive.
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FIG. 9.
Amino acid sufficiency modulates
insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes.
3T3-L1 adipocytes expressing the GLUT4 reporter were cultured in MEM
with the indicated concentrations of amino acids for 36 h and
serum starved during the last 12 h. Cells were stimulated with 160 nM insulin for the indicated times, then chilled, stained for
externalized Myc epitope tag, and analyzed by flow cytometry to
determine the relative proportion of GLUT4 at the cell surface in each
sample. MEM with 2× amino acids approximates the amino acid
concentrations found in DMEM (see Materials and Methods). Culture of
the cells in media with poor amino acid availability results in reduced
translocation of GLUT4 to the cell surface after insulin stimulation.
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FIG. 10.
Rapamycin treatment diminishes insulin-stimulated
GLUT4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes
expressing the GLUT4 reporter were cultured in DMEM with the indicated
concentrations of rapamycin for 36 h and starved for the final
12 h of this period. Cells were then stimulated with 160 nM
insulin for the indicated amounts of time, chilled, stained to detect
externalized Myc epitope, and subjected to FACS to determine the
relative proportion of GLUT4 at the cell surface in each sample. In the
presence of increasing concentrations of rapamycin, GLUT4 was
translocated to progressively fewer degrees by insulin stimulation.
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DISCUSSION |
We have shown that insulin triggers rapid exocytosis of
GLUT4 in 3T3-L1 adipocytes and preadipocytes and in CHO cells, but not
in NIH 3T3 cells. This conclusion is based on studies using a novel,
FACS-based assay to measure changes in the proportion of GLUT4 present
at the plasma membrane and is supported by subcellular fractionation
data. This action of insulin is blocked by either of two structurally
dissimilar phosphatidylinositol-3-kinase inhibitors in 3T3-L1 cells at
all times during differentiation, suggesting that identical
signaling mechanisms are involved. Moreover, insulin stimulates
GLUT4 externalization with identical, biphasic kinetics at all times
during 3T3-L1 differentiation, and we demonstrate reinternalization and
recycling of the reporter protein as well. Strikingly, we find that
when CHO cells are cultured identically to 3T3-L1 adipocytes (in DMEM),
the kinetics and initial magnitude of insulin-stimulated GLUT4
redistribution are similar in both cell types. In contrast, when CHO
cells are cultured in their usual medium (F12), GLUT4 is only minimally
distributed to a highly insulin-responsive compartment, assessed
kinetically. The presence of GLUT4 in this rapidly mobilized
compartment correlates with a basal-state redistribution of GLUT4 out
of the perinuclear region and into punctate structures in the
periphery. We next demonstrate that the difference in GLUT4 targeting
in these two media is, at least in part, due to a difference in amino
acid concentrations. Thus, our data indicate that in CHO cells
cultured with abundant amino acids, GLUT4 accumulates in a peripheral
compartment that is rapidly mobilized after insulin addition.
Conversely, in low amino acid concentrations, GLUT4 may be targeted
primarily to the endosomal system or the trans-Golgi reticulum.
We show that rapamycin can inhibit the ability of
amino acids to cause GLUT4 accumulation in a highly
insulin-responsive compartment in CHO cells. Finally, we find that
amino acid concentrations also modulate GLUT4 trafficking in 3T3-L1
adipocytes and that this effect is also rapamycin sensitive.
Assay for GLUT4 trafficking at the cell surface.
Several
assays for GLUT4 trafficking at the cell surface have been described.
The subcellular fractionation protocol pioneered by Cushman and
colleagues provided the first evidence that glucose uptake is regulated
by redistribution of glucose transporters to the plasma membrane
(15, 102). However, the use of subcellular fractionation
to measure cell surface GLUT4 is laborious, and accurate quantitation
has been difficult because of cross-contamination of plasma membrane
fractions (35). The use of photoactivatable bismannose
compounds to selectively tag cell surface glucose transporters allowed
improved quantitation as well as measurement of steady-state internalization and externalization rate constants in the basal and
insulin-stimulated states (10, 14, 35, 36, 85, 113). Yet
this approach, too, is laborious and requires quantitative immunoprecipitation and analysis by SDS-PAGE. The preparation of plasma
membrane sheets, followed by immunostaining for GLUT4 and fluorescence
microscopy, is at best semiquantitative (50, 59, 63, 79).
Expression of an exogenous, tagged GLUT4 reporter offers greater
flexibility in detection and quantitation. Ebina and others
have shown
that an epitope tag in the first exofacial loop allows
detection of a
GLUT4 reporter on the surface of intact cells and
that changes in the
amount present can be easily quantified (
44,
75,
108).
Other investigators fused GLUT4 to GFP and observed
insulin-regulated
trafficking in individual cells by fluorescence
microscopy (
18,
67,
104). We and others have described the
use of these tags in
combination (
54; J. S. Bogan and H. F.
Lodish,
Abstr. 38th Annu. Meet. Am. Soc. Cell Biol., abstr. L65,
1998). The
level of expression of such reporter proteins is critical;
significant
overexpression of a GLUT4 reporter in primary rat
adipose cells results
in saturation of the trafficking mechanism,
with decreased insulin
responsiveness (
2). Indeed, significant
overexpression in
3T3-L1 adipocytes might be one reason that insulin
stimulated a
relatively low increase in cell surface epitope-tagged
GLUT4 in the
original study (
44).
We present here the first detailed characterization of a GLUT4 reporter
with both an exofacial epitope tag and GFP fused to
the cytosolic tail.
The assay that we describe represents a significant
advance over
previous metrics because it allows accurate quantification
of changes
in the proportion
rather than the amount
of GLUT4 that
is present at
the cell surface. These measurements are made on
a cell-by-cell basis
using flow cytometry, with the result that
alterations in cell surface
GLUT4 targeting are determined with
high specificity and precision.
Importantly, we show that this
reporter protein codistributes with
native GLUT4, that native
GLUT4 coimmunopurifies with
vesicles containing the reporter,
and that the reporter is
reinternalized after insulin removal
and recycles to the plasma
membrane upon restimulation. The time
course for GLUT4
reinternalization is slightly prolonged in 3T3-L1
adipocytes
compared to fibroblasts; this may be because adipocytes
express a
greater number of insulin receptors, which are endocytosed
with bound insulin (
78). Thus, insulin removal may not
effectively
stop insulin signaling in adipocytes. We have been careful
to
isolate a stable population of cells that express a moderate amount
of the reporter protein so as to avoid saturation of the trafficking
mechanism. The use of flow cytometry, as well as the presence
of
several tandem epitope tags, nonetheless enables us to measure
small
amounts of cell surface and total reporter. Finally, our
method allows
rapid analysis of multiple samples, making possible
the detailed
kinetic studies that we present
here.
Kinetics of GLUT4 mobilization after insulin addition.
Using
this novel, FACS-based assay to measure the relative proportion of
GLUT4 at the cell surface, we present detailed kinetic data
characterizing the insulin responsiveness of various cell types. In
3T3-L1 preadipocytes and adipocytes as well as in CHO cells, insulin
causes a rapid ~5-fold increase in plasma membrane GLUT4, peaking at
about 5 min. Cell surface GLUT4 then declines until a steady-state
level is reached at about 15 min. We also detected this transient
overshoot in cell surface GLUT4 using uninfected 3T3-L1 adipocytes and
subcellular fractionation to study the endogenous protein. In the
fractionation data, the peak occurs slightly later (7 min), possibly
because of the difficulty in synchronizing the responses of a large
number of cells (several 10-cm dishes) required for biochemical
analysis. By comparison, the FACS-based assay is done in a
six-well-plate format. It seems more likely that all of the cells in a
single well will behave as a synchronized cohort; if so, the FACS data
may be the more accurate kinetic measurement.
A second possible explanation for the slightly faster kinetics
that we observed using the FACS-based assay compared to
subcellular
fractionation could be transient trafficking of GLUT4
through
caveolae. GLUT4 present in these Triton-insoluble plasma
membrane
domains might not be detected using our fractionation and
immunoblotting
protocol, yet an externalized Myc epitope tag would
presumably
still be detected by cell surface staining of intact cells.
Whether
or not GLUT4 traffics through caveolae remains uncertain, and
data have been reported both in favor of and against this
possibility
(
26,
47,
86). Recent work indicates that
mobilization of
GLUT4 requires signaling through a CAP-Cbl complex
that localizes
transiently in Triton-insoluble, caveolin-enriched
plasma membrane
subdomains after insulin stimulation
(
6). These data are broadly
consistent with the
notion that GLUT4 may also traffic transiently
through such domains
during insulin-stimulated exocytosis. Such
a phenomenon might
also help to explain observations that GLUT4
may under some
circumstances be present in the plasma membrane
without a corresponding
increase in glucose uptake (
33,
49,
106,
111).
Our data could be consistent with a biphasic effect on either GLUT4
exocytosis or endocytosis. We favor the former interpretation
because
of biochemical and immunoelectron microscopy data indicating
that, in
adipocytes, GLUT4 is sequestered from endosomes into
a highly
insulin-responsive, TfnR-negative and CD-M6PR-negative
pool (
1,
32,
46,
55,
57,
64,
65,
76). Insulin
acts primarily to mobilize
this sequestered pool of GLUT4 to the
cell surface. Thus, in the
absence of insulin, the exocytosis
rate is limited by sequestration and
accumulation of GLUT4 in
the insulin-responsive compartment. After
insulin addition, the
GLUT4 that has accumulated in this compartment is
mobilized, and
the compartment itself is depleted of GLUT4.
Importantly, the
rate of GLUT4 exocytosis in the steady-state presence
of insulin
is now limited at some other step in the recycling pathway;
this
could be trafficking from endosomes to the insulin-responsive
compartment or directly to the cell surface (
37,
114).
Recent
analyses also suggest the presence of a unique GLUT4 storage
compartment
and suggest that traffic through this compartment to the
highly
insulin-responsive exocytic compartment may be rate limiting for
exocytosis in the steady-state presence of insulin (
55,
56).
This notion may fit with other recent work demonstrating
that
GFP-tagged GLUT4 proteins move to the cell surface directly from
the perinuclear region, which may be a storage compartment, in
3T3-L1
adipocytes (
70). In either case, the relative amount
of
GLUT4 present in the highly insulin-responsive compartment
of
unstimulated cells can be assessed indirectly, as the amount
of
GLUT4 translocated immediately (in the first 5 min) after insulin
addition (i.e., before steady state is
reached).
The above reasoning forms the rationale for our focus on the biphasic
kinetics of GLUT4 translocation in insulin-responsive
cells. The first
phase of the response (the overshoot before the
steady state)
represents mobilization of GLUT4 that has accumulated
in the
insulin-responsive pool. The second phase (the steady state
in the
presence of insulin, after 15 to 20 min) is determined
by trafficking
rates that do not inform us of the initial size
of the
insulin-responsive pool. The initial overshoot of the steady-state
GLUT4 distribution after insulin stimulation was predicted by
mathematical analysis, but measurement of GLUT4 or IRAP in plasma
membranes by subcellular fractionation or photolabeling did not
convincingly demonstrate its occurrence (
14,
37,
74,
81,
85,
111). Likewise, older studies of glucose uptake and cytochalasin
B binding failed to detect a biphasic response (
49,
52,
53).
Our observation of this phenomenon may reflect an improved
sensitivity
of our FACS assay. However, we were also able to detect
this initial
overshoot in cell surface GLUT4 using uninfected 3T3-L1
adipocytes
and subcellular fractionation. We do not know why this
response
has not been detected previously, though clearly the details
of
insulin stimulation are important. In experiments examining native
GLUT4, we used high concentrations of insulin added from a prewarmed
3× stock to simultaneously and maximally stimulate all of the
cells in
the population. We would probably have not stimulated
cells this way
were it not for our FACS results, which we sought
to confirm. Another
consideration is that the divergent results
may be due to differences
between 3T3-L1 cells and primary rat
adipocytes, which were used for
many previous kinetic studies.
Our measurement of the initial rate of
GLUT4 externalization is
more rapid than that reported by Clark et al.
(
14) and Patki
et al. (
70) but is quite
similar to data of Satoh et al. (
14,
70,
85). Indeed,
careful examination of the latter group's
data suggests a slight
overshoot of the insulin-stimulated steady-state
response in primary
rat adipocytes, though at the time this appears
to have been attributed
to uncertainty in the measurement (see
Fig.
6A of Satoh et al.
[
85]).
Cell type specificity of GLUT4 trafficking.
Using the assay
described here, we initially observed highly insulin-responsive GLUT4
trafficking in differentiated and undifferentiated 3T3-L1 cells and in
CHO cells, leading us to suggest that machinery required for
insulin-responsive GLUT4 trafficking might not be exclusive to adipose
and muscle (J. S. Bogan and H. F. Lodish, Abstr. 38th Annu.
Meet. Am. Soc. Cell Biol., abstr. L65, 1998). Very recently, similar
results led others to conclude that undifferentiated fibroblasts
possess the requisite mechanism (54). Our data demonstrate that this is not the case. We find that a highly insulin-responsive pool containing GLUT4 is present in fully differentiated 3T3-L1 adipocytes but absent in NIH 3T3 cells. This conclusion is based as much on the presence of the overshoot as it is on the magnitude of
the increase in cell surface GLUT4 after insulin addition to 3T3-L1 adipocytes. After insulin treatment of NIH 3T3 cells, we observed neither the overshoot nor a large increase in cell surface GLUT4, consistent with the generally held notion that a highly insulin-responsive mechanism is not present ubiquitously (16, 19,
27, 34, 38, 39, 83, 89, 107).
The situation in 3T3-L1 preadipocytes and in CHO cells is more
complicated. We observed highly insulin-responsive GLUT4 trafficking,
at least to some degree, in "undifferentiated" 3T3-L1
preadipocytes.
The overall fold increase in GLUT4 at the cell
surface is less
in these cells than in day 2 3T3-L1 cells or in fully
differentiated
3T3-L1 adipocytes, yet we consistently observed an
overshoot of
the final steady-state response in the presence of insulin
(Fig.
2b,
3a,
4a,
4b, and
4c). This result does not arise from culture
of the cells in fetal bovine serum rather than calf serum (Fig.
4c). We
also consistently observed greater sequestration of the
GLUT4 reporter
on day 2 of differentiation (and subsequently)
than in the
undifferentiated cells (Fig.
2b and
3a). It is possible
that two
mechanisms are operative: one for basal sequestration
that is active in
3T3-L1 adipocytes but not active in 3T3-L1 preadipocytes
and another
that is responsible for the overshoot that is active
in both 3T3-L1
adipocytes and preadipocytes, but not in NIH 3T3
cells. However, a
simpler explanation is that GLUT4 undergoes
partial targeting to a
highly insulin-responsive compartment in
the 3T3-L1 preadipocytes,
sufficient to cause the overshoot but
not sufficient to cause
significant basal sequestration (i.e.,
by drawing enough GLUT4 out of
the endosomal system). We hypothesize
that such a mechanism becomes
more active at day 2 of differentiation
and is then sufficient to
deplete GLUT4 from endosomes. This would
result in greater fold
translocation of GLUT4 to the cell surface
on day 2 because of
increased sequestration in the basal state,
consistent with our
data. Such an interpretation might also be
compatible with findings
that a biochemically detectable population
of highly insulin-responsive
vesicles first develops in 3T3-L1
cells at 2 to 3 days after induction
of differentiation (
19).
Of course, there is also likely
to be significant variation among
3T3-L1 cell lines used in different
laboratories.
The idea of partial sorting may also apply to CHO cells, which appear
to possess a highly insulin-sensitive trafficking mechanism,
but which
do not generally translocate GLUT4 by the same fold
increase seen in
fully differentiated 3T3-L1 adipocytes (
4,
16,
43,
44,
93). Importantly, CHO cells have several adipocyte-like
features, and the observation of a highly insulin-responsive GLUT4
trafficking mechanism in these cells does not imply that such
a
mechanism is present in all cell types. CHO-K1 cells transfected
with
the
3-adrenergic receptor accumulate triglyceride droplets
when
cultured in differentiation medium similar to that used for
3T3-L1
cells (
25). The untransfected CHO cells
constitutively
express hormone-sensitive lipase and
peroxisome proliferator-activated
receptor
(PPAR
), a major
regulator of adipose differentiation;
PPAR
expression is upregulated
in the presence of the
3-adrenergic
receptor and differentiation
medium. Thus, CHO cells have several
adipocyte-like characteristics,
and the notion that CHO cells
can mobilize GLUT4 from an
adipocyte-like, highly insulin-responsive
compartment is not
inconsistent with reports finding that heterogeneous
expression of
GLUT4 usually results in intracellular sequestration
without insulin
responsiveness (
4,
16,
27,
34,
39,
89,
107).
Amino acid concentrations regulate GLUT4 trafficking.
We
hypothesize that the mechanism for sorting and retaining GLUT4 in a
highly insulin-responsive compartment is somewhat less efficient in CHO
cells than in 3T3-L1 adipocytes, but that it is otherwise essentially
the same and that both cell types can be considered models for primary
adipocytes. 3T3-L1 adipocytes are the better model, based on expression
of known adipocyte marker proteins. Despite this distinction, amino
acid concentrations (and rapamycin treatment) likely alter the same
step(s) in the GLUT4 recycling pathway in both cell types. If so, then
the simplest explanation to encompass the data that we present is that
amino acid concentrations (and rapamycin) alter the rate of GLUT4
movement from the highly insulin-responsive compartment to the cell
surface in both the absence and presence of insulin. In CHO cells, most GLUT4 recycles via the endosomal system. Yet in sufficient amino acids,
traffic through the highly insulin-responsive compartment becomes
significant and is detectable (Fig. 5 and 7). In contrast, in 3T3-L1
adipocytes, the sorting-retention machinery is efficient enough to
cause some GLUT4 accumulation inand trafficking througha highly
insulin-responsive compartment even in low concentrations of amino
acids. Thus, in the presence of low amino acid concentrations or
rapamycin, the overshoot remains, though the overall fold increase in
cell surface GLUT4 is decreased (Fig. 9 and 10).
Our kinetic and microscopy data suggest that amino acid concentrations
regulate the accumulation of GLUT4 in a peripheral,
highly
insulin-responsive compartment in CHO cells. Thus, in the
presence of
high amino acid concentrations, GLUT4 is concentrated
in the periphery
and insulin stimulation results in an initial,
marked overshoot of the
steady-state proportion of GLUT4 at the
cell surface. In low amino acid
concentrations, much GLUT4 remains
in a perinuclear or trans-Golgi
location, and the kinetics of
externalization are more modest and
exhibit no overshoot of the
final, steady-state response. We do not
know if this peripheral,
insulin-mobilizable compartment in CHO cells
is the same as the
highly insulin-responsive compartment in 3T3-L1
cells. Indeed,
immunofluorescence microscopy in primary and cultured
adipocytes
suggests that TfnR-negative compartments containing GLUT4
are
present both in the periphery and in a perinuclear location
(
7,
60,
61). Perinuclear GLUT4 may be in a storage
compartment
and appears to require intact actin and microtubule
networks for
mobilization (
55,
56,
70). It may be that
insulin stimulates
both release of a GLUT4-tethering mechanism and
fusion of vesicles
containing GLUT4 with the plasma membrane and that
low amino acid
concentrations (or rapamycin) inhibit the latter but not
the former.
If such an explanation is correct, it would suggest that
the peripheral
vesicles that we observed in CHO cells cultured in DMEM
may be
docked but not fused at the plasma
membrane.
That amino acid abundance (or rapamycin treatment) would secondarily
regulate a step in the GLUT4 recycling pathway that is
also controlled
by insulin fits well with other data. Rapamycin
appears to inhibit mTOR
kinase activity quite specifically and
mimics amino acid starvation in
both yeast and mammalian cells;
in
Saccharomyces cerevisiae,
Tor protein activates metabolic pathways
for glucose utilization
(
9,
29; reviewed in reference
88).
In
mammalian cells, insulin is well known to signal through
phosphatidylinositol-3-kinase
and protein kinase B (PKB [Akt]) to
phosphorylate p70 S6 kinase
and eIF-4E BP1, and this effect is
sensitive to both amino acid
sufficiency and rapamycin (
28,
58,
90,
94). Paradoxically,
amino acids appear to inhibit
insulin-stimulated phosphorylation
of IRS-1 and IRS-2 and inhibit
phosphatidylinositol-3-kinase activity
(
71). This latter
effect may result from mTOR-mediated serine
phosphorylation and
subsequent proteasomal degradation of IRS
proteins (
30,
31,
72). A rapamycin-sensitive pathway is
also important in
controlling expression of the p85
regulatory
subunit of
phosphatidylinositol-3-kinase in muscle (
80). Other
data
indicate that rapamycin and nutrient insufficiency decrease
signaling
through atypical protein kinase C (
69,
115). Since
both
PKB and atypical protein kinase C isoforms have been implicated
in
regulation of GLUT4 trafficking, either or both of these pathways
could
be important for the response of GLUT4 to amino acid concentrations
that we describe (
17). Our data do not contradict previous
work
showing that short-term rapamycin treatment has no effect on
insulin-stimulated
glucose uptake (
21). Rather, we find an
effect of longer-term
amino acid starvation or rapamycin treatment on
GLUT4 distribution
in unstimulated cells. Thus, it seems more likely
that amino acids
and rapamycin are regulating some transcriptional or
translational
output, which in turn alters the distribution of GLUT4 in
the
basal state. Further characterization of this mechanism will be
the
subject of much future
work.
| |
ACKNOWLEDGMENTS |
We thank Zhijun Luo and Joseph Avruch for the
GLUT4myc cDNA, Bill Schiemann for the pCS2+MT vector,
Maureen Charron for antisera, and Glenn Paradis for assistance with
flow cytometry and sorting. We thank Toshio Kitamura for the pMX
retrovirus vector, Merav Socolovsky and Garry Nolan for retrovirus
packaging cell lines, and David Hirsch, Roger Lawrence, and Monty
Kreiger for the CHO cell line expressing the murine ecotropic
retrovirus receptor. We thank Natalie Hendon for assistance with tissue
culture and Jen Cook-Chrysos for help with figures. We thank three
anonymous reviewers for helpful comments on the manuscript.
This work was supported by NIH grants K11 DK02371 to J.S.B. and R37
DK47618 to H.F.L., as well as by a grant from the American Diabetes
Association to J.S.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Whitehead
Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA
02142-1479. Phone: (617) 258-5216. Fax: (617) 258-6768. E-mail:
lodish{at}wi.mit.edu.
| |
REFERENCES |
| 1.
|
Aledo, J. C.,
L. Lavoie,
A. Volchuk,
S. R. Keller,
A. Klip, and H. S. Hundal.
1997.
Identification and characterization of two distinct intracellular GLUT4 pools in rat skeletal muscle: evidence for an endosomal and an insulin-sensitive GLUT4 compartment.
Biochem. J.
325:727-732.
|
| 2.
|
Al-Hasani, H.,
D. R. Yver, and S. W. Cushman.
1999.
Overexpression of the glucose transporter GLUT4 in adipose cells interferes with insulin-stimulated translocation.
FEBS Lett.
460:338-342[CrossRef][Medline].
|
| 3.
|
Araki, S.,
J. Yang,
M. Hashiramoto,
Y. Tamori,
M. Kasuga, and G. D. Holman.
1996.
Subcellular trafficking kinetics of GLUT4 mutated at the N- and C-terminal.
Biochem. J.
315:153-159.
|
| 4.
|
Asano, T.,
K. Takata,
H. Katagiri,
K. Tsukuda,
J. L. Lin,
H. Ishihara,
K. Inukai,
H. Hirano,
Y. Yazaki, and Y. Oka.
1992.
Domains responsible for the differential targeting of glucose transporter isoforms.
J. Biol. Chem.
267:19636-19641[Abstract/Free Full Text].
|
| 5.
|
Baker, B. W.,
D. Boettiger,
E. Spooncer, and J. D. Norton.
1992.
Efficient retroviral-mediated gene transfer into human B lymphoblastoid cells expressing mouse ecotropic viral receptor.
Nucleic Acids Res.
20:5234[Free Full Text].
|
| 6.
|
Baumann, C. A.,
V. Ribon,
M. Kanzaki,
D. C. Thurmond,
S. Mora,
S. Shigematsu,
P. E. Bickel,
J. E. Pessin, and A. R. Saltiel.
2000.
CAP defines a second signalling pathway required for insulin-stimulated glucose transport.
Nature
407:202-207[CrossRef][Medline].
|
| 7.
|
Bogan, J. S., and H. F. Lodish.
1999.
Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous ACRP30 and GLUT4.
J. Cell Biol.
146:609-620[Abstract/Free Full Text].
|
| 8.
|
Bradley, R. L., and B. Cheatham.
1999.
Regulation of ob gene expression and leptin secretion by insulin and dexamethasone in rat adipocytes.
Diabetes
48:272-278[Abstract].
|
| 9.
|
Burnett, P. E.,
R. K. Barrow,
N. A. Cohen,
S. H. Snyder, and D. M. Sabatini.
1998.
RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.
Proc. Natl. Acad. Sci. USA
95:1432-1437[Abstract/Free Full Text].
|
| 10.
|
Calderhead, D. M.,
K. Kitagawa,
L. I. Tanner,
G. D. Holman, and G. E. Lienhard.
1990.
Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes.
J. Biol. Chem.
265:13801-13808.
|
| 11.
|
Charron, M. J.,
F. C. Brosius III,
S. L. Alper, and H. F. Lodish.
1989.
A glucose transport protein expressed predominately in insulin-responsive tissues.
Proc. Natl. Acad. Sci. USA
86:2535-2539[Abstract/Free Full Text].
|
| 12.
|
Charron, M. J.,
E. B. Katz, and A. L. Olson.
1999.
GLUT4 gene regulation and manipulation.
J. Biol. Chem.
274:3253-3256[Free Full Text].
|
| 13.
|
Cheatham, B.,
C. J. Vlahos,
L. Cheatham,
L. Wang,
J. Blenis, and C. R. Kahn.
1994.
Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.
Mol. Cell. Biol.
14:4902-4911[Abstract/Free Full Text].
|
| 14.
|
Clark, A. E.,
G. D. Holman, and I. J. Kozka.
1991.
Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells.
Biochem. J.
278:235-241.
|
| 15.
|
Cushman, S. W., and L. J. Wardzala.
1980.
Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell: apparent translocation of intracellular transport systems to the plasma membrane.
J. Biol. Chem.
255:4758-4762[Free Full Text].
|
| 16.
|
Czech, M. P.,
A. Chawla,
C. W. Woon,
J. Buxton,
M. Armoni,
W. Tang,
M. Joly, and S. Corvera.
1993.
Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.
J. Cell Biol.
123:127-135[Abstract/Free Full Text].
|
| 17.
|
Czech, M. P., and S. Corvera.
1999.
Signaling mechanisms that regulate glucose transport.
J. Biol. Chem.
274:1865-1868[Free Full Text].
|
| 18.
|
Dobson, S. P.,
C. Livingstone,
G. W. Gould, and J. M. Tavare.
1996.
Dynamics of insulin-stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein.
FEBS Lett.
393:179-184[CrossRef][Medline].
|
| 19.
|
El-Jack, A. K.,
K. V. Kandror, and P. F. Pilch.
1999.
The formation of an insulin-responsive vesicular cargo compartment is an early event in 3T3-L1 adipocyte differentiation.
Mol. Biol. Cell
10:1581-1594[Abstract/Free Full Text].
|
| 20.
|
Filippis, A.,
S. Clark, and J. Proietto.
1998.
Possible role for gp160 in constitutive but not insulin-stimulated GLUT4 trafficking: dissociation of gp160 and GLUT4 localization.
Biochem. J.
330:405-411.
|
| 21.
|
Fingar, D. C.,
S. F. Hausdorff,
J. Blenis, and M. J. Birnbaum.
1993.
Dissociation of pp70 ribosomal protein S6 kinase from insulin-stimulated glucose transport in 3T3-L1 adipocytes.
J. Biol. Chem.
268:3005-3008[Abstract/Free Full Text].
|
| 22.
|
Frost, S. C., and M. D. Lane.
1985.
Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes.
J. Biol. Chem.
260:2646-2652[Abstract/Free Full Text].
|
| 23.
|
Garza, L. A., and M. J. Birnbaum.
2000.
Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes.
J. Biol. Chem.
275:2560-2567[Abstract/Free Full Text].
|
| 24.
|
Green, H., and O. Kehinde.
1975.
An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion.
Cell
5:19-27[CrossRef][Medline].
|
| 25.
|
Gros, J.,
C. C. Gerhardt, and A. D. Strosberg.
1999.
Expression of human (beta)3-adrenergic receptor induces adipocyte-like features in CHO/K1 fibroblasts.
J. Cell Sci.
112:3791-3797[Abstract].
|
| 26.
|
Gustavsson, J.,
S. Parpal, and P. Stralfors.
1996.
Insulin-stimulated glucose uptake involves the transition of glucose transporters to a caveolae-rich fraction within the plasma membrane: implications for type II diabetes.
Mol. Med.
2:367-372[Medline].
|
| 27.
|
Haney, P. M.,
J. W. Slot,
R. C. Piper,
D. E. James, and M. Mueckler.
1991.
Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type.
J. Cell Biol.
114:689-699[Abstract/Free Full Text].
|
| 28.
|
Hara, K.,
K. Yonezawa,
Q. P. Weng,
M. T. Kozlowski,
C. Belham, and J. Avruch.
1998.
Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism.
J. Biol. Chem.
273:14484-14494[Abstract/Free Full Text]. (Erratum, 273:22160.)
|
| 29.
|
Hardwick, J. S.,
F. G. Kuruvilla,
J. K. Tong,
A. F. Shamji, and S. L. Schreiber.
1999.
Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.
Proc. Natl. Acad. Sci. USA
96:14866-14870[Abstract/Free Full Text].
|
| 30.
|
Hartman, M. E.,
M. Villela-Bach,
J. Chen, and G. G. Freund.
2001.
FRAP-Dependent serine phosphorylation of IRS-1 inhibits IRS-1 tyrosine phosphorylation.
Biochem. Biophys. Res. Commun.
280:776-781[CrossRef][Medline].
|
| 31.
|
Haruta, T.,
T. Uno,
J. Kawahara,
A. Takano,
K. Egawa,
P. M. Sharma,
J. M. Olefsky, and M. Kobayashi.
2000.
A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1.
Mol. Endocrinol.
14:783-794[Abstract/Free Full Text].
|
| 32.
|
Hashiramoto, M., and D. E. James.
2000.
Characterization of insulin-responsive GLUT4 storage vesicles isolated from 3T3-L1 adipocytes.
Mol. Cell. Biol.
20:416-427[Abstract/Free Full Text].
|
| 33.
|
Hausdorff, S. F.,
D. C. Fingar,
K. Morioka,
L. A. Garza,
E. L. Whiteman,
S. A. Summers, and M. J. Birnbaum.
1999.
Identification of wortmannin-sensitive targets in 3T3-L1 adipocytes: dissociation of insulin-stimulated glucose uptake and glut4 translocation.
J. Biol. Chem.
274:24677-24684[Abstract/Free Full Text].
|
| 34.
|
Herman, G. A.,
F. Bonzelius,
A. M. Cieutat, and R. B. Kelly.
1994.
A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4.
Proc. Natl. Acad. Sci. USA
91:12750-12754[Abstract/Free Full Text].
|
| 35.
|
Holman, G. D., and S. W. Cushman.
1996.
Subcellular trafficking of GLUT4 in insulin target cells.
Semin. Cell Dev. Biol.
7:259-268[CrossRef].
|
| 36.
|
Holman, G. D.,
I. J. Kozka,
A. E. Clark,
C. J. Flower,
J. Saltis,
A. D. Habberfield,
I. A. Simpson, and S. W. Cushman.
1990.
Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel: correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester.
J. Biol. Chem.
265:18172-18179[Abstract/Free Full Text].
|
| 37.
|
Holman, G. D.,
L. Lo Leggio, and S. W. Cushman.
1994.
Insulin-stimulated GLUT4 glucose transporter recycling: a problem in membrane protein subcellular trafficking through multiple pools.
J. Biol. Chem.
269:17516-17524[Abstract/Free Full Text].
|
| 38.
|
Hudson, A. W.,
D. C. Fingar,
G. A. Seidner,
G. Griffiths,
B. Burke, and M. J. Birnbaum.
1993.
Targeting of the "insulin-responsive" glucose transporter (GLUT4) to the regulated secretory pathway in PC12 cells.
J. Cell Biol.
122:579-588[Abstract/Free Full Text].
|
| 39.
|
Hudson, A. W.,
M. Ruiz, and M. J. Birnbaum.
1992.
Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts.
J. Cell Biol.
116:785-797[Abstract/Free Full Text].
|
| 40.
|
Ishii, K.,
H. Hayashi,
M. Todaka,
S. Kamohara,
F. Kanai,
H. Jinnouchi,
L. Wang, and Y. Ebina.
1995.
Possible domains responsible for intracellular targeting and insulin-dependent translocation of glucose transporter type 4.
Biochem. J.
309:813-823.
|
| 41.
|
Jhun, B. H.,
A. L. Rampal,
H. Liu,
M. Lachaal, and C. Y. Jung.
1992.
Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes: evidence of constitutive GLUT4 recycling.
J. Biol. Chem.
267:17710-17715[Abstract/Free Full Text].
|
| 42.
|
Jiang, T.,
G. Sweeney,
M. T. Rudolf,
A. Klip,
A. Traynor-Kaplan, and R. Y. Tsien.
1998.
Membrane-permeant esters of phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem.
273:11017-11024[Abstract/Free Full Text].
|
| 43.
|
Johnson, A. O.,
A. Subtil,
R. Petrush,
K. Kobylarz,
S. R. Keller, and T. E. McGraw.
1998.
Identification of an insulin-responsive, slow endocytic recycling mechanism in Chinese hamster ovary cells.
J. Biol. Chem.
273:17968-17977[Abstract/Free Full Text].
|
| 44.
|
Kanai, F.,
Y. Nishioka,
H. Hayashi,
S. Kamohara,
M. Todaka, and Y. Ebina.
1993.
Direct demonstration of insulin-induced GLUT4 translocation to the surface of intact cells by insertion of a c-myc epitope into an exofacial GLUT4 domain.
J. Biol. Chem.
268:14523-14526[Abstract/Free Full Text].
|
| 45.
|
Kandror, K. V.
1999.
Insulin regulation of protein traffic in rat adipose cells.
J. Biol. Chem.
274:25210-25217[Abstract/Free Full Text].
|
| 46.
|
Kandror, K. V., and P. F. Pilch.
1998.
Multiple endosomal recycling pathways in rat adipose cells.
Biochem. J.
331:829-835.
|
| 47.
|
Kandror, K. V.,
J. M. Stephens, and P. F. Pilch.
1995.
Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.
J. Cell Biol.
129:999-1006[Abstract/Free Full Text].
|
| 48.
|
Kao, A. W.,
B. P. Ceresa,
S. R. Santeler, and J. E. Pessin.
1998.
Expression of a dominant interfering dynamin mutant in 3T3L1 adipocytes inhibits GLUT4 endocytosis without affecting insulin signaling.
J. Biol. Chem.
273:25450-25457[Abstract/Free Full Text].
|
| 49.
|
Karnieli, E.,
M. J. Zarnowski,
P. J. Hissin,
I. A. Simpson,
L. B. Salans, and S. W. Cushman.
1981.
Insulin-stimulated translocation of glucose transport systems in the isolated rat adipose cell: time course, reversal, insulin concentration dependency, and relationship to glucose transport activity.
J. Biol. Chem.
256:4772-4777[Free Full Text].
|
| 50.
|
Katagiri, H.,
T. Asano,
H. Ishihara,
K. Inukai,
Y. Shibasaki,
M. Kikuchi,
Y. Yazaki, and Y. Oka.
1996.
Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.
J. Biol. Chem.
271:16987-16990[Abstract/Free Full Text].
|
| 51.
|
Kinoshita, S.,
B. K. Chen,
H. Kaneshima, and G. P. Nolan.
1998.
Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells.
Cell
95:595-604[CrossRef][Medline].
|
| 52.
|
Kohanski, R. A.,
S. C. Frost, and M. D. Lane.
1986.
Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes.
J. Biol. Chem.
261:12272-12281[Abstract/Free Full Text].
|
| 53.
|
Kuroda, M.,
R. C. Honnor,
S. W. Cushman,
C. Londos, and I. A. Simpson.
1987.
Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte: cAMP-independent effects of lipolytic and antilipolytic agents.
J. Biol. Chem.
262:245-253[Abstract/Free Full Text].
|
| 54.
|
Lampson, M. A.,
A. Racz,
S. W. Cushman, and T. E. McGraw.
2000.
Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts.
J. Cell Sci.
113:4065-4076[Abstract].
|
| 55.
|
Lee, W.,
J. Ryu,
R. P. Souto,
P. F. Pilch, and C. Y. Jung.
1999.
Separation and partial characterization of three distinct intracellular GLUT4 compartments in rat adipocytes: subcellular fractionation without homogenization.
J. Biol. Chem.
274:37755-37762[Abstract/Free Full Text].
|
| 56.
|
Lee, W.,
J. Ryu,
R. A. Spangler, and C. Y. Jung.
2000.
Modulation of GLUT4 and GLUT1 recycling by insulin in rat adipocytes: kinetic analysis based on the involvement of multiple intracellular compartments.
Biochemistry
39:9358-9366[CrossRef][Medline].
|
| 57.
|
Livingstone, C.,
D. E. James,
J. E. Rice,
D. Hanpeter, and G. W. Gould.
1996.
Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes.
Biochem. J.
315:487-495.
|
| 58.
|
Long, W.,
L. Saffer,
L. Wei, and E. J. Barrett.
2000.
Amino acids regulate skeletal muscle PHAS-I and p70 S6-kinase phosphorylation independently of insulin.
Am. J. Physiol. Endocrinol. Metab.
279:E301-E306[Abstract/Free Full Text].
|
| 59.
|
Macaulay, S. L.,
D. R. Hewish,
K. H. Gough,
V. Stoichevska,
S. F. MacPherson,
M. Jagadish, and C. W. Ward.
1997.
Functional studies in 3T3L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation.
Biochem. J.
324:217-224.
|
| 60.
|
Malide, D., and S. W. Cushman.
1997.
Morphological effects of wortmannin on the endosomal system and GLUT4-containing compartments in rat adipose cells.
J. Cell Sci.
110:2795-2806[Abstract].
|
| 61.
|
Malide, D.,
N. K. Dwyer,
E. J. Blanchette-Mackie, and S. W. Cushman.
1997.
Immunocytochemical evidence that GLUT4 resides in a specialized translocation post-endosomal VAMP2-positive compartment in rat adipose cells in the absence of insulin.
J. Histochem. Cytochem.
45:1083-1096[Abstract/Free Full Text].
|
| 62.
|
Malide, D.,
J. F. St-Denis,
S. R. Keller, and S. W. Cushman.
1997.
vp165 and GLUT4 share similar vesicle pools along their trafficking pathways in rat adipose cells.
FEBS Lett.
409:461-468[CrossRef][Medline].
|
| 63.
|
Marsh, B. J.,
R. A. Alm,
S. R. McIntosh, and D. E. James.
1995.
Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes.
J. Cell Biol.
130:1081-1091[Abstract/Free Full Text].
|
| 64.
|
Martin, S.,
C. A. Millar,
C. T. Lyttle,
T. Meerloo,
B. J. Marsh,
G. W. Gould, and D. E. James.
2000.
Effects of insulin on intracellular GLUT4 vesicles in adipocytes: evidence for a secretory mode of regulation.
J. Cell Sci.
113(Pt. 19):3427-3438[Abstract].
|
| 65.
|
Martin, S.,
J. E. Rice,
G. W. Gould,
S. R. Keller,
J. W. Slot, and D. E. James.
1997.
The glucose transporter GLUT4 and the aminopeptidase vp165 colocalise in tubulo-vesicular elements in adipocytes and cardiomyocytes.
J. Cell Sci.
110:2281-2291[Abstract].
|
| 66.
|
Melvin, D. R.,
B. J. Marsh,
A. R. Walmsley,
D. E. James, and G. W. Gould.
1999.
Analysis of amino and carboxy terminal GLUT-4 targeting motifs in 3T3-L1 adipocytes using an endosomal ablation technique.
Biochemistry
38:1456-1462[CrossRef][Medline].
|
| 67.
|
Oatey, P. B.,
D. H. Van Weering,
S. P. Dobson,
G. W. Gould, and J. M. Tavare.
1997.
GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein.
Biochem. J.
327:637-642.
|
| 68.
|
Onishi, M.,
S. Kinoshita,
Y. Morikawa,
A. Shibuya,
J. Phillips,
L. L. Lanier,
D. M. Gorman,
G. P. Nolan,
A. Miyajima, and T. Kitamura.
1996.
Applications of retrovirus-mediated expression cloning.
Exp. Hematol.
24:324-329[Medline].
|
| 69.
|
Parekh, D.,
W. Ziegler,
K. Yonezawa,
K. Hara, and P. J. Parker.
1999.
Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon.
J. Biol. Chem.
274:34758-34764[Abstract/Free Full Text].
|
| 70.
|
Patki, V. V.,
J. Buxton,
A. Chawla,
L. Lifshitz,
K. Fogarty,
W. Carrington,
R. Tuft, and S. Corvera.
2001.
Insulin action on GLUT4 traffic visualized in single 3T3-L1 adipocytes by using ultra-fast microscopy.
Mol. Biol. Cell
12:129-141[Abstract/Free Full Text].
|
| 71.
|
Patti, M. E.,
E. Brambilla,
L. Luzi,
E. J. Landaker, and C. R. Kahn.
1998.
Bidirectional modulation of insulin action by amino acids.
J. Clin. Investig.
101:1519-1529[Medline].
|
| 72.
|
Pederson, T. M.,
D. L. Kramer, and C. M. Rondinone.
2001.
Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation.
Diabetes
50:24-31[Abstract/Free Full Text].
|
| 73.
|
Pessin, J. E.,
D. C. Thurmond,
J. S. Elmendorf,
K. J. Coker, and S. Okada.
1999.
Molecular basis of insulin-stimulated GLUT4 vesicle trafficking: location! location! location!
J. Biol. Chem.
274:2593-2596[Free Full Text].
|
| 74.
|
Piper, R. C.,
L. J. Hess, and D. E. James.
1991.
Differential sorting of two glucose transporters expressed in insulin-sensitive cells.
Am. J. Physiol.
260:C570-C580[Abstract/Free Full Text].
|
| 75.
|
Quon, M. J.,
M. Guerre-Millo,
M. J. Zarnowski,
A. J. Butte,
M. Em,
S. W. Cushman, and S. I. Taylor.
1994.
Tyrosine kinase-deficient mutant human insulin receptors (Met1153 Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4.
Proc. Natl. Acad. Sci. USA
91:5587-5591[Abstract/Free Full Text].
|
| 76.
|
Ramm, G.,
J. W. Slot,
D. E. James, and W. Stoorvogel.
2000.
Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-Golgi network in rat adipocytes.
Mol. Biol. Cell
11:4079-4091[Abstract/Free Full Text].
|
| 77.
|
Rea, S., and D. E. James.
1997.
Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles.
Diabetes
46:1667-1677[Abstract].
|
| 78.
|
Reed, B. C., and M. D. Lane.
1980.
Insulin receptor synthesis and turnover in differentiating 3T3-L1 preadipocytes.
Proc. Natl. Acad. Sci. USA
77:285-289[Abstract/Free Full Text].
|
| 79.
|
Robinson, L. J.,
S. Pang,
D. S. Harris,
J. Heuser, and D. E. James.
1992.
Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilzed 3T3-L1 adipocytes: effects of ATP, insulin, and GTPS and localization of GLUT4 to clathrin lattices.
J. Cell Biol.
117:1181-1196[Abstract/Free Full Text].
|
| 80.
|
Roques, M., and H. Vidal.
1999.
A phosphatidylinositol 3-kinase/p70 ribosomal S6 protein kinase pathway is required for the regulation by insulin of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase gene expression in human muscle cells.
J. Biol. Chem.
274:34005-34010[Abstract/Free Full Text].
|
| 81.
|
Ross, S. A.,
J. J. Herbst,
S. R. Keller, and G. E. Lienhard.
1997.
Trafficking kinetics of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes.
Biochem. Biophys. Res. Commun.
239:247-251[CrossRef][Medline].
|
| 82.
|
Ross, S. A.,
S. R. Keller, and G. E. Lienhard.
1998.
Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells.
Biochem. J.
330:1003-1008.
|
| 83.
|
Ross, S. A.,
H. M. Scott,
N. J. Morris,
W. Y. Leung,
F. Mao,
G. E. Lienhard, and S. R. Keller.
1996.
Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes.
J. Biol. Chem.
271:3328-3332[Abstract/Free Full Text].
|
| 84.
|
Saltiel, A. R.
2001.
New perspectives into the molecular pathogenesis and treatment of type 2 diabetes.
Cell
104:517-529[CrossRef][Medline].
|
| 85.
|
Satoh, S.,
H. Nishimura,
A. E. Clark,
I. J. Kozka,
S. J. Vannucci,
I. A. Simpson,
M. J. Quon,
S. W. Cushman, and G. D. Holman.
1993.
Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells: evidence that exocytosis is a critical site of hormone action.
J. Biol. Chem.
268:17820-17829[Abstract/Free Full Text].
|
| 86.
|
Scherer, P. E.,
M. P. Lisanti,
G. Baldini,
M. Sargiacomo,
C. C. Mastick, and H. F. Lodish.
1994.
Induction of caveolin during adipogenesis and association of GLUT4 with caveolin-rich vesicles.
J. Cell Biol.
127:1233-1243[Abstract/Free Full Text].
|
| 87.
|
Scherer, P. E.,
S. Williams,
M. Fogliano,
G. Baldini, and H. F. Lodish.
1995.
A novel serum protein similar to C1q, produced exclusively in adipocytes.
J. Biol. Chem.
270:26746-26749[Abstract/Free Full Text].
|
| 88.
|
Schmelzle, T., and M. N. Hall.
2000.
TOR, a central controller of cell growth.
Cell
103:253-262[CrossRef][Medline].
|
| 89.
|
Schurmann, A.,
I. Monden,
H. G. Joost, and K. Keller.
1992.
Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells.
Biochim. Biophys. Acta
1131:245-252[Medline].
|
| 90.
|
Scott, P. H.,
G. J. Brunn,
A. D. Kohn,
R. A. Roth, and J. C. Lawrence.
1998.
Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway.
Proc. Natl. Acad. Sci. USA
95:7772-7777[Abstract/Free Full Text].
|
| 91.
|
Shao, D., and M. A. Lazar.
1997.
Peroxisome proliferator activated receptor gamma, CCAAT/enhancer-binding protein alpha, and cell cycle status regulate the commitment to adipocyte differentiation.
J. Biol. Chem.
272:21473-21478[Abstract/Free Full Text].
|
| 92.
|
Shapiro, L., and P. E. Scherer.
1998.
The crystal structure of a complement-1q family protein suggests an evolutionary link to tumor necrosis factor.
Curr. Biol.
8:335-338[CrossRef][Medline].
|
| 93.
|
Shibasaki, Y.,
T. Asano,
J. L. Lin,
K. Tsukuda,
H. Katagiri,
H. Ishihara,
Y. Yazaki, and Y. Oka.
1992.
Two glucose transporter isoforms are sorted differentially and are expressed in distinct cellular compartments.
Biochem. J.
281:829-834.
|
| 94.
|
Shigemitsu, K.,
Y. Tsujishita,
K. Hara,
M. Nanahoshi,
J. Avruch, and K. Yonezawa.
1999.
Regulation of translational effectors by amino acid and mammalian target of rapamycin signaling pathways: possible involvement of autophagy in cultured hepatoma cells.
J. Biol. Chem.
274:1058-1065[Abstract/Free Full Text].
|
| 95.
|
Simpson, F.,
J. P. Whitehead, and D. E. James.
2001.
GLUT4at the crossroads between membrane trafficking and signal transduction.
Traffic
2:2-11[CrossRef][Medline].
|
| 96.
|
Slot, J. W.,
H. J. Geuze,
S. Gigengack,
D. E. James, and G. E. Lienhard.
1991.
Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat.
Proc. Natl. Acad. Sci. USA
88:7815-7819[Abstract/Free Full Text].
|
| 97.
|
Slot, J. W.,
H. J. Geuze,
S. Gigengack,
G. E. Lienhard, and D. E. James.
1991.
Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.
J. Cell Biol.
113:123-135[Abstract/Free Full Text].
|
| 98.
|
Smith, R. M.,
M. J. Charron,
N. Shah,
H. F. Lodish, and L. Jarett.
1991.
Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4.
Proc. Natl. Acad. Sci. USA
88:6893-6897[Abstract/Free Full Text].
|
| 99.
|
Socolovsky, M.,
I. Dusanter-Fourt, and H. F. Lodish.
1997.
The prolactin receptor and severely truncated erythropoietin receptors support differentiation of erythroid progenitors.
J. Biol. Chem.
272:14009-14012[Abstract/Free Full Text].
|
| 100.
|
Subtil, A.,
M. A. Lampson,
S. R. Keller, and T. E. McGraw.
2000.
Characterization of the insulin-regulated endocytic recycling mechanism in 3T3-L1 adipocytes using a novel reporter molecule.
J. Biol. Chem.
275:4787-4795[Abstract/Free Full Text].
|
| 101.
|
Sumitani, S.,
T. Ramlal,
R. Somwar,
S. R. Keller, and A. Klip.
1997.
Insulin regulation and selective segregation with glucose transporter-4 of the membrane aminopeptidase vp165 in rat skeletal muscle cells.
Endocrinology
138:1029-1034[Abstract/Free Full Text].
|
| 102.
|
Suzuki, K., and T. Kono.
1980.
Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site.
Proc. Natl. Acad. Sci. USA
77:2542-2545[Abstract/Free Full Text].
|
| 103.
|
Swift, S. E.,
J. B. Lorens,
P. Achacoso, and G. P. Nolan.
1998.
Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems, p. 10.17.14-10.17.29.
In
J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology, vol. 2. John Wiley and Sons, Inc., New York, N.Y.
|
| 104.
|
Thurmond, D. C.,
B. P. Ceresa,
S. Okada,
J. S. Elmendorf,
K. Coker, and J. E. Pessin.
1998.
Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes.
J. Biol. Chem.
273:33876-33883[Abstract/Free Full Text].
|
| 105.
|
Todaka, M.,
H. Hayashi,
T. Imanaka,
Y. Mitani,
S. Kamohara,
K. Kishi,
K. Tamaoka,
F. Kanai,
M. Shichiri,
N. Morii,
S. Narumiya, and Y. Ebina.
1996.
Roles of insulin, guanosine 5'-[gamma-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation.
Biochem. J.
315:875-882.
|
| 106.
|
Vannucci, S. J.,
H. Nishimura,
S. Satoh,
S. W. Cushman,
G. D. Holman, and I. A. Simpson.
1992.
Cell surface accessibility of GLUT4 glucose transporters in insulin-stimulated rat adipose cells: modulation by isoprenaline and adenosine.
Biochem. J.
288:325-330.
|
| 107.
|
Verhey, K. J.,
S. F. Hausdorff, and M. J. Birnbaum.
1993.
Identification of the carboxy terminus as important for the isoform-specific subcellular targeting of glucose transporter proteins.
J. Cell Biol.
123:137-147[Abstract/Free Full Text].
|
| 108.
|
Wang, Q.,
Z. Khayat,
K. Kishi,
Y. Ebina, and A. Klip.
1998.
GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay.
FEBS Lett.
427:193-197[CrossRef][Medline].
|
| 109.
|
Wei, M. L.,
F. Bonzelius,
R. M. Scully,
R. B. Kelly, and G. A. Herman.
1998.
GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.
J. Cell Biol.
140:565-575[Abstract/Free Full Text].
|
| 110.
|
Wu, J. C.,
G. Merlino, and N. Fausto.
1994.
Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha.
Proc. Natl. Acad. Sci. USA
91:674-678[Abstract/Free Full Text].
|
| 111.
|
Yang, J.,
A. E. Clark,
R. Harrison,
I. J. Kozka, and G. D. Holman.
1992.
Trafficking of glucose transporters in 3T3-L1 cells: inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins.
Biochem. J.
281:809-817.
|
| 112.
|
Yang, J.,
A. E. Clark,
I. J. Kozka,
S. W. Cushman, and G. D. Holman.
1992.
Development of an intracellular pool of glucose transporters in 3T3-L1 cells.
J. Biol. Chem.
267:10393-10399[Abstract/Free Full Text].
|
| 113.
|
Yang, J., and G. D. Holman.
1993.
Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells.
J. Biol. Chem.
268:4600-4603[Abstract/Free Full Text].
|
| 114.
|
Yeh, J. I.,
K. J. Verhey, and M. J. Birnbaum.
1995.
Kinetic analysis of glucose transporter trafficking in fibroblasts and adipocytes.
Biochemistry
34:15523-15531[CrossRef][Medline].
|
| 115.
|
Ziegler, W. H.,
D. B. Parekh,
J. A. Le Good,
R. D. Whelan,
J. J. Kelly,
M. Frech,
B. A. Hemmings, and P. J. Parker.
1999.
Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex.
Curr. Biol.
9:522-529[CrossRef][Medline].
|
Molecular and Cellular Biology, July 2001, p. 4785-4806, Vol. 21, No. 14
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.14.4785-4806.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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-
Shewan, A. M., van Dam, E. M., Martin, S., Luen, T. B., Hong, W., Bryant, N. J., James, D. E.
(2003). GLUT4 Recycles via a trans-Golgi Network (TGN) Subdomain Enriched in Syntaxins 6 and 16 But Not TGN38: Involvement of an Acidic Targeting Motif. Mol. Biol. Cell
14: 973-986
[Abstract]
[Full Text]
-
Xu, Z., Kandror, K. V.
(2002). Translocation of Small Preformed Vesicles Is Responsible for the Insulin Activation of Glucose Transport in Adipose Cells. EVIDENCE FROM THE IN VITRO RECONSTITUTION ASSAY. J. Biol. Chem.
277: 47972-47975
[Abstract]
[Full Text]
-
Crespo, J. L., Hall, M. N.
(2002). Elucidating TOR Signaling and Rapamycin Action: Lessons from Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev.
66: 579-591
[Abstract]
[Full Text]
-
Zeigerer, A., Lampson, M. A., Karylowski, O., Sabatini, D. D., Adesnik, M., Ren, M., McGraw, T. E.
(2002). GLUT4 Retention in Adipocytes Requires Two Intracellular Insulin-regulated Transport Steps. Mol. Biol. Cell
13: 2421-2435
[Abstract]
[Full Text]
-
Kanzaki, M., Watson, R. T., Khan, A. H., Pessin, J. E.
(2001). Insulin Stimulates Actin Comet Tails on Intracellular GLUT4-containing Compartments in Differentiated 3T3L1 Adipocytes. J. Biol. Chem.
276: 49331-49336
[Abstract]
[Full Text]
-
Huang, J., Imamura, T., Olefsky, J. M.
(2001). Insulin can regulate GLUT4 internalization by signaling to Rab5 and the motor protein dynein. Proc. Natl. Acad. Sci. USA
98: 13084-13089
[Abstract]
[Full Text]
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Abstract
Introduction
