REVIEWS

REGULATED TRANSPORT OF THE

GLUCOSE TRANSPORTER GLUT4
Nia J. Bryant, Roland Govers and David E. James
In muscle and fat cells, insulin stimulates the delivery of the glucose transporter GLUT4 from an
intracellular location to the cell surface, where it facilitates the reduction of plasma glucose levels.
Understanding the molecular mechanisms that mediate this translocation event involves integrating
our knowledge of two fundamental processes — the signal transduction pathways that are
triggered when insulin binds to its receptor and the membrane transport events that need to be
modified to divert GLUT4 from intracellular storage to an active plasma membrane shuttle service.

FACILITATIVE SUGAR
TRANSPORTER
A polytopic membrane protein
that transports sugars down a
concentration gradient in an
energy-independent manner.
TYPE II DIABETES
Also known as non-insulin-
dependent diabetes or maturity
onset diabetes.
Garvan Institute of Medical
Research, 384 Victoria Road,
Darlinghurst, New South
Wales 2010, Australia.
Correspondence to D.E.J.
e-mail:
d.james@garvan.org.au
DOI: 10.1038/nrm782
Glucose is a fundamental source of energy for all
eukaryotic cells. In humans, although all cells use glu-
cose for their energy needs, the main consumer under
basal conditions is the brain, which accounts for as
much as 80% of whole-body consumption. The energy
is provided by the breakdown of endogenous glycogen
stores that are primarily in the liver. These whole-body
energy stores are replenished from glucose in the diet,
which, after being digested and absorbed across the gut
wall, is distributed among the various tissues of the body
(reviewed in REF. 1). This distribution process involves a
family of transport proteins — called GLUTs — which
act as shuttles to move sugar across the cell surface.
These polytopic membrane proteins (FIG. 1) form an
aqueous pore across the membrane through which
glucose can move. A large family of FACILITATIVE SUGAR
TRANSPORTERS exists in mammals, the individual mem-
bers of which differ in their tissue distribution and
kinetic properties, as well as in their intracellular local-
ization. The latter property is of particular interest for
glucose transport in specialized cell types, and provides
the basis for polarized glucose transport in epithelial
cells, such as those in the gut, or for acute regulation of
glucose transport, as is observed in muscle and fat cells
after a meal. Many mammalian tissues, such as the
brain, have a constitutively high glucose requirement
and have been endowed with transporters that are con-
stitutively targeted to the cell surface (for example,
GLUTs 1–3). By contrast, certain tissues, such as muscle
and adipose tissue, have acquired a highly specialized
glucose-transport system, the activity of which can be
rapidly upregulated to allow these tissues to increase
their rate of glucose transport by 10–40-fold within
minutes of exposure to a particular stimulus (reviewed
in REF. 1). This system is crucial during exercise, when
the metabolic demands of skeletal muscle can increase
more than 100-fold, and during the absorptive period
(after a meal), to facilitate the rapid insulin-dependent
storage of glucose in muscle and adipose tissue, so pre-
venting large fluctuations in blood glucose levels.
Dysfunctional glucose uptake into muscle and fat cells
contributes to the onset of TYPE II DIABETES (BOX 1).
In 1980, it was reported that, in rat adipocytes,
insulin triggers the movement of the sugar transporter
that is found in these cells from an intracellular store to
the plasma membrane2,3. This translocation hypothesis
was later confirmed when GLUT4 was identified as the
main glucose transporter in these cells. GLUT4, which
is expressed primarily in muscle and fat cells, is found in
a complex intracellular tubulo–vesicular network that is
connected to the endosomal–trans-Golgi network
(TGN) system. In the absence of stimulation, GLUT4 is
almost completely excluded from the plasma mem-
brane (FIG. 2). The addition of insulin, or exercise in the
case of muscle cells, causes GLUT4 to shift from its
intracellular location to the plasma membrane (FIG. 2).
Several observations indicate that GLUT4 has a crucial
role in whole-body glucose homeostasis. First, insulin-
stimulated glucose transport is an important rate-limit-
ing step for glucose metabolism in both muscle and fat

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REVIEWS

Sugar moiety an insulin-regulated step(s). Although many important

Plasma
membrane
Cytoplasm NH2
COOH
H
Figure 1 | Schematic representation of the GLUT family of proteins. The GLUT family of
proteins comprises 13 members at present, which are predicted to span the membrane 12 times
with both amino- and carboxyl-termini located in the cytosol. On the basis of sequence homology
and structural similarity, three subclasses of sugar transporters have been defined: Class I (GLUTs
1–4) are glucose transporters; Class II (GLUTs 5, 7, 9 and 11) are fructose transporters; and Class
III (GLUTs 6, 8, 10,12 and HMIT1) are structurally atypical members of the GLUT family, which are
poorly defined at present. The diagram shows a homology plot between GLUT1 and GLUT4.
Residues that are unique to GLUT4 are shown in red.
tissue, and is severely disrupted in type II diabetes1 (BOX 1);
second, disruption of GLUT4 expression in mice results
in insulin resistance4; and overexpression of GLUT4
ameliorates diabetes in the DB/DB MOUSE model5.
Analysing the molecular and cellular regulation of
GLUT4 transport not only promises to provide new
insights into protein sorting, but could also yield new
targets for therapeutic intervention in what could well
be one of the most prevalent diseases that we will have
to confront in the future.
Understanding the regulation of GLUT4 and glucose
transport has proved to be extremely challenging, prin-
cipally because it involves several signal-transduction
pathways that are superimposed on a complex series of
vesicle transport processes. Insulin binds to a surface
receptor on muscle and fat cells and triggers a cascade of
signalling events (BOX 2) that culminates in GLUT4
translocation. Studies of this process have been carried
out using two approaches.‘Outside–in’ approaches have
largely focused on mapping insulin-specific signalling
pathways in muscle and fat cells with the view to identi-
fying downstream targets that directly control GLUT4
translocation. Conversely,‘inside–out’ approaches have
used cell-biological studies to map the intracellular
transport itinerary of GLUT4 with the aim of identifying
signalling molecules that are integral to the insulin reg-
ulation of GLUT4 translocation have been identified
(BOX 2), any convergence between these two approaches
remains to be achieved. In this review, we focus on our
cell-biological understanding of GLUT4 transport,
and highlight potential regulatory sites of the insulin-
signalling cascade.
GLUT4 transport
GLUT4 is found in many organelles, including the
plasma membrane, sorting endosomes, recycling endo-
somes, the TGN and vesicles that mediate the transport
of GLUT4 between these compartments (FIG. 3).
Presumably this localization represents a complex and
dynamic transport itinerary, and it raises several impor-
tant questions. How does GLUT4 transport from one
organelle to another, what is the relationship between
these pathways and the intracellular sequestration of
GLUT4 in basal cells, and which of these steps does
insulin influence to affect GLUT4 exocytosis?
In non-stimulated adipocytes, the rate of GLUT4 exo-
cytosis is 10-fold slower than that of the transferrin recep-
tor (TfR) — one of the most well-studied constitutive
recycling proteins in mammalian cells6,7. To account for
this, GLUT4 must be selectively retained in one of its
intracellular locations, packaged into a specialized com-
partment that remains static in the absence of insulin, or
involved in a dynamic intracellular transport loop that
excludes it from recycling endosomal vesicles. Current
evidence favours a role for all three mechanisms, which
emphasizes the complexity of this process. Another pro-
tein, the insulin-responsive aminopeptidase (IRAP),
which was recently described as a receptor for angiotensin
IV (REF. 8), colocalizes with GLUT4 and is transported in a
very similar manner9. Below, we discuss studies concern-
ing the transport of either GLUT4 or IRAP, with a partic-
ular focus on adipocytes and muscle cells. We also com-
pare this with transport in cells such as fibroblasts that are
not, or only mildly, responsive to insulin, as key differ-
ences have been identified that provide new insights into
our understanding of insulin action.
Endosomal sorting of GLUT4
Morphological studies in both muscle and fat cells
indicate that, although there is some overlap of GLUT4
with markers of the endocytic system such as the TfR, a

Box 1 | Type II diabetes

DB/DB MOUSE
A genetic mouse model of type
II diabetes and obesity. The
defect has been mapped to the
gene for the leptin receptor.
The prevalence of type II diabetes is increasing at an alarming rate. In 1998, 143 million people worldwide suffered from
this disease, and it is likely that this number will double over the next 20–30 years71. The incidence of type II diabetes
increases sharply with age, and it is highly prevalent in certain ethnic groups. For example, 10–30% of Australian
aborigines are currently thought to have type II diabetes, and this number is predicted to increase to more than 50% in
the next ten years. The disease is characterized by defective insulin action, a condition that is referred to as insulin
resistance. Insulin resistance is characterized by dysfunctional glucose uptake into muscle and adipose tissue, in
conjunction with an oversupply of glucose from the liver, which results in high circulating plasma glucose levels. This
causes many of the complications of type II diabetes, including eye, nerve and kidney disease. The highest contributor
to morbidity and mortality in type II diabetes is heart disease and, strikingly, type II diabetes is one of the main causes
of heart disease in the Western world.

268 | APRIL 2002 | VOLUME 3 www.nature.com/reviews/molcellbio


EVANESCENCE WAVE
MICROSCOPY
A technique in which only
fluorophores within a
100–220 nm field above a glass
coverslip are excited, which
allows localization of molecules
very close to the cell surface.
ATRIAL CARDIOMYOCYTE
A heart muscle cell.
AP-1
(Adaptor protein complex 1).
Adaptor proteins link cargo
molecules on membranes with
coat proteins such as clathrin.
Several classes of adaptor
proteins have been identified
and shown to be involved in
different transport steps. AP-1 is
thought to regulate transport
from the trans-Golgi network to
endosomes.
SNARE
(soluble N-ethylmaleimide
sensitive factor attachment
protein receptor). A family of
membrane-tethered coiled-coil
proteins that regulate fusion
reactions and target specificity in
the vacuolar system. They can be
divided into v-SNAREs (vesicle)
and t-SNAREs (target) on the
basis of their localization, or into
Q-SNAREs and R-SNAREs on
the basis of a highly conserved
amino acid.
CONVERTASE
An enzyme that is responsible
for protein activation through
proteolytic activity.
COAT-ASSOCIATED PROTEIN
A protein that links cargo
molecules to vesicle coats.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
significant pool of GLUT4 is not localized to
endosomes10. Endosomes can be chemically ablated on
uptake of horseradish peroxidase (HRP)-conjugated
transferrin11. This procedure can be used to determine
the proportion of a protein that is localized to the endo-
somal system, and has shown that only 30–40% of
GLUT4 is found in endosomes under basal conditions11.
Furthermore, chemical ablation of endosomes does not
block insulin-stimulated GLUT4 translocation in
adipocytes12. So, although insulin has a modest effect on
general recycling through the endosomal system, which
results in the translocation of many molecules — includ-
ing the TfR — to the plasma membrane, endosomes do
not seem to be the main insulin-sensitive GLUT4 storage
compartment. Intriguingly, in fibroblasts, most GLUT4
and IRAP colocalizes with the TfR in endosomes13. This
might explain the relatively small insulin effect that is
observed in these cells (twofold increase), and also indi-
cates that the transport of GLUT4 is more specialized in
bona fide insulin-responsive cell types. Nevertheless,
there is evidence for a selective retention mechanism of
GLUT4 and IRAP in fibroblasts, but this is clearly insuffi-
cient to generate the robust insulin effect that is observed
in adipocytes. Intriguingly, studies that use EVANESCENCE
13
WAVE MICROSCOPY in 3T3-L1 fibroblasts , and an in vitro
assay that reconstitutes budding of transport vesicles
from endosomes in Chinese hamster ovary (CHO)
cells14, have shown that GLUT4 is packaged into endoso-
mal transport vesicles that are distinct from those that
contain the TfR. However, in fibroblasts, the GLUT4
transport vesicles that bud from endosomes are very
short-lived, presumably because they fuse rapidly with
the plasma membrane, even in the absence of insulin.
This is clearly not the case in adipocytes, though, as there
is little exocytosis of GLUT4 under basal conditions6.
Collectively, these studies implicate an important role for
the segregation of GLUT4 from the endosomal system in
the insulin-responsive transport of GLUT4.
The role of the trans-Golgi network
What is the fate of GLUT4-containing endosomal trans-
port vesicles in adipocytes? One possibility is that they
are somehow retained in the cytosol in the absence of
insulin, and are discharged in response to insulin. This
simple model, however, overlooks the fact that a propor-
tion of GLUT4, which does not represent newly synthe-
sized protein, is present in the TGN of both muscle and
fat cells15,16. Interestingly, recent evidence indicates that
GLUT4 recycles rapidly between the TGN and endo-
somes. First, morphological studies in ATRIAL CARDIOMY-
OCYTES indicate that 60% of all the GLUT4 expressed in
this cell type is localized to secretory granules that con-
tain atrial natriuretic factor (ANF)17. GLUT4 seems to
enter these granules at the TGN, and as the localization
of GLUT4 here is not blocked by protein-synthesis
inhibitors, these studies indicate that a GLUT4 recycling
pathway, possibly from endosomes, merges with the
secretory pathway at the TGN. Second, GLUT4 has been
shown to localize to adaptor-protein-1 (AP-1)positive,
clathrin-coated, vesicles in the vicinity of the TGN in
adipocytes18. Third, by following the internalization of
REVIEWS
– Insulin + Insulin
Figure 2 | Insulin triggers the translocation of GLUT4 from
an intracellular location to the plasma membrane of
adipocytes. The figure shows a confocal image of 3T3-L1
adipocytes incubated either with (right panel), or without (left
panel) 100 nM insulin for 15 mins. The location of GLUT4 in
these cells is shown using an antibody that specifically
recognizes GLUT4 and a secondary antibody conjugated to
Alexa-488 (shown in green). Confocal-laser-scanned sections
were obtained from the base of the cells to the perinuclear
region, which were then stacked to create a three-dimensional
reconstruction. Images courtesy of Timo Meerloo, Garvan
Institute of Medical Research, Darlinghurst, Australia.
GLUT4 from the cell surface of adipocytes, it has been
shown that the transporter is transported through endo-
somes into a perinuclear compartment that is distinct
from recycling endosomes10. Using a similar approach,
we have recently shown that this perinuclear compart-
ment represents a subdomain of the TGN that also
contains the SNARE proteins Syntaxin 6 and Syntaxin 16
(A. Shewan, S. Martin, D. E. J., unpublished observa-
tions). The transport of GLUT4 between endosomes
and the TGN is regulated by a unique acidic targeting
motif in the carboxyl terminus of GLUT4 (REF. 19).
Intriguingly, the transport of other proteins, such as the
pro-protein CONVERTASES furin and PC6B, between endo-
somes and the TGN is also regulated by acidic targeting
motifs20,21. The COAT-ASSOCIATED PROTEIN phosphofurin
acidic cluster sorting protein 1 (PACS1) has been found
to bind to the acidic motif in the furin tail22. So far, we
have been unable to detect an interaction between
PACS1 and GLUT4 (S. Rea, D. E. J., unpublished obser-
vations), which indicates that other, related coat proteins
might function in this specific region of the cell.
The recycling of membrane proteins through the
TGN is unusual in that most endosomal proteins do
not take this route. However, several molecules, includ-
ing certain bacterial toxins23, mannose-6-phosphate
receptors, TGN38 and pro-protein convertases
(reviewed in REF. 20), have been shown to follow this
pathway. Once in the TGN, these molecules are sorted
to one of many different destinations — this is an
important function of this organelle. For example,
Shiga toxin is transported to the endoplasmic reticu-
lum (ER), the mannose-6-phosphate receptors are
transported back to endosomes, and the pro-protein
convertases enter the secretory pathway. Once GLUT4
arrives at the TGN, its fate is uncertain. Despite the fact
VOLUME 3 | APRIL 2002 | 2 6 9
REVIEWS

ARNO
(ARF nucleotide binding-site
opener). This activates ADP-
ribosylating factors (ARFs),
which are known to have a role
in protein sorting and vesicle
budding.
γ-EAR-CONTAINING
This represents a protein
domain within the γ-subunit of
coat adaptor proteins.
that GLUT4 re-enters the secretory pathway in atrial
cardiomyocytes, it is subsequently retrieved from these
granules before they are delivered to the cell surface,
possibly by an AP-1-mediated pathway17. Consistent
with this, GLUT4 does not colocalize with other secre-
tory proteins, such as the 30 kDa adipocyte comple-
ment-related protein (ACRP30), leptin or adipsin, in
adipocytes24–26. Nevertheless, it seems evident that tran-
sit through the TGN probably precedes the packaging
of GLUT4 into its insulin-responsive compartment
because prolonged incubation of adipocytes at 19°C —
a temperature that blocks exit from the TGN —
inhibits insulin action27.
The TGN is clearly a complex and central sorting sta-
tion in which key sorting decisions are made. Many coat-
protein complexes, including AP-1, AP-3, AP-4, as well as
the Golgi-localized, γ-EAR-CONTAINING, ARF-binding (GGA)
family of coat proteins28 have been localized to the TGN
and might regulate transport into or out of this organelle.
Moreover, these coats are multisubunit protein com-
plexes, and it has been shown that unique isoforms of just
one component of a particular coat is sufficient to gener-
ate cell-type specific sorting. At least a portion of GLUT4
must be delivered back to endosomes to account for the
relatively large pool (~30–40%) that is found in this com-
partment. AP-1 coated vesicles have been proposed to

Box 2 | Insulin signalling pathways that control glucose transport in muscle and fat cells
At least two discrete signalling pathways have been implicated
– Insulin Flotillin
in insulin-regulated GLUT4 translocation. The first involves
Insulin receptor Lipid raft
the lipid kinase phosphatidylinositol 3-kinase (PI3K), and the
second involves the proto-oncoprotein c-Cbl. Insulin binds to
its receptor — a heterotetramer that is comprised of two
α- and two β-subunits — on the surface of target cells. This
binding induces a conformational change in the receptor, and
leads to activation of its tyrosine-kinase domain, which is TC10
located within the intracellular portion of its β-subunits. On GDP
activation, the receptor phosphorylates several proximal PDK CAP
substrates, including members of the insulin-receptor- IRS-1
substrate family (IRS-1 and IRS-2 being the most important Cbl
in muscle and fat cells) and c-Cbl. Tyrosine-phosphorylated PI3K
AKT
IRS proteins, which are thought to be held in close proximity PKCζ
to the plasma membrane through association with the
underlying cytoskeleton, recruit more effector molecules,
+ Insulin Insulin
such as PI3K, to this location. Substantial evidence indicates
that the Class 1a PI3K might have an important role in
insulin-stimulated GLUT4 translocation, although a role for Polyphosphoinositides
other PI3K isoforms cannot be excluded. Two important
targets of PI3K in muscle and fat cells that have been shown to
have a role in insulin-stimulated GLUT4 translocation are the
serine/threonine kinase Akt/protein kinase B (PKB) and the
atypical protein kinase C (PKC) isoform, PKCζ. PI3K PI3K PDK TC10
CAP
activates Akt by generating polyphosphoinositides in the GTP
AKT PKCζ C3G
inner leaflet of the plasma membrane. This acts as a docking IRS-1
Cbl
site for Akt through its pleckstrin homology domain, thereby CrkII
bringing it in close proximity to its upstream regulatory
kinase, phosphatidylinositol-dependent kinase-1 (PDK-1).
The mechanism of activation of PKCζ, although not clear,
PKCζ
might involve its recruitment to intracellular membranes, and AKT
indeed it has been shown to be present in intracellular
GLUT4-containing vesicles. Although Akt and PKCζ have
both been implicated in insulin action, there are numerous
downstream targets of PI3K — including proteins such as GLUT4 translocation
ARNO that have a role in membrane transport — that might
also be involved in the insulin regulation of GLUT4
translocation. The second, putative signalling pathway that
has been shown to have a role in insulin-stimulated GLUT4
translocation operates independently of PI3K and involves a dimeric complex that comprises c-Cbl and the c-Cbl-associated protein CAP. Intriguingly,
whereas many growth factors trigger the activation of PI3K, Akt and PKCζ in many cell types, aspects of the c-Cbl–CAP pathway, including the tyrosine
phosphorylation and the expression of CAP, seem to be unique to muscle and fat cells. Insulin triggers the movement of this dimeric c-Cbl–CAP complex
into cell-surface lipid rafts through association with the raft protein flotillin. Inhibition of this process inhibits insulin-stimulated GLUT4 translocation in
adipocytes72. Tyrosine-phosphorylated c-Cbl then recruits a complex of CrkII, an adaptor protein, and C3G into lipid rafts. C3G is a guanine-nucleotide-
exchange factor for the Rho-like GTPase TC10. Because TC10 is constitutively localized to lipid rafts, this catalyses GTP loading and, consequently,
activation of TC10.

270 | APRIL 2002 | VOLUME 3 www.nature.com/reviews/molcellbio


CD-MPR have a role in sorting GLUT4 to endosomes. This would
(Cation-dependent mannose-6- explain the fact that GLUT4 colocalizes with molecules,
phosphate receptor). This such as the cation-dependent mannose-6-phosphate
protein shuttles between the
receptor, (CD-MPR)18 that also follow this route.
trans-Golgi network and
endosomes.
GLUT4 storage vesicles
Despite the fact that GLUT4 is obviously engaged in a
recycling loop between endosomes and the TGN, there is
clear evidence for the existence of a more static secretory
pool of GLUT4 that can move directly to the cell surface
in response to insulin. Using both morphological and
biochemical methods, a discrete population of small
(50 nm diameter) vesicles have been identified29–31. These
vesicles exclude other recycling proteins, such as the TfR
and the CD-MPR, and are highly responsive to insulin.
Importantly, these vesicles contain the v-SNARE, vesi-
cle associated membrane protein (VAMP2) — the
same v-SNARE that is found in synaptic vesicles and
aquaporin-2-containing vesicles — which indicates a
generic role for this molecule in regulated exocytosis in
many cell types. This v-SNARE has been shown to form
a complex with the t-SNAREs Syntaxin 4 and SNAP23
(BOX 3), which are highly enriched in the plasma mem-
brane of fat and muscle cells. The identification of these
SNAREs has provided important clues about the mech-
anism of GLUT4 translocation.
A model for GLUT4 transport
Several models, based on the studies described above,
have been proposed to explain the transport of GLUT4.
1 Basal
+ Insulin
2 GLUT4 to the cell surface, but not other recycling pro-
4 teins such as GLUT1. These data argue strongly in
5 3
6
7 TGN pools of GLUT4 would become depleted and all
8
9 endosomal system. This TGN–endosomal recycling
10
11 12
0 5 10 15 20 25 30 35
GLUT4 distribution (%)
Figure 3 | Relative GLUT4 distribution throughout organelles of cells from non-stimulated
and insulin-stimulated brown adipose tissue. Cryosections of brown adipose tissue were
immunolabelled with anti-GLUT4 antibody and gold-conjugated Protein A. Gold particles were
counted and assigned to the following organelles: (1) trans-Golgi network (TGN); (2) tubulo–vesicular
(T–V) elements located underneath the plasma membrane; (3) clusters of T–V elements; (4) T–V
elements distributed throughout the cytoplasm; (5) T–V elements connected or close to late
endosomal vacuoles (6); (7) T–V elements connected or close to early endosomal vacuoles (8); (9)
non-coated invaginations of the plasma membrane; (10) coated pits and vesicles; (11) plasma
membrane; (12) cytoplasm. The graph (right) shows the relative distribution of GLUT4 throughout
these organelles. Reproduced with permission from REF. 16 ©1991 The Rockefeller University Press.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
REVIEWS
These include retention mechanisms, dynamic sorting
events and the packaging of GLUT4 into a more sta-
tionary population of secretory-type vesicles. It now
seems likely that these different models are not mutually
exclusive, and indeed facets of each of them must be
incorporated into a working model. We propose such a
model in FIG. 4. This model accommodates many of the
apparently contradictory observations, and proposes
that GLUT4 transport is controlled by retention mecha-
nisms and dynamic sorting, as well as by being pack-
aged into a more stationary population of secretory-
type vesicles. The main feature of this model is that
GLUT4 is selectively targeted to an intracellular trans-
port loop between the TGN and the endosomes (cycle 2
in FIG. 4). The entry of GLUT4 into this intracellular,
seemingly futile, cycle probably excludes it from the cell-
surface recycling pathway (cycle 1 in FIG. 4). This would
account for the very low levels of GLUT4 at the cell sur-
face in basal adipocytes compared with other proteins
such as the TfR that do not enter this cycle. An essential
feature of this model is that there is an intracellular store
of GLUT4 that represents a slowly exchanging pool that
moves between the TGN and endosomes.
This intracellular store can presumably fuse with
either endosomes (in the absence of insulin), or with
the cell surface (in the presence of insulin). Several
lines of evidence support the existence of this unique
pool of GLUT4 vesicles and the idea that it can fuse
directly with the cell surface in response to insulin. In
particular, studies of the SNARE proteins that are
involved in the docking and fusion of GLUT4 storage
vesicles (GSVs) with the cell surface (BOX 3) have been
most enlightening. Disrupting the function of the
Syntaxin 4–SNAP23–VAMP2 SNARE complex selec-
tively inhibits the insulin-stimulated translocation of
favour of a model in which a population of vesicles is
ready to move directly to the cell surface. Once formed,
it is highly unlikely that these vesicles remain static in
the absence of an insulin signal, as the endosomal and
the GLUT4 would be present in GSVs if this were the
case. It therefore seems likely that the GSVs slowly fuse
with endosomes, and allow GLUT4 to re-enter the
pathway is not unique to insulin-responsive cells, but
probably exists in all cell types. Numerous examples of
proteins that are transported to the cell surface from an
intracellular pool in response to external stimuli have
been identified (TABLE 1). For example, aquaporin-2 — a
water channel that is normally found in the TGN region
of renal epithelial cells — translocates to the plasma
membrane in response to the peptide hormone vaso-
pressin. Intriguingly, the SNARE complex that controls
GLUT4 translocation is also responsible for the translo-
cation of aquaporin-2.
A similar TGN–endosomal transport pathway has
been described in the budding yeast Saccharomyces cere-
visiae to account for the upregulation of amino-acid
permeases on the cell surface that occurs in response to
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REVIEWS

Box 3 | The SNARE hypothesis

The multitude of membrane transport events that occurs in eukaryotic cells are controlled by families of proteins known
as SNAREs and SNARE-associated proteins. v-SNAREs (membrane proteins that are found in transport vesicles) bind in
a highly specific manner to t-SNAREs (membrane proteins that are found on the relevant target membrane). The
formation of a stable, ternary complex between the correct set of SNARE proteins brings transport vesicles and target
membranes into close proximity, and ultimately leads to their fusion. Although the precise role of SNAREs in membrane
docking and fusion is still debated, these molecules and their associated proteins clearly have an important role in
membrane fusion. Membrane fusion can be broken down into the three distinct stages, as outlined in the figure.
Vesicle tethering. The small GTPase Rab family of proteins is responsible for tethering the transport vesicles to the
appropriate target membrane. Rab proteins bind to specific transport vesicles and seem to function — through their
GTPase activity — as molecular switches, to recruit cytosolic effector molecules that are required for vesicle tethering to
docking sites on the appropriate target membrane (reviewed in REF. 74).
Vesicle docking. After a transport vesicle is tethered to its target membrane, the formation of a stable ternary SNARE
complex docks the transport vesicle onto the target membrane. The Sec1-like/Munc18 (SM) family adds a further level of
regulation to membrane fusion at this stage. SM proteins seem to have both a positive and negative role in SNARE-
complex assembly. These proteins bind tightly to t-SNARE molecules and prevent ternary-complex formation. However,
their binding also seems to be required to activate the t-SNARE for entry into the ternary complex. The formation of a
stable SNARE complex completes the docking stage of vesicular transport.
Membrane fusion. The docked vesicle fuses with the target membrane, where it delivers its contents. Every SNARE-
dependent fusion event that has so far been identified to date requires the NEM (N-ethylmaleimide) sensitive factor
(NSF) and its binding partner α-SNAP, but their precise role remains unclear.
The SNARE, Rab and SM protein families are all highly conserved throughout evolution, as well as throughout the cell. A
situation is emerging in many cellular systems, in which different members of these families mark different transport
vesicles and target (or acceptor) membranes. The coordination of the various families of proteins that are involved in
membrane fusion results in a highly regulated process.

t-SNARES

Target
membrane
SNAP23
v-SNARE
Rab
Tether
Tethering Docking Fusion

Transport vesicle

external growth conditions. On rich nitrogen sources,
the general amino-acid permease Gap1 is transported to
the vacuole, where it is degraded. By contrast, when cells
are grown on low nitrogen sources, Gap1 is transported
from an intracellular storage pool to the cell surface32.
Intriguingly, a di-leucine-containing motif in the car-
boxyl-terminus of Gap1, which is required for its regu-
lated transport, resembles a motif that is required for
the insulin-sensitive transport of GLUT4 (REF. 33). The
regulated transport of Gap1 is controlled by the addi-
tion of ubiquitin to the amino terminus of Gap1, which
seems to occur in the TGN. This is intriguing, as it has
been reported that GLUT4 is modified by the addition
of the ubiquitin-like molecule sentrin, also known as
SUMO1, in muscle cells34. So, it is tempting to speculate
that there might be a role for sumoylation and/or ubiq-
uitylation in regulating the transport of GLUT4
between the TGN and endosomes. Ubiquitylation has
recently been shown to regulate the entry of membrane
GTPγS
proteins into multivesicular bodies, which targets them
A non-hydrolysable analogue of for degradation35. Intriguingly, chronic insulin treat-
GTP. ment markedly reduces the stability of GLUT4 in
adipocytes36. It remains to be seen if this is linked to its
ubiquitylation or if there is a role for this process in
insulin resistance.
The futile cycle that is depicted in FIG. 4 might explain
several observations that are related to GLUT4 trans-
port. First, such a cycle might provide the basis for the
considerable increase in the rate of GLUT4 exocytosis
— compared with that of other proteins — in response
to insulin. This would explain the very large increase in
cell-surface levels of GLUT4. Second, it might explain
how different stimuli mobilize discrete intracellular
pools of GLUT4. Most notably, in skeletal muscle exer-
cise causes a large increase in GLUT4 translocation to
the plasma membrane, mainly from the endosomal
pool rather than the GSVs (REF. 37). Similar observa-
tions have been made using other agonists such as GTPγS
(REF. 38). Intriguingly, the regulation of GLUT4 move-
ment from these different compartments seems to be
quite unique. Whereas wortmannin, which inhibits
phosphatidylinositol 3-kinase (PI3K) activity, completely
inhibits insulin-stimulated GLUT4 translocation, it has
no effect on the translocation of GLUT4 that occurs in

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Plasma membrane
Cytoplasm
Cycle 1
Early
Munc18c
endosome
Syntaxin4
+ Insulin Recycling
SNAP23 endosome
VAMP2
GLUT4
storage Cycle 2
Transport
vesicles vesicle
Trans-Golgi network
Figure 4 | A model that depicts the transport of GLUT4 in insulin-responsive cells. The
model depicts two main intracellular-recycling pathways: cycle 1, between the cell surface and
endosomes; and cycle 2, between the trans-Golgi network (TGN) and endosomes. GLUT4
transport is intricately controlled at several points along these cycles. On entry into the endosomal
system, GLUT4 is selectively retained at the expense of other recycling transport, such as the
transferrin receptor that constitutively moves through cycle 1. This retention mechanism might
predispose GLUT4 for sorting into transport vesicles that bud slowly from the endosome and that
are targeted to the TGN. GLUT4 is sorted into a secretory pathway in the TGN. This sorting step
probably involves a specialized population of secretory vesicles that excludes other secretory
cargo, and that does not fuse constitutively with the plasma membrane. Vesicles that emerge from
this sorting step, which we have previously referred to as GLUT4 storage vesicles or GSVs, might
constitute most of the GLUT4 that is excluded from the endosomal system. In the absence of
insulin, GSVs might slowly fuse with endosomes, thereby accounting for the presence of a
significant but small pool of GLUT4 in endosomes, even in the absence of insulin. Insulin would
then shift GLUT4 from this TGN–endosome cycle to a pathway that takes GLUT4 directly to the
cell surface. The inset shows the SNARE proteins that are thought to regulate docking and fusion
of GSVs with the cell surface (reviewed in REF. 73). The t-SNAREs Syntaxin 4 and SNAP23 in the
plasma membrane of fat and muscle cells form a ternary complex with the v-SNARE VAMP2,
which is present on GSVs. Munc18c has been identified as the SM (Sec1-like/Munc18 family)
protein (BOX 3) that controls the formation of this ternary complex.
response to exercise or GTPγS (REF. 39). Furthermore, in
adipocytes, overexpression of constitutively active
mutants of a downstream target of PI3K, Akt, stimulate
the exocytosis of GSVs but not the endosomal pool40.
These studies indicate that the exocytic cues that regu-
late the movement of GLUT4 from different locations
are quite distinct, and so specificity is probably achieved
by the use of a combination of discrete pools of intracel-
lular GLUT4, each of which are coupled to unique regu-
latory mechanisms. These studies also indicate that, at
least as far as insulin action is concerned, the regulation
might be quite similar in both muscle and fat cells.
Intrinsic activation
Can translocation of GLUT4 to the plasma membrane
account for the stimulatory effects of insulin on glucose
transport in muscle and fat cells, or is its ability to trans-
port glucose also subject to regulation? Several recent
studies have shown that insulin-stimulated transport
and GLUT4 translocation can be dissociated from each
other under certain conditions, which indicates that
there might be further means of regulating the transport
properties of GLUT4 — a phenomenon previously
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
REVIEWS
referred to as ‘intrinsic activation’. First, kinetic studies in
L6 myotubes have indicated that the insulin-dependent
arrival of GLUT4 at the cell surface precedes the increase
in glucose uptake by several minutes41. Intriguingly, a
similar difference is not observed in adipocytes42,43,
which raises the possibility that transporters that translo-
cate more slowly might account for the increase in glu-
cose uptake in L6 cells. Second, a discrepancy in the
dose-response effects of wortmannin on insulin-stimu-
lated glucose transport compared with GLUT4 translo-
cation have been observed in both 3T3-L1 adipocytes44
and L6 cells45. In both of these studies, glucose uptake
was inhibited at a much lower dose of wortmannin than
GLUT4 translocation, which indicates that these two
processes are clearly dissociated. Finally, an inhibitor of
the mitogen-activated protein kinase (MAPK) isoform
p38 inhibits insulin-stimulated glucose uptake without
any apparent effect on GLUT4 translocation46. In addi-
tion to these studies, several agents such as leptin47, iso-
proterenol48 and dibutyryl cyclic AMP49 decrease glucose
uptake, whereas cycloheximide50 and adenosine48
increase glucose uptake without affecting the amount of
GLUT4 at the plasma membrane. An important limita-
tion of the intrinsic-activation hypothesis is that a plau-
sible biochemical mechanism for intrinsic activation of
GLUT4 is yet to be described. It is most likely that intrin-
sic activation involves some type of covalent or struc-
tural change in GLUT4. Several possible mechanisms,
such as phosphorylation51, nucleotide binding52 and (at
least in the case of GLUT1) the formation of homo-
oligomers53 have been proposed. Moreover, it has been
reported that GLUT4 can be detected in both clathrin-
coated pits54 and caveolae55 at the cell surface in
adipocytes, and it is possible that within these subdo-
mains, the structure of GLUT4, and consequently its
activity, is constrained in some way.
Integrating the transport with the signals
As discussed above, there are several steps that are
involved in maintaining the intracellular pool of
GLUT4 in the absence of insulin, any one of which
could be a target of insulin action. GLUT4 is trans-
ported between several intracellular compartments
even in the absence of insulin, and this alone involves
selective retention mechanisms, vesicle-budding reac-
tions that involve the binding of coat proteins such as
AP-1 to GLUT4, the movement of vesicles along
cytoskeletal elements and the docking and fusion of
transport vesicles with their relevant target membrane.
Organelles that are potential targets of insulin action
include the plasma membrane, endosomes and the
TGN, which shows that a vast amount of transport
machinery is involved. An important question is: which
step does insulin modulate to increase GLUT4 translo-
cation to the cell surface? The lack of in vitro assays that
recapitulate some of these stages of GLUT4 transport
has been a major limitation in answering this and other
questions. Recently, an assay for the in vitro fusion of
intracellular vesicles that contain GLUT4 with plasma
membranes has been described56. Using this system, it was
shown that insulin modulates targets in both the vesicle
VOLUME 3 | APRIL 2002 | 2 7 3
REVIEWS

Table 1 | Proteins that translocate after stimulus (in addition to GLUT4 and IRAP)
Name Cell type Stimulus Intracellular Remarks References
localization
General amino acid S. cerevisiae Poor nitrogen source Golgi Sec13 dependent 32
permease Gap1 (ammonia/urea)
Aquaporin-1 Rat peritoneal Hyperosmotic stimulus Endosomal 75
mesothelial cells
Cholangiocytes Secretin Unknown Inhibited at 20ºC and by colchicine 76
Aquaporin-2 Renal epithelium Arginine vasopressin/ trans-Golgi Translocation blocked at 20ºC and by bafilomycin 77–84
(renal inner medullary forskolin network A1; VAMP2, Syntaxin-4 and SNAP23 probably
collecting-duct cells) (TGN) involved; cyclic AMP and PKA involved (PKA-mediated
phosphorylation of AQP-2 is probably required for
translocation); mutation of phosphorylation site
Ser256 blocks translocation; might involve
heterotrimeric G proteins (Gαi); okadaic acid induces
translocation independent of AQP-2 phosphorylation;
AQP-2 recycles in absence and presence of stimulus
Epithelial Na Renal epithelium cAMP agonists Unknown Process inhibited at 15ºC; PPPXY 85,86
channel (ENaC) sequence is involved
Na+-K+-ATPase Kidney epithelium Insulin/arginine Unknown Translocation accompanied by subunit 87–91
vasopressin dephosphorylation (insulin + AVP); inhibited by
wortmannin (insulin)
Skeletal muscle Exercise/insulin Unknown
Na+/H+ exchanger Renal and intestinal bFGF Recycling Blocked by PI3K inhibitors 92,93
NHE3 epithelial cells endosomes
Calcium channel Neuronal cells IGF-1, PDGF, head Unknown Translocation is wortmannin sensitive 94,95
GRC activator (neuropeptide)
N-type calcium channel Neuronal cell KCl, ionomycin, Unknown Translocation is BFA-insensitive 96
PKC activation
8 pS chloride channel Renal epithelium PKA activation Unknown Translocation is BFA-sensitive 97
H+/K+-ATPase Gastric parietal cells Histamine Vesicles 98
Menkes protein Ubiquitously Copper TGN 99,100
MNK expressed
GABA transporter GAT1 Neuronal cells PKC activation (PMA) Unknown 101
Glutamate Neuronal cells PDGF Unknown Translocation inhibited by wortmannin and 102
transporter EAAC1 LY 294002, not by PKC inhibitor BisII
Flt3 ligand (growth T lymphocytes Bone-marrow failure Perinuclear Translocation not due to de novo protein 103,104
factor for (chemotherapy); synthesis
haematopoietic cells) IL-2, -4, -7, -15
κ opioid Magnocellular Salt loading AVP-containing Occurs during neuropeptide release; removed from 105
receptor KOR1 neurosecretory secretory plasma membrane within 1 hr of stimulation
neurons vesicles
bFGF, basic fibroblast growth factor; IGF, insulin growth factor; IL, interleukin; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase
A; PKC, protein kinase C; BFA, brefeldin A.

and the plasma membranes. So, these data support the
notion that there are several signalling pathways that
converge on different aspects of GLUT4 transport. In
support of this, whereas Akt is activated at the plasma
membrane, another downstream target of PI3K, protein
kinase Cζ (PKCζ), is selectively activated in
endosomes57. So, it might be of interest to look for spe-
cific substrates of each of these kinases at these discrete
cellular locations.
The most likely targets of insulin action at the cell
surface are the SNARE proteins (BOX 3). Several proteins
have been implicated in regulating the formation of the
ternary complex — which consists of Syntaxin 4,
SNAP23 and VAMP2 — in response to insulin. For
example, Synip binds to Syntaxin 4 and prevents
VAMP2, but not SNAP23, binding. The association of
Synip with Syntaxin 4 is reduced on stimulation with
insulin58, but how this dissociation is achieved remains
unknown. Similarly, the VAMP2-binding proteins
pantophysin59 and vesicle-associated protein 33
(VAP33)60 have been proposed to prevent the entry of
the v-SNARE into the ternary complex in the absence of
insulin, but again, the signal that transduces this is not
known. Intriguingly, insulin stimulates the GTP-loading
of Rab4, and GTP–Rab4 is known to bind Syntaxin 4
(REFS 61,62). Furthermore, insulin or overexpression of
PKCζ induces serine phosphorylation of VAMP2 in pri-
mary cultures of rat skeletal muscle63. So, we can imag-
ine that GLUT4 vesicles that are formed from either
endosomes or the TGN constantly sample the cell sur-
face, but that their fusion is limited by the availability of
tethering and/or docking sites. Insulin might overcome

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 REVIEWS

BREFELDIN A
A fungal metabolite that affects
membrane transport and the
structure of the Golgi apparatus.
this barrier by modulating auxiliary regulatory proteins
such as the Rab proteins or Synip.
If each of the signalling pathways that are implicated
in insulin action (BOX 2) were assigned discrete functions
in the GLUT4-recruitment process, we would predict
that activation of each signalling intermediate on its
own would have little or no effect compared with that of
insulin. However, in the case of the TC10 pathway this
does not seem to be the case. Overexpression of consti-
tutively active forms of PI3K (REF. 64), Akt65 or PKC63, but
not TC10 (REF. 66), has a robust stimulatory effect on
GLUT4 translocation that is similar to that observed
with insulin. One interpretation of these data is that the
TC10 pathway regulates a factor, or process, that is per-
missive for GLUT4 translocation to the cell surface. One
such process that has recently been proposed is the reg-
ulation of the actin cytoskeleton67, which is consistent
with the generalized role of Rho family members in
actin rearrangement. Considerable evidence supports a
role for the actin cytoskeleton in insulin-stimulated glu-
cose transport. Agents that depolymerize actin inhibit
GLUT4 translocation68 and, although controversial, it
has been suggested that insulin might modulate the cor-
tical actin cytoskeleton in adipocytes69. This leads to a
model in which actin might be involved in tethering the
GLUT4 vesicles at the cell surface, and this might pre-
cede the docking/fusion step. More recently, it has been
shown that insulin stimulates the formation of actin
tails that are associated with GLUT4-containing mem-
branes70, which raises the possibility that actin might be
involved in propelling the vesicles towards the cell sur-
face. In either case, we can imagine that this step, which
is regulated by the TC10 pathway, might not be suffi-
cient to activate GLUT4 translocation, and this might
also explain why activation of either the Akt or PKC
pathways on their own might overcome the need for
this pathway. So, until the function of TC10 has been
more clearly defined, with particular attention to the
identification of its downstream targets in muscle and
fat cells, it is difficult to assign an important role for this
pathway in insulin action.
Conclusions and perspectives
So, to return to the central questions that we posed at
the beginning of this review: how is GLUT4 transported
from one organelle to another, and what is the rela-
tionship between these pathways and the intracellular
sequestration of GLUT4 in the absence of insulin? The
intracellular movement of GLUT4 is complex, and
involves many organelles and perhaps also a unique
storage compartment — GSVs. In the absence of
insulin, GLUT4 is trapped in an intracellular circuit
between endosomes and the TGN as a diversionary
tactic to avoid appearing at the cell surface. Elements of
this system are absent from ‘non-insulin-responsive’ cell
types. So, the adaptations that occur during muscle and
fat differentiation to allow the entry of GLUT4 into this
intracellular loop are clearly an important area for
future study.
We imagine that the complex transport itinerary of
GLUT4 is governed by the protein encountering differ-
ent coat complexes throughout the cell. Some of these
we know, including AP-2 at the cell surface, and AP-1 at
the TGN. But the coats that regulate transport between
endosomes and the TGN are not yet known. These are
probably somewhat specialized, perhaps by being
expressed uniquely in insulin-responsive cells and/or by
being resistant to the effects of BREFELDIN A (BFA).
In addition to GLUT4, GSVs also contain IRAP and
VAMP2. The identification of the latter has allowed
huge inroads to our understanding of the docking and
fusion of GSVs with the plasma membrane. The
SNAREs and some of their associated proteins are now
known. Such discoveries will provide a template for the
discovery of new molecules that might be unique to the
insulin-stimulated transport of GLUT4 in muscle and
fat cells. A large gap in our current knowledge of the
exocytosis of GLUT4 is the identity of the Rab protein
that is involved in delivery of the transport to the cell
surface. Although Rab4 has been implicated in this
process, it might be involved in less specialized aspects
of the transport itinerary of GLUT4, in which case the
Rab that is responsible for the delivery of GSVs to the
plasma membrane remains to be identified.
Perhaps the ultimate question is, what does insulin
do? Without more complete answers to the above two
questions, this question will probably remain unan-
swered. Although our knowledge of signalling has
advanced tremendously over the past few years, we have
not, as yet, identified the intersection point of the
insulin-signalling pathway with the GLUT4 transport
pathway. This intersection point might be governed by
coat proteins, cytoskeletal elements, the SNARE pro-
teins or, more probably, a combination of all three.
Identification of this intersection point will require a
convergence of different approaches. New approaches
such as DNA microarrays will provide knowledge of the
genes that are uniquely expressed in muscle and fat cells,
and will offer new insights into the dynamic and regu-
lated characteristics of GLUT4 transport. Finally, the
development of in vitro assays that reconstitute various
aspects of insulin-stimulated GLUT4 translocation will
be required for the discovery and characterization of
key molecules that are involved in this process. All of
this knowledge will contribute to our understanding of
both cell biology and type II diabetes.

1. Shepherd, P. R. & Kahn, B. B. Glucose transporters and
insulin action — implications for insulin resistance and
diabetes mellitus. N. Engl. J. Med. 341, 248–257 (1999).
2. Suzuki, K. & Kono, T. 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 (1980).
3. Cushman, S. W. & Wardzala, L. J. 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
(1980).
4. Stenbit, A. E. et al. GLUT4 heterozygous knockout mice
develop muscle insulin resistance and diabetes. Nature
Med. 3, 1096–1101 (1997).
5. Brozinick, J. T. Jr et al. GLUT4 overexpression in db/db mice
dose-dependently ameliorates diabetes but is not a lifelong
cure. Diabetes 50, 593–600 (2001).
6. Holman, G. D., Leggio, L. L. & Cushman, S. W. Insulin-
stimulated GLUT4 glucose transporter recycling. A problem
in membrane protein subcellular trafficking through multiple

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY VOLUME 3 | APRIL 2002 | 2 7 5

REVIEWS
pools. J. Biol. Chem. 269, 17516–17524 (1994).
This study used mathematical analysis to show that
the intracellular transport of GLUT4 could not be
explained by a simple two-compartment model,
which indicates that GLUT4 is partitioned within the
cell among at least two separate compartments.
7. Tanner, L. I. & Lienhard, G. E. Insulin elicits a redistribution of
transferrin receptors in 3T3-L1 adipocytes through an
increase in the rate constant for receptor externalization.
J. Biol. Chem. 262, 8975–8980 (1987).
8. Albiston, A. L. et al. Evidence that the angiotensin IV (AT4)
receptor is the enzyme insulin regulated aminopeptidase.
J. Biol. Chem. 13, 13 (2001).
9. Ross, S. A. et al. Characterization of the insulin-regulated
membrane aminopeptidase in 3T3-L1 adipocytes. J. Biol.
Chem. 271, 3328–3332 (1996).
10. Palacios, S. et al. Recycling of the insulin-sensitive glucose
transporter GLUT4. Access of surface internalized GLUT4
molecules to the perinuclear storage compartment is
mediated by the Phe5–Gln6–Gln7–Ile8 motif. J. Biol. Chem.
276, 3371–3383 (2001).
11. Martin, S. et al. The glucose transporter (GLUT-4) and
vesicle-associated membrane protein-2 (VAMP-2) are
segregated from recycling endosomes in insulin-sensitive
cells. J. Cell Biol. 134, 625–635 (1996).
Using a chemical technique to ablate endosomes, this
paper was one of the first to show that a significant
pool of GLUT4 is excluded from endosomes and that,
together with the v-SNARE VAMP2, this might
demarcate a separate secretory compartment.
12. Martin, L. B. et al. Vesicle-associated membrane protein 2
plays a specific role in the insulin-dependent trafficking of the
facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes.
J. Biol. Chem. 273, 1444–1452 (1998).
13. Lampson, M. A. et al. Insulin-regulated release from the
endosomal recycling compartment is regulated by budding
of specialized vesicles. Mol. Biol. Cell 12, 3489–3501
(2001).
Using quantitative immunofluorescence-microscopy
techniques, this study provides evidence that GLUT4
is retained in recycling endosomes in CHO cells, and
moves to the cell surface in transport vesicles that are
distinct from those that contain the TfR.
14. Lim, S. N. et al. Identification of discrete classes of
endosome-derived small vesicles as a major cellular pool for
recycling membrane proteins. Mol. Biol. Cell 12, 981–995
(2001).
In vitro reconstitution experiments were used to show
that GLUT4 and the TfR are sorted into different
endosomally derived vesicles in CHO cells.
15. Slot, J. W. et al. Translocation of the glucose transporter
GLUT4 in cardiac myocytes of the rat. Proc. Natl Acad. Sci.
USA 88, 7815–7819 (1991).
16. Slot, J. W. et al. Immuno-localization of the insulin
regulatable glucose transporter in brown adipose tissue of
the rat. J. Cell Biol. 113, 123–135 (1991).
This was the first immunocytochemical analysis of
GLUT4 in insulin-sensitive cells and showed that
insulin caused a striking redistribution of GLUT4 from
intracellular tubulo–vesicular elements to the plasma
membrane.
17. Slot, J. W. et al. Glucose transporter (GLUT-4) is targeted to
secretory granules in rat atrial cardiomyocytes. J. Cell Biol.
137, 1–12 (1997).
18. Martin, S. et al. Biogenesis of insulin-responsive GLUT4
vesicles is independent of brefeldin A-sensitive trafficking.
Traffic 1, 652–660 (2000).
19. Shewan, A. M. et al. The cytosolic C-terminus of the glucose
transporter GLUT4 contains an acidic cluster endosomal
targeting motif distal to the dileucine signal. Biochem. J.
350, 99–107 (2000).
20. Molloy, S. S. et al. Bi-cycling the furin pathway: from TGN
localization to pathogen activation and embryogenesis.
Trends Cell Biol. 9, 28–35 (1999).
21. Xiang, Y. et al. The PC6B cytoplasmic domain contains two
acidic clusters that direct sorting to distinct trans-Golgi
network/endosomal compartments. Mol. Biol. Cell 11,
1257–1273 (2000).
22. Wan, L. et al. PACS-1 defines a novel gene family of
cytosolic sorting proteins required for trans-Golgi network
localization. Cell 94, 205–216 (1998).
23. Mallard, F. et al. Direct pathway from early/recycling
endosomes to the Golgi apparatus revealed through the
study of shiga toxin B-fragment transport. J. Cell Biol. 143,
973–990 (1998).
24. Barr, V. A. et al. Insulin stimulates both leptin secretion and
production by rat white adipose tissue. Endocrinology 138,
4463–4472 (1997).
25. Bogan, J. S. & Lodish, H. F. Two compartments for insulin-
stimulated exocytosis in 3T3-L1 adipocytes defined by
276 | APRIL 2002 | VOLUME 3
endogenous ACRP30 and GLUT4. J. Cell Biol. 146,
609–620 (1999).
26. Millar, C. A. et al. Adipsin and the glucose transporter
GLUT4 traffic to the cell surface via independent pathways
in adipocytes. Traffic 1, 141–151 (2000).
27. Robinson, L. J. & James, D. E. Insulin-regulated sorting of
glucose transporters in 3T3-L1 adipocytes. Am. J. Physiol.
263, E383–E393 (1992).
28. Robinson, M. S. & Bonifacino, J. S. Adaptor-related
proteins. Curr. Opin. Cell Biol. 13, 444–453 (2001).
29. Hashiramoto, M. & James, D. E. Characterization of insulin-
responsive GLUT4 storage vesicles isolated from 3T3-L1
adipocytes. Mol. Cell. Biol. 20, 416–427 (2000).
30. Ramm, G. et al. 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 (2000).
31. Kandror, K. V. & Pilch, P. F. Compartmentalization of protein
traffic in insulin-sensitive cells. Am. J. Physiol. 271, E1–E14
(1996).
32. Roberg, K. J., Rowley, N. & Kaiser, C. A. Physiological
regulation of membrane protein sorting late in the secretory
pathway of Saccharomyces cerevisiae. J. Cell Biol. 137,
1469–1482 (1997).
33. Hein, C. & Andre, B. A C-terminal di-leucine motif and
nearby sequences are required for NH4+-induced
inactivation and degradation of the general amino acid
permease, Gap1p, of Saccharomyces cerevisiae.
Mol. Microbiol. 24, 607–616 (1997).
34. Giorgino, F. et al. The sentrin-conjugating enzyme mUbc9
interacts with GLUT4 and GLUT1 glucose transporters and
regulates transporter levels in skeletal muscle cells.
Proc. Natl Acad. Sci. USA 97, 1125–1130 (2000).
35. Piper, R. C. & Luzio, J. P. Late endosomes: sorting and
partitioning in multivesicular bodies. Traffic 2, 612–621
(2001).
36. Sargeant, R. J. & Paquet, M. R. Effect of insulin on the rates
of synthesis and degradation of GLUT1 and GLUT4 glucose
transporters in 3T3-L1 adipocytes. Biochem. J. 290,
913–919 (1993).
37. Ploug, T. et al. Analysis of GLUT4 distribution in whole
skeletal muscle fibers: identification of distinct storage
compartments that are recruited by insulin and muscle
contractions. J. Cell Biol. 142, 1429–1446 (1998).
This study provided the first quantitative analysis of
the distribution of GLUT4 in skeletal muscle after
activation by either insulin, exercise or exercise plus
insulin.
38. Millar, C. A. et al. Differential regulation of secretory
compartments containing the insulin-responsive glucose
transporter 4 in 3T3-L1 adipocytes. Mol. Biol. Cell 10,
3675–3688 (1999).
This paper shows that in 3T3-L1 adipocytes, GLUT4
movement to the cell surface can be triggered from
different intracellular pools.
39. Brozinick, J. T. Jr & Birnbaum, M. J. Insulin, but not
contraction, activates Akt/PKB in isolated rat skeletal
muscle. J. Biol. Chem. 273, 14679–14682 (1998).
40. Foran, P. G. et al. Protein kinase B stimulates the
translocation of GLUT4 but not GLUT1 or transferrin
receptors in 3T3-L1 adipocytes by a pathway involving
SNAP-23, synaptobrevin-2, and/or cellubrevin.
J. Biol. Chem. 274, 28087–28095 (1999).
41. Somwar, R. et al. GLUT4 translocation precedes the
stimulation of glucose uptake by insulin in muscle cells:
potential activation of GLUT4 via p38 mitogen-activated
protein kinase. Biochem. J. 359, 639–649 (2001).
42. Karnielli, E. et al. Insulin-stimulated translocation of glucose
transport systems in the isolated rat adipose cell.
J. Biol. Chem. 256, 4772–4777 (1981).
43. Molero, J. C. et al. Nocodazole inhibits insulin-stimulated
glucose transport in 3T3-L1 adipocytes via a microtubule-
independent mechanism. J. Biol. Chem. 276, 43829–43835
(2001).
44. Hausdorff, S. F. et al. Identification of wortmannin-sensitive
targets in 3T3-L1 adipocytes. Dissociation of insulin-
stimulated glucose uptake and GLUT4 translocation.
J. Biol. Chem. 274, 24677–24684 (1999).
45. Somwar, R. et al. Differential effects of phosphatidylinositol
3-kinase inhibition on intracellular signals regulating GLUT4
translocation and glucose transport. J. Biol. Chem. 276,
46079–46087 (2001).
46. Sweeney, G. et al. An inhibitor of p38 mitogen-activated
protein kinase prevents insulin-stimulated glucose transport
but not glucose transporter translocation in 3T3-L1
adipocytes and L6 myotubes. J. Biol. Chem. 274,
10071–10078 (1999).
47. Sweeney, G. et al. High leptin levels acutely inhibit insulin-
stimulated glucose uptake without affecting glucose
transporter 4 translocation in L6 rat skeletal muscle cells.
Endocrinology 142, 4806–4812 (2001).
48. Joost, H. G. et al. Insulin-stimulated glucose transport in rat
adipose cells. Modulation of transporter intrinsic activity by
isoproterenol and adenosine. J. Biol. Chem. 261,
10033–10036 (1986).
49. Lawrence, J. C. et al. GLUT4 facilitates insulin stimulation
and cAMP-mediated inhibition of glucose transport.
Proc. Natl Acad. Sci. USA 89, 3493–3497 (1992).
50. Clancy, B. M. et al. Protein synthesis inhibitors activate
glucose transport without increasing plasma membrane
glucose transporters in 3T3-L1 adipocytes. J. Biol. Chem.
266, 10122–10130 (1991).
51. James, D. E., Hiken, J. & Lawrence, J. C. Isoproterenol
stimulates phosphorylation of the insulin-regulatable glucose
transporter in rat adipocytes. Proc. Natl Acad. Sci. USA 86,
8368–8372 (1989).
52. Piper, R. C. et al. GLUT4 phosphorylation and inhibition of
glucose transport by dibutyryl cAMP. J. Biol. Chem. 268,
16557–16563 (1993).
53. Zottola, R. J. et al. Glucose transporter function is controlled
by transporter oligomeric structure. A single, intramolecular
disulfide promotes GLUT1 tetramerization. Biochemistry 34,
9734–9747 (1995).
54. Robinson, L. J. et al. Translocation of the glucose
transporter (GLUT4) to the cell surface in permeabilized
3T3-L1 adipocytes: effects of ATP, insulin and GTPγS and
localization of GLUT4 to clathrin lattices. J. Cell Biol. 117,
1181–1196 (1992).
55. Ros-Baro, A. et al. Lipid rafts are required for GLUT4
internalization in adipose cells. Proc. Natl Acad. Sci. USA
98, 12050–12055 (2001).
56. Inoue, G., Cheatham, B. & Kahn, C. R. Development of an
in vitro reconstitution assay for glucose transporter 4
translocation. Proc. Natl Acad. Sci. USA 96, 14919–14924
(1999).
This paper was the first to show that it is feasible to
reconstitute the docking and fusion of intracellular
GLUT4 vesicles with the plasma membrane in vitro.
57. Sanchez, P. et al. Localization of atypical protein kinase C
isoforms into lysosome-targeted endosomes through
interaction with p62. Mol. Cell. Biol. 18, 3069–3080 (1998).
58. Min, J. et al. Synip: a novel insulin-regulated syntaxin 4-
binding protein mediating GLUT4 translocation in
adipocytes. Mol. Cell 3, 751–760 (1999).
59. Brooks, C. C. et al. Pantophysin is a phosphoprotein
component of adipocyte transport vesicles and associates
with GLUT4-containing vesicles. J. Biol. Chem. 275,
2029–2036 (2000).
60. Foster, L. J. et al. A functional role for VAP-33 in insulin-
stimulated GLUT4 traffic. Traffic 1, 512–521 (2000).
61. Shibata, H., Omata, W. & Kojima, I. Insulin stimulates
guanine nucleotide exchange on Rab4 via a wortmannin-
sensitive signaling pathway in rat adipocytes. J. Biol. Chem.
272, 14542–14546 (1997).
62. Li, L. et al. Direct interaction of Rab4 with syntaxin 4.
J. Biol. Chem. 276, 5265–5273 (2001).
63. Braiman, L. et al. Activation of protein kinase Cζ induces
serine phosphorylation of VAMP2 in the GLUT4
compartment and increases glucose transport in skeletal
muscle. Mol. Cell. Biol. 21, 7852–7861 (2001).
64. Martin, S. S. et al. Activated phosphatidylinositol 3-kinase is
sufficient to mediate actin rearrangement and GLUT4
translocation in 3T3-L1 adipocytes. J. Biol. Chem. 271,
17605–17608 (1996).
65. Kohn, A. D. et al. Expression of a constitutively active Akt
Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose
uptake and glucose transporter 4 translocation.
J. Biol. Chem. 271, 31372–31378 (1996).
66. Chiang, S. H. et al. Insulin-stimulated GLUT4 translocation
requires the CAP-dependent activation of TC10.
Nature 410, 944–948 (2001).
67. Tong, P. et al. Insulin-induced cortical actin remodeling
promotes GLUT4 insertion at muscle cell membrane ruffles.
J. Clin. Invest. 108, 371–381 (2001).
68. Emoto, M., Langille, S. E. & Czech, M. P. A role for kinesin in
insulin-stimulated GLUT4 glucose transporter translocation
in 3T3-L1 adipocytes. J. Biol. Chem. 276, 10677–10682
(2001).
69. Kanzaki, M. & Pessin, J. E. Insulin-stimulated GLUT4
translocation in adipocytes is dependent upon cortical actin
remodeling. J. Biol. Chem. 276, 42436–42444 (2001).
70. Kanzaki, M. et al. Insulin stimulates actin comet tails on
intracellular GLUT4-containing compartments in
differentiated 3T3L1 adipocytes. J. Biol. Chem. 276,
49331–49336 (2001).
71. Harris, M. I. et al. Prevalence of diabetes, impaired fasting
glucose, and impaired glucose tolerance in U.S. adults. The
Third National Health and Nutrition Examination Survey,
1988–1994. Diabetes Care 21, 518–524 (1998).
72. Watson, R. T. et al. Lipid raft microdomain
www.nature.com/reviews/molcellbio

compartmentalization of TC10 is required for insulin
signaling and GLUT4 translocation. J. Cell Biol. 154,
829–840 (2001).
73. Foster, L. J. & Klip, A. Mechanism and regulation of GLUT-4
vesicle fusion in muscle and fat cells. Am. J. Physiol. Cell
Physiol. 279, C877–C890 (2000).
74. Zerial, M. & McBride, H. Rab proteins as membrane
organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001).
75. Kuboshima, S. et al. Hyperosmotic stimuli induces
recruitment of aquaporin-1 to plasma membrane in cultured
rat peritoneal mesothelial cells. Adv. Perit. Dial. 17, 47–52
(2001).
76. Marinelli, R. A. et al. Secretin promotes osmotic water
transport in rat cholangiocytes by increasing aquaporin-1
water channels in plasma membrane. Evidence for a
secretin-induced vesicular translocation of aquaporin-1.
J. Biol. Chem. 272, 12984–12988 (1997).
77. Kamsteeg, E. J. et al. The subcellular localization of an
aquaporin-2 tetramer depends on the stoichiometry of
phosphorylated and nonphosphorylated monomers.
J. Cell Biol. 151, 919–930 (2000).
78. Gustafson, C. E. et al. Recycling of AQP2 occurs through a
temperature- and bafilomycin-sensitive trans-Golgi-
associated compartment. Am. J. Physiol. Renal Physiol.
278, F317–F326 (2000).
79. Mandon, B. et al. Syntaxin-4 is localized to the apical
plasma membrane of rat renal collecting duct cells: possible
role in aquaporin-2 trafficking. J. Clin. Invest. 98, 906–913
(1996).
80. Valenti, G. et al. The phosphatase inhibitor okadaic acid
induces AQP2 translocation independently from AQP2
phosphorylation in renal collecting duct cells. J. Cell Sci.
113, 1985–1992 (2000).
81. Klussmann, E. et al. Protein kinase A anchoring proteins are
required for vasopressin-mediated translocation of
aquaporin-2 into cell membranes of renal principal cells.
J. Biol. Chem. 274, 4934–4938 (1999).
82. Katsura, T. et al. Protein kinase A phosphorylation is involved
in regulated exocytosis of aquaporin-2 in transfected
LLC-PK1 cells. Am. J. Physiol. 272, F817–F822 (1997).
83. Valenti, G. et al. A heterotrimeric G protein of the G i family is
required for cAMP- triggered trafficking of aquaporin 2 in
kidney epithelial cells. J. Biol. Chem. 273, 22627–22634
(1998).
84. Mulders, S. M. et al. An aquaporin-2 water channel mutant
which causes autosomal dominant nephrogenic diabetes
insipidus is retained in the Golgi complex. J. Clin. Invest.
102, 57–66 (1998).
85. Snyder, P. M. Liddle’s syndrome mutations disrupt
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
cAMP-mediated translocation of the epithelial Na+ channel
to the cell surface. J. Clin. Invest. 105, 45–53 (2000).
86. Loffing, J. et al. Aldosterone induces rapid apical
translocation of ENaC in early portion of renal collecting
system: possible role of SGK. Am. J. Physiol. Renal. Physiol.
280, F675–F682 (2001).
87. Djelidi, S. et al. Basolateral translocation by vasopressin of
the aldosterone-induced pool of latent Na-K-ATPases is
accompanied by α1 subunit dephosphorylation: study in a
new aldosterone-sensitive rat cortical collecting duct cell
line. J. Am. Soc. Nephrol. 12, 1805–1818 (2001).
88. Juel, C., Nielsen, J. J. & Bangsbo, J. Exercise-induced
translocation of Na+-K+ pump subunits to the plasma
membrane in human skeletal muscle. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 278, R1107–R1110 (2000).
89. Lavoie, L. et al. Insulin-induced translocation of Na+-K+-
ATPase subunits to the plasma membrane is muscle fiber
type specific. Am. J. Physiol. 270, C1421– C1429 (1996).
90. Omatsu-Kanbe, M. & Kitasato, H. Insulin stimulates the
translocation of Na+/K+-dependent ATPase molecules from
intracellular stores to the plasma membrane in frog skeletal
muscle. Biochem. J. 272, 727–733 (1990).
91. Aledo, J. C. & Hundal, H. S. Sedimentation and
immunological analyses of GLUT4 and α2-Na,K-ATPase
subunit-containing vesicles from rat skeletal muscle:
evidence for segregation. FEBS Lett. 376, 211–215 (1995).
92. Janecki, A. J. et al. Basic fibroblast growth factor stimulates
surface expression and activity of Na+/H+ exchanger NHE3
via mechanism involving phosphatidylinositol 3-kinase.
J. Biol. Chem. 275, 8133–8142 (2000).
93. D’Souza, S. et al. The epithelial sodium-hydrogen antiporter
Na+/H+ exchanger 3 accumulates and is functional in
recycling endosomes. J. Biol. Chem. 273, 2035–2043
(1998).
94. Boels, K. et al. The neuropeptide head activator induces
activation and translocation of the growth-factor-regulated
Ca2+-permeable channel GRC. J. Cell Sci. 114, 3599–3606
(2001).
95. Kanzaki, M. et al. Translocation of a calcium-permeable
cation channel induced by insulin-like growth factor-1.
Nature Cell Biol. 1, 165–170 (1999).
96. Passafaro, M. et al. N-type Ca2+ channels are present in
secretory granules and are transiently translocated to the
plasma membrane during regulated exocytosis. J. Biol.
Chem. 271, 30096–30104 (1996).
97. Shintani, Y. & Marunaka, Y. Regulation of chloride channel
trafficking by cyclic AMP via protein kinase A-independent
pathway in A6 renal epithelial cells. Biochem. Biophys. Res.
Commun. 223, 234–239 (1996).
REVIEWS
98. Agnew, B. J. et al. Cytological transformations associated
with parietal cell stimulation: critical steps in the activation
cascade. J. Cell Sci. 112, 2639–2646 (1999).
99. Petris, M. J. & Mercer, J. F. The Menkes protein (ATP7A;
MNK) cycles via the plasma membrane both in basal and
elevated extracellular copper using a C-terminal di-leucine
endocytic signal. Hum. Mol. Genet. 8, 2107–2115 (1999).
100. Yamaguchi, Y. et al. Biochemical characterization and
intracellular localization of the Menkes disease protein.
Proc. Natl Acad. Sci. USA 93, 14030–14035 (1996).
101. Quick, M. W. et al. Second messengers, trafficking-related
proteins, and amino acid residues that contribute to the
functional regulation of the rat brain GABA transporter
GAT1. J. Neurosci. 17, 2967–2979 (1997).
102. Sims, K. D., Straff, D. J. & Robinson, M. B. Platelet-derived
growth factor rapidly increases activity and cell surface
expression of the EAAC1 subtype of glutamate transporter
through activation of phosphatidylinositol 3-kinase.
J. Biol. Chem. 275, 5228–5237 (2000).
103. Chklovskaia, E. et al. Mechanism of flt3 ligand expression in
bone marrow failure: translocation from intracellular stores to
the surface of T lymphocytes after chemotherapy-induced
suppression of hematopoiesis. Blood 93, 2595–2604
(1999).
104. Chklovskaia, E. et al. Cell-surface trafficking and release of
flt3 ligand from T lymphocytes is induced by common
cytokine receptor γ-chain signaling and inhibited by
cyclosporin A. Blood 97, 1027–1034 (2001).
105. Shuster, S. J. et al. Stimulus-dependent translocation of
κ opioid receptors to the plasma membrane. J. Neurosci.
19, 2658–2664 (1999).
Online links
DATABASES
The following terms in this article are linked online to:
Interpro: http://www.ebi.ac.uk/interpro/
pleckstrin homology domain
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink
Akt | TfR
OMIM: http://www.ncbi.nlm.nih.gov/Omim
Type II diabetes
Swiss-Prot: http://www.expasy.ch/
ACRP30 | adipsin | ANF | c-Cbl | Gap1 | GLUT1 | GLUT4 | GLUT5 |
GLUT8 | GLUT9 | GLUT10 | GLUT11 | IRS-1 | IRS-2 | p38 | PKCζ |
Rab4 | sentrin | SNAP23 | Syntaxin 4 | Syntaxin 6 | Syntaxin 16 |
Synip | TC10 | TGN38 | VAMP2 | VAP33
Access to this interactive links box is free online.
VOLUME 3 | APRIL 2002 | 2 7 7