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Diabetes & Metabolism 40 (2014) 400–410

Review

Molecular mechanisms of GLUT4 regulation in adipocytes

Mécanismes moléculaires de la régulation de GLUT4 dans l’adipocyte
R. Govers ∗
UMR Inserm U1062, INRA 1260, Aix Marseille University, Campus Timone, Faculty of Medicine, 27, boulevard Jean-Moulin, 13385 Marseille Cedex 5, France
Received 9 December 2013; received in revised form 24 January 2014; accepted 26 January 2014
Available online 20 March 2014

Abstract
Insulin resistance is strongly linked to type 2 diabetes and associated with a reduced uptake of glucose by muscle and adipose tissue. The
transporter that is responsible for this uptake and whose function is disturbed in insulin resistance and type 2 diabetes is GLUT4. In the non-
stimulated state, GLUT4 is efficiently sequestered intracellularly. This retention prevents GLUT4 from reaching the cell surface and transporting
glucose into muscle and fat cells when blood glucose levels are low. After a meal when blood glucose levels rise, insulin is secreted by the
pancreas, which, upon binding to its receptor, triggers an intracellular signaling cascade, leading to the translocation of GLUT4 from intracellular
compartments to the cell surface, resulting in glucose uptake and normalization of the blood glucose levels. Its regulation is dominated by its
localization, efficient intracellular retention and sensitivity to insulin and contraction, which makes GLUT4 an interesting and unique molecule.
These aspects of the intracellular regulation of GLUT4 are described in this review.
© 2014 Elsevier Masson SAS. All rights reserved.

Keywords: GLUT4; Intracellular traffic; Translocation; Regulated exocytosis; Adipocyte

1. GLUT4 in the context of insulin resistance and type 2
diabetes
In healthy individuals, despite alternating periods of fasting
and feeding, plasma glucose levels are mostly remarkably well
maintained within a narrow range due to the action of the hor-
mones insulin and glucagon. In contrast, type 2 diabetes (T2D)
patients suffer from hyperglycemia that is caused by insulin
resistance in combination with insulin levels that are too low to
compensate for this resistance. In insulin resistance, the tissues
that are implicated in glucose homeostasis and that are sensitive
to insulin (i.e. liver, muscle and adipose tissue), no longer effi-
ciently respond to insulin. Hence, at normal insulin levels, the
liver continues to synthesize and release glucose, while mus-
cle and adipose tissue take up less glucose from the blood. It
is widely accepted that this uptake of glucose is mediated by
the insulin-sensitive transporter GLUcose Transporter member
4 (GLUT4). In muscle, this transporter also takes up glucose
upon muscle contraction.
∗
Tel.: +33 4 91 29 40 96; fax: +33 4 91 78 21 01.
E-mail address: Roland.Govers@univ-amu.fr
GLUT4 is one of the 14 members of the GLUT/SLC2A fam-
ily of facilitative transmembrane hexose transporters. In addition
to the well-described expression of GLUT4 in skeletal mus-
cle, cardiomyocytes, and adipose tissue, this transporter is also
expressed in certain regions of the brain [1], in kidney [2], and
in several tumors, including multiple myeloma [3] and breast
cancer cells [4]. The cellular regulation of GLUT4 has been
best studied in muscle and adipose tissue and has appeared to
be largely similar in these two tissues. Nevertheless, several
minor and major differences exist. In addition to the contraction-
induced GLUT4-mediated glucose uptake that is specific for
muscle, there also exists a tissue-specific regulation of GLUT4
expression. This is evident for example in T2D, where there is
a reduction in GLUT4 expression (at both mRNA and protein
levels) in adipocytes but not in muscle cells [5]. This may not
seem to matter much as in healthy individuals only 15% of the
blood glucose is absorbed by adipose tissue and the remaining
85% by muscle. However, in adipose tissue, GLUT4 function
appears to be extremely important and to play a pivotal role in
glucose-sensing, as its expression level in this tissue is positively
correlated with insulin-sensitivity in muscle and liver, mainly via

1262-3636/$ – see front matter © 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.diabet.2014.01.005
 R. Govers / Diabetes & Metabolism 40 (2014) 400–410
Fig. 1. GLUT4 translocation in response to insulin stimulation. 3T3-L1 adipocytes were incubated for 20 minutes in the absence (“basal” state, left panel) or presence
of 100 nM insulin (“insulin”, right panel). Cells were then immunolabeled for GLUT4 and analyzed by immunofluorescence microscopy. Of note is the predominant
intracellular (perinuclear) GLUT4 localization in basal adipocytes and cell surface GLUT4 localization in insulin-stimulated cells. Bar: 10 ␮m.
the GLUT4 activity-dependent secretion of adipokines that act
on these two tissues [6,7].
2. Intracellular localization of GLUT4
The intracellular localization of GLUT4 plays a crucial
role in its regulation [8–10] and is disturbed in T2D [11–13].
In non-stimulated (basal) cells, GLUT4 is efficiently retained
intracellularly, resulting in low cell surface GLUT4 levels,
while insulin reduces this retention, thereby largely increas-
ing the amount of GLUT4 at the cell surface (Fig. 1). How
GLUT4 is retained is still largely unclear. GLUT4 may be
selectively retained in one or several of its intracellular loca-
tions, packaged into a specific insulin-sensitive compartment
that remains static in the absence of insulin, or trafficking within
a dynamic intracellular transport loop that excludes it from
recycling endosomal vesicles. These three different possibili-
ties are not mutually exclusive and various pieces of evidence
exist that support all three scenarios. For decades, researchers
have been investigating the nature of the cellular compartments
that are implicated in intracellular GLUT4 retention and that
are sensitive to insulin stimulation (in adipocytes and mus-
cle) and contraction (in muscle). These compartments have
been collectively termed GLUT4 storage compartments (GSCs)
and insulin-responsive vesicles (IRVs). More specifically, the
non-endosomal vesicular structures that are particularly sen-
sitive to insulin have been termed GLUT4 storage vesicles
(GSVs) and specialized GLUT4 vesicles [14–16]. In addition
to its storage compartments, GLUT4 is also present in several
other cell compartments. These include the plasma membrane,
where GLUT4 is transporting glucose, and organelles impli-
cated in the targeting of GLUT4 into its storage compartments
(Fig. 2). In the following sections, each of these compartments
is described in detail as well as the molecular elements that
regulate GLUT4 in these compartments of which some are sen-
sitive to insulin stimulation. Note that most studies described in
this review are based on the use of the 3T3-L1 cell line. These
401
cells can be efficiently differentiated into adipocytes in vitro,
express GLUT4 upon differentiation and respond well to insulin
[17].
2.1. GLUT4 storage vesicles (GSVs)
GSVs are currently widely accepted to be non-endosomal
insulin-sensitive GLUT4-containing vesicles that are relatively
small in size (50–70 nm) [18], smaller than for example endo-
somal vesicles [19], and sensitive to insulin stimulation and
contraction. GSVs are considered to contain up to half of the
total cellular GLUT4 pool as ∼ 50% of GLUT4 is localized in
a non-endosomal cell fraction [15,20]. Proteins that are consid-
ered to be enriched, but not uniquely expressed, in GSVs include
GLUT4, insulin-regulated aminopeptidase (IRAP) [21], sortilin
(a transmembrane protein involved in the exit of proteins from
the TGN) [22], low-density lipoprotein receptor-related protein
1 (LRP1, implicated in many physiological processes) [23], and
VAMP2 [24].
The biogenesis of GSVs appears to be cell-specific as the
expression of GLUT4 in fibroblasts (cells that normally do not
express GLUT4) leads to the localization of GLUT4 uniquely
in endosomal structures and not in GSVs [25]. It has been sug-
gested that the formation of GSVs or the traffic of GLUT4
into GSVs is primarily induced by the interaction between
transmembrane protein sortilin and GLUT4 and that IRAP
may be facilitative in this process. Accordingly, the absence
of GSVs in fibroblasts is correlated with the absence of sor-
tilin. Moreover, the ectopic coexpression of sortilin with GLUT4
in these cells leads to a shift in the localization of GLUT4
from larger structures into small vesicles (presumably GSVs
or GSV-like vesicles), stabilization of GLUT4 (preventing its
rapid degradation), and an enhancement of insulin-induced
GLUT4 translocation [22,26]. In contrast, the knockdown of
sortilin in adipocytes decreases the amount of GLUT4 in small
vesicles (likely to be GSVs), reduces GLUT4 stability, and
reduces insulin-induced GLUT4 translocation [22]. Intriguingly,
402 R. Govers / Diabetes & Metabolism 40 (2014) 400–410

Fig. 2. Model for intracellular GLUT4 traffic. In non-stimulated adipocytes (left panel), GLUT4 (depicted as triangles) is largely excluded from the plasma membrane
and is predominantly present and retained within GSVs and (subdomains of) recycling endosomes. The TGN generates the GSVs and together with the GSVs and
recycling endosomes, forms an intracellular trafficking loop that contributes to GLUT4 retention. Upon insulin stimulation (right panel), GSVs fuse with the plasma
membrane while also GLUT4 retention in recycling endosomes is reduced, leading to the redistribution of GLUT4 to the plasma membrane. As long as the insulin
stimulus is present, the recruited GLUT4 molecules continue to recycle between the plasma membrane and endosomes, without being sorted into new GSVs. The
thickness of each arrow reflects the relative contribution of the pathway to the intracellular GLUT4 traffic. For reasons of simplicity, the GLUT4 molecules present in
the biosynthetic pathway (up to Golgi stacks) have been omitted. PM: plasma membrane; EE: early (sorting) endosome; RE: recycling endosome; TGN: trans-Golgi
network; GSVs: GLUT4 storage vesicles.

in insulin resistance and diabetes, sortilin levels are reduced
[27,28]. This may imply a role for sortilin in the associated
impairment in GLUT4 translocation. How sortilin is involved
in the formation of GSVs remains uncertain, but it is possible
that an interaction between the lumenal domains of sortilin and
GLUT4 plays a role [29]. IRAP has also been suggested to
be implicated in the sorting of GLUT4 into GSVs or in the
biogenesis of GSVs (thereby contributing to GLUT4 reten-
tion) as IRAP knockdown in vitro induces a redistribution
of GLUT4 towards endosomes (that serve as an alternative
trafficking route when the traffic of GLUT4 to GSVs is dis-
rupted [30]), concomitant with a modest increase in cell surface
GLUT4 levels [21]. IRAP ablation in vivo has been described
to decrease GLUT4 expression in muscle and adipose tissue
[31]. This supports the idea that IRAP facilitates the sorting of
GLUT4 into GSVs as the absence of GLUT4 from GSVs is
known to reduce the stability of the GLUT4 molecule and to
decrease GLUT4 expression levels [22]. Possibly, IRAP facil-
itates the targeting of GLUT4 into GSVs by means of the
interaction between their lumenal domains [26]. Apparently, the
various GSV proteins (including LRP-1 [23]) may be present
in (large) multimeric protein complexes. Knockdown of sev-
eral of these proteins individually leads to a more pronounced
degradation of the other GSV components. Possibly, the GSV
components become complexed in the compartment from which
the GSVs are formed (likely the TGN; Fig. 2), subsequently
triggering the formation of subdomains within this compart-
ment and their budding into GSVs. Sortilin could be implicated
in this process which is in line with its role in the segrega-
tion of regulated from constitutive secretory proteins at the
TGN [32].
In the basal state, GSVs are largely refrained from fusion with
the plasma membrane. What is preventing this fusion is unclear
at present though various scenarios have been proposed, which
are not necessarily mutually exclusive. Possibly, in the absence
of insulin stimulation, the GSVs, which may be mobile and to
some extent traffic close to the plasma membrane, do not have
the proper signals that allow them to fuse with the plasma mem-
brane. Also, in the basal state, the plasma membrane itself is not
“activated” and hence incapable of receiving incoming GSVs
[33]. Alternatively, a large part of the GSVs may be attached
to an immobile intracellular matrix that renders them relatively
static in the absence of insulin. Various proteins have been sug-
gested to be implicated in this form of retention. One is called
TUG (a putative Tether, containing a UBX domain, for GLUT4),
identified by a functional genetic screen [34]. TUG (reviewed
in detail in [35]) was found to interact directly with GLUT4
uniquely in the absence of insulin and has been proposed to
tether GLUT4 vesicles intracellularly. Insulin would release this
tether, possibly via an intramolecular cleavage within TUG [36]
(see also Section 2.5). Given the presence of TUG in an early
(pre-TGN) Golgi compartment where it is involved in Golgi
organization [37] and its interaction with Golgin-160, known
to play a role in intracellular GLUT4 traffic [36,38], TUG may
 R. Govers / Diabetes & Metabolism 40 (2014) 400–410
sequester GSVs by linking them to the Golgi matrix. Alterna-
tively, the data on TUG are consistent with a role for TUG in
the targeting of GLUT4 to GSVs [39]. In line with both pos-
sible roles of TUG are the findings that disruption of cellular
TUG function in vitro increases the amount of GLUT4 in a con-
stitutive endosome-plasma membrane recycling pathway at the
expense of GLUT4 in GSVs (as in normal insulin-stimulated
cells) [39] and that disruption of TUG function in vivo reduces
fasting plasma glucose and insulin levels in mice [40].
No matter what the exact mechanism is by which the GSVs
are sequestrated, it needs to be sensitive to insulin regulation. In
support of exocytosis of the entire GSV upon insulin stimulation
are the findings that insulin reduces the amount of these vesi-
cles within the cytoplasm without altering the concentration of
GLUT4 in the GSVs that remain [41] and that the GSVs them-
selves physically fuse with the plasma membrane [42]. GSV
exocytosis occurs in a highly regulated fashion in that the amount
of GSVs that are recruited correlates with the strength of the
insulin stimulus [43,44].
2.2. Presence of GLUT4 in endosomes
Several pieces of evidence exist that indicate that a part of
the cellular GLUT4 pool is located in endosomal (non-GSV)
compartments and that also this GLUT4 pool is retained intra-
cellularly and sensitive to insulin stimulation. First, in cultured
adipocytes, about half of the total cellular GLUT4 pool is
present in endosomal compartments [15,20]. Given the fact that
GLUT4 is hardly recycling towards the plasma membrane in
non-stimulated adipocytes [43], a retention mechanism has to be
present in endosomes. Moreover, upon insulin stimulation, much
more than 50% of the total cellular GLUT4 pool participates in
cell surface recycling [43], indicating that both endosomal and
non-endosomal GLUT4 pools contribute. This is further sup-
ported by morphological and cell fractionation studies that have
demonstrated that GLUT4 translocates from both GSVs and
endosomes [41,45]. Second, acutely inhibiting protein traffic
from endosomes in living cells reduces insulin-induced GLUT4
translocation by half [20,46]. Third, in adipocytes, a mutated
GLUT4 molecule that is exclusively present in endosomal com-
partments, is still efficiently retained, albeit to a somewhat lesser
extent when compared with wild-type GLUT4 [43]. Moreover,
this molecule still translocates upon insulin stimulation [43].
Fourth, in fibroblast-like preadipocytes and CHO cells, GLUT4
is exclusively present in endosomes [20]. However, in these
cells GLUT4 is retained intracellularly and also more sensi-
tive to insulin stimulation when compared with the transferrin
receptor (TfR), a bona fide endosomal marker that recycles con-
stitutively [47,48]. Nevertheless, also here, its retention and
insulin-sensitivity are reduced when compared with GLUT4
in adipocytes. Finally, prolonged (but not acute) insulin stim-
ulation induces the fusion of GLUT4-containing vesicles with
the plasma membrane that are relatively large in size and that
resemble endosomal vesicles rather than GSVs, suggesting not
only that insulin recruits GLUT4 from both GSVs and endo-
somes but also that there is a temporal shift in the recruitment
from both storage compartments [19]. Possibly related to these
403
findings is that a single insulin stimulation of cardiac myocytes
reduces GLUT4 content uniquely in non-endosomal structures,
while repeated insulin stimulations reduce GLUT4 content
in both non-endosomal as well as endosomal compartments,
again arguing in favor of the recruitment of endosomal GLUT4
[49].
Taken together, these findings indicate that a GLUT4 reten-
tion mechanism exists in endosomal compartments but that
a highly efficient intracellular GLUT4 retention requires the
presence of GLUT4 in non-endosomal structures as well. The
mechanism implicated in the retention of GLUT4 in endosomes
is unknown. As far as the insulin-induced recruitment of GLUT4
is concerned, endosomes may not be the principle GLUT4 donor
site, but may provide the cell system with additional GLUT4
under conditions of high demand, for example under prolonged
insulin stimulations [19] or under repeated stimulations [49].
Upon prolonged insulin stimulation, relatively more intracel-
lular GLUT4 is in endosomes and less in GSVs [20,45] (Fig. 2).
Presumably, GLUT4 that is initially recruited from GSVs is
internalized and immediately recycled back to the cell surface
in vesicles that are likely to be distinct from GSVs and presum-
ably derived from recycling and/or early endosomes. This is
supported by data that suggest that the GLUT4-containing vesi-
cles that fuse with the plasma membrane upon prolonged insulin
stimulation are of endosomal origin [19]. It implies that the same
initially recruited GLUT4 molecules remain recycling, uniquely
between the plasma membrane and endosomes, and fits with the
finding that the amount of VAMP2-decorated GSVs decrease
with insulin, while the amount of GLUT4 in the remaining
(non-recruited) GSVs remains unchanged [41]. Moreover, it
explains why the GLUT4 molecules that recycle between the
cell surface and intracellular compartments in insulin-stimulated
adipocytes do not readily mix with the GLUT4 molecules that
remain retained intracellularly, in particular under conditions of
low to moderate insulin stimulation [43,50]. Taken together, this
has several important consequences. It means that insulin only
transiently activates the GSV release mechanism. It also implies
that in insulin-stimulated cells, the intracellular traffic in general
or specifically that of GLUT4 between endosomes and GSVs,
between endosomes and the TGN (that generates the GSVs), or
between the TGN and GSVs, is disrupted.
2.3. Involvement of the TGN (sub)compartment in GLUT4
traffic
The presence of GLUT4 in the TGN has been suggested on
several occasions and is likely to play a role in its intracellu-
lar regulation. At the morphological level, a significant amount
of GLUT4 in adipocytes is localized in vesicles that lie in the
vicinity of the TGN [51,52], where it colocalizes to a large extent
with the cation-dependent mannose 6-phosphate receptor (CD-
MPR), a protein that recycles between the TGN and endosomes
[52] and with adaptor protein-1 (AP-1), known to be involved in
the exit of proteins from the TGN [53], but not with TGN proteins
TGN38 and furin. Given that the TGN is subcompartmentalized
[54–56], GLUT4 is likely to be present in a specific subdomain
of the TGN [14]. This GLUT4 pool is not depleted upon insulin
404 R. Govers / Diabetes & Metabolism 40 (2014) 400–410
stimulation [52], suggesting an insulin-independent equilibrium
between in- and out-going GLUT4 and a possible involvement
of this GLUT4 pool in an intracellular cycle between TGN and
endosomes (Fig. 2).
Next, it has been shown that a large amount of GLUT4 is
recycling via the TGN in cardiomyocytes [57], while IRAP,
GLUT4 fellow traveller, has been shown to traffic through the
TGN after retrieval from the plasma membrane in adipocytes
[58].
Finally, various Golgi/TGN proteins have been shown to be
implicated in intracellular GLUT4 traffic. Sortilin [22] and GGA
proteins [59,60], involved in the exit of certain proteins from the
TGN, have a clear role in the retention of GLUT4, likely by reg-
ulating the traffic of GLUT4 from the TGN into the GSVs. Also
the TGN t-SNARE proteins syntaxin 6 and syntaxin 16 have
been indicated to be involved in GLUT4 traffic (see Section 2.8
“SNARE proteins”) and may be colocalizing with GLUT4 in the
TGN subdomain that is distinct from the one containing TGN38.
Other Golgi proteins that have been found to be involved in
GLUT4 traffic include Golgin-97 [61], Golgin-160 [38] and
the IRAP-interacting proteins p115 [62] and tankyrase [63].
The Golgi matrix protein Golgin-160 may play a role in GSV
sequestration by linking GSVs to the Golgi matrix as this protein
has been shown to interact with the GLUT4/GSV-sequestering
protein TUG [36] (see Section 2.1).
In conclusion, GLUT4 is present in the TGN. Its presence
is most likely related to the role of the TGN in the biogenesis
of GSVs and in the targeting of GLUT4 and other proteins into
these vesicles. The GSV components are delivered to the TGN
via recycling endosomes, a transport step involving syntaxins 6
and 16, CHC22 (at least in vertebrate cells) [64,65] and possibly
also Rab11 (see Section 2.7 “Rab proteins”).
2.4. Major insulin actions on GLUT4 traffic
Insulin has been reported to display several effects on
GLUT4. First of all, insulin releases GLUT4 from its static com-
partment, where it is relatively immobile. This likely represents
the release of GSVs and results in a two- to three-fold increase
in the amount of GLUT4 vesicles that are mobile [66,67] and
in the redistribution of GLUT4 from a perinuclear location
towards the periphery of the cell, where it accumulates near
the plasma membrane [19,30]. Probably related to this release
is the insulin-induced increase in mean GLUT4 vesicle speed
[67]. In insulin-resistant adipocytes, this insulin effect is partially
impaired [30].
Insulin also displays several major effects at the cell surface.
Insulin enhances the process of the docking and fusion of
GLUT4 vesicles with the plasma membrane. More precisely,
insulin stimulation results in an increase in the halting of
GLUT4 vesicles at the plasma membrane, more efficient tether-
ing and docking (reducing un-tethering and un-docking), and a
reduced docking dwell time, concomitant with an enhancement
of their fusion with the plasma membrane [16,68,69]. The
fusion of GLUT4 vesicles with the plasma membrane appears
to be the major insulin-sensitive step in the process of tethering,
docking and fusion [70] and likely implicates the activation of a
plasma membrane component [33]. Interestingly, these effects
of insulin on GLUT4 at the plasma membrane are impaired
in adipocytes rendered insulin-resistant in vitro as well as in
adipocytes obtained from insulin-resistant human subjects
[30,71]. Functionally, these two insulin actions (GSV brake
release and stimulation of GSV tethering, docking and fusion)
lead to an insulin dose-dependent increase in the size of the cell
surface recycling GLUT4 pool [43,44,72] and in an increased
GLUT4 exocytosis rate in the recycling pool [44].
Another action of insulin that may contribute to increased cell
surface GLUT4 levels in adipocytes is its effect on GLUT4 endo-
cytosis, as insulin may modestly decrease GLUT4 endocytosis
rates. Nevertheless, this insulin action remains controversial
[73–76]. The insulin-induced reduction in GLUT4 endocytosis
may be linked to the existence of multiple GLUT4 internaliza-
tion pathways. In adipocytes, GLUT4 has been demonstrated
to be internalized by both AP-2/clathrin- and cholesterol-
dependent mechanisms that act independently [76,77], whereas
the constitutively recycling TfR is internalized uniquely via the
clathrin-dependent pathway. Insulin has been shown to induce
a shift in GLUT4 internalization from one mechanism to the
other [76]. If the kinetics of both pathways is distinct, this could
explain the effect of insulin on the GLUT4 internalization rate.
Alternatively (or possibly linked), insulin may affect GLUT4
internalization kinetics via its effect on the clustering of GLUT4
molecules at the plasma membrane [78,79].
2.5. Insulin receptor signaling pathways implicated in
GLUT4 translocation
The binding of insulin to the insulin receptor (IR) leads to
structural changes within the receptor that induce the trans-
phosphorylation of various tyrosine residues. This results in
the recruitment of IR substrates to the IR, amongst which IR
substrate 1 (IRS1), and their subsequent tyrosine phosphory-
lation. Phosphorylated IRS1 activates PI 3-kinase in the inner
leaflet of the plasma membrane [80] leading to phosphoryla-
tion of the inositol ring of phosphatidylinositol and the local
formation of the lipid second messenger PIP3. This on its turn
serves as a recruitment platform for phosphoinositide-dependent
kinase-1 (PDK1) and serine/threonine protein kinase B (PKB,
also known as Akt2 [81]). PKB then becomes phosphorylated by
PDK1 as well as by mammalian target of rapamycin complex-
2 (mTORC2), which results in its activation. Activated kinase
PKB also binds to intracellular vesicles that contain GLUT4
[82] and phosphorylates a multitude of substrates. While PKB is
considered to be a key player in insulin-induced GLUT4 translo-
cation [83], PKB-independent pathways exist that contribute to
the full effect of insulin on the redistribution of GLUT4 to the
plasma membrane [84]. For instance, PDK1 not only phosphory-
lates and activates PKB but also the atypical PKC isoforms ␭ and
␨ [85–88]. While both are implicated in GLUT4 translocation,
their downstream effectors are currently unknown [88]. Other
PKB-independent pathways involved in GLUT4 translocation
include phospholipase C (PLC) that interacts with and is acti-
vated by the IR [89], and the IRS1/PI 3-kinase/PKB-independent
TC10 pathway [90]. TC10 is a Rho family member GTPase and
 R. Govers / Diabetes & Metabolism 40 (2014) 400–410
regulates the activity of the exocyst, implicated in the targeting
of secretory vesicles, via its interaction with exocyst subunit
Exo70 [91]. In particular the TC10␣ isoform appears to be
implicated in GLUT4 translocation [92]. Together with a part
of the cellular IR pool, TC10␣ resides to a large extent in lipid
rafts [93]. It has been suggested to be regulated by a CrkII-C3G
complex that binds indirectly to the IR via CAP, APS, and Cbl
[94,95]. However, subsequent siRNA experiments have chal-
lenged this mode of regulation [96]. More recent data indicate
that TC10␣ activity is likely to be regulated via its phosphoryla-
tion and that insulin induces TC10␣ phosphorylation by CDK5
[93]. The role of TC10␣ in GLUT4 translocation likely implies
the exocyst [91,97], but also the cleavage of TUG, required for
the insulin-induced reduction in GSV sequestration [36], as well
as the insulin-induced rearrangement of cortical actin [93], con-
sistent with the role of the cytoskeleton and associated proteins
in GLUT4 translocation (elegantly reviewed in [98]).
In insulin resistance, impaired GLUT4 translocation has
been attributed to defects in signaling by IRS1 [99,100], PKB
[101,102], effectors downstream of PKB [103], and TC10
[104,105]. Additional studies are needed to fully elucidate the
mechanisms by which insulin resistance impinges on these (and
other) signaling pathways involved in GLUT4 translocation.
2.6. PKB substrates
Of the PKB substrates, Akt substrate of 160 kDa (AS160; also
known as Tbc1d4) is best known and has been shown to be a key
modulator of GLUT4 translocation [106,107]. AS160 has been
the first molecule that directly links insulin signaling to intra-
cellular GLUT4 traffic and hence has gotten a lot of attention.
AS160 is a Rab-GTPase-activating protein (GAP [108]) and
active in its dephosphorylated form. Insulin stimulation induces
the phosphorylation of AS160 on at least 5 residues, which
renders its GAP domain inactive [109]. This PKB-mediated
phosphorylation event is facilitated by scaffold protein ClipR-59
[110] and leads to an increase in the GTP-bound (active) form of
the AS160 Rab substrate(s) and in the redistribution of GLUT4
vesicles towards the cell surface. AS160 can interact with GSV
compounds IRAP [24] and LRP1 [23] and is present on GLUT4-
containing vesicles but dissociates from these vesicles upon
insulin stimulation [24]. This suggests that its inhibitory action
on GLUT4 translocation in the absence of insulin requires its
presence on GSVs, maintaining a GSV-associated Rab in its
GDP-bound inactive state. A mutated phosphorylation-deficient
AS160 molecule is constitutively active and induces a reduction
in both Rab activity and insulin-induced GLUT4 transloca-
tion [109]. This effect on GLUT4 translocation is dependent
on a functional Rab-GAP domain within AS160, demonstrat-
ing that its activity towards Rab proteins is required for its
role in GLUT4 traffic. AS160 knockdown induces a moder-
ate increase in basal cell surface GLUT4 levels [24,111,112],
that cannot be rescued by the expression of an AS160 mutant
lacking a functional GAP domain, confirming once more that
the GAP activity is required for AS160 function [111]. Taken
together, these data indicate that AS160 (and hence also its
upstream regulator PKB) plays a crucial role both in the intra-
405
cellular retention of GLUT4 and in the insulin-induced release
of this retention, leading to the accumulation of GLUT4 near
the plasma membrane [30,84]. In addition, AS160 has been
found to play a role in the docking of GLUT4 vesicles with
the plasma membrane [69,70]. This is likely related to the
presence of AS160 at the plasma membrane where it binds to
negatively charged phospholipids [113]. The fact that AS160
knockdown only partially releases GLUT4 retention while
insulin-sensitivity is maintained [112,114] indicates that insulin
induces GLUT4 translocation by both AS160-dependent and
-independent mechanisms. Another indication that supports a
role for an AS160/PKB-independent signaling step in GLUT4
translocation comes from studies that demonstrate that the final
insulin-enhanced GSV-plasma membrane fusion event is PI 3-
kinase-dependent but PKB/AS160-independent [69,84]. Given
that the major insulin action involves this fusion step [70], it is
evident that AS160 is not the (only) master director of GLUT4
translocation. At present, the molecular mechanism by which
insulin enhances fusion rates is unknown and remains to be
elucidated.
Several studies have demonstrated that AS160 is not the
only effector molecule by which PKB regulates GLUT4 traf-
fic [84,112]. Accordingly, other PKB substrates have been
identified that are implicated in insulin-induced GLUT4 translo-
cation. These include motor protein myosin Va, that associates
with the actin cytoskeleton upon its PKB-mediated phosphor-
ylation [115], Grp1, a guanine nucleotide exchange factor
(GEF) for ADP-ribosylation factor 6 (ARF6) [116], also
involved in GLUT4 translocation [117], GSV- and plasma
membrane-associated lipid-binding protein CDP138 [118], syn-
taxin 4-interacting protein Synip, negatively regulating GLUT4
translocation at the GSV-plasma membrane docking step
[119–121], and lipid kinase PIKfyve [122].
2.7. Rab proteins
Recently, several studies have focused on the identification
and functional characterization of the Rab proteins that are
potential substrates of AS160. In adipocytes, the Rab protein
that is currently considered to be the main substrate of AS160
is Rab10. In basal (non-stimulated) adipocytes, a constitutively
active Rab10 mutant increases GLUT4 cell surface levels, while
knockdown of Rab10, but not that of other in vitro AS160 Rab
substrates, inhibits GLUT4 translocation [123,124]. Moreover,
the increase in cell surface GLUT4 levels in basal adipocytes due
to AS160 knockdown is reduced by a simultaneous knockdown
of Rab10 [114,123] and further enhanced by Rab10 overexpress-
ion [114]. This indicates that in basal cells, Rab10 is the Rab
protein maintained in the GDP-bound (inactive) form by AS160
to impose GLUT4 retention. Rab10 probably acts at the level
of the GSVs because neither knockdown nor overexpression of
Rab10 has any effect on cell surface TfR levels [114,123], while
GLUT4-positive TfR-negative vesicles that fuse with the plasma
membrane in response to insulin (i.e. GSVs) are predominantly
loaded with Rab10 [42,125]. Also in support is the recent identi-
fication of Dennd4C as the guanine nucleotide exchange factor
(GEF) for Rab10 (generating the GTP-bound form of Rab10)
406 R. Govers / Diabetes & Metabolism 40 (2014) 400–410
and its implication in GLUT4 translocation [126]. Dennd4C
is constitutively active and not sensitive to insulin stimula-
tion, implying that insulin regulates Rab10 exclusively via
AS160 [114]. The increase in basal cell surface GLUT4 levels
induced by Rab10 overexpression, AS160 knockdown, or by the
combination of both can be further enhanced by insulin [114],
suggesting the existence of a Rab10/AS160-independent insulin
signaling pathway that contributes to GLUT4 translocation. This
is further supported by studies based on total internal reflection
fluorescence (TIRF), a technique that enables the visualization
of labeled proteins at the cell surface or just beneath (at a distance
of maximum 200 nanometers from the plasma membrane), that
have suggested that Rab10 regulates the recruitment of GLUT4
vesicles to the plasma membrane but not their fusion [114].
While Rab10 is the only Rab protein identified on fusion-
competent TfR-negative GLUT4 vesicles (GSVs), other Rab
proteins also seem to be involved in GLUT4 translocation.
Rab14 has been indicated to be present on endosome-derived
GLUT4 vesicles and to be involved in their traffic. The nature of
the acceptor compartment for the Rab14-decorated GLUT4 vesi-
cles is unclear at present as these vesicles have been suggested
to be destined for delivery to the plasma membrane [42], GSVs
[114] and TGN [127]. Overexpression of a constitutively active
Rab14 mutant inhibits GLUT4 translocation [127]. Remark-
ably, Rab14 knockdown also decreases GLUT4 translocation,
but unlike Rab10, does not reduce the increased basal cell surface
GLUT4 levels in AS160-depleted cells, implying that Rab14 is
not likely to be regulated by AS160 [114]. In support of a role
for Rab10 and Rab14 in distinct pathways is the largely addi-
tive effect of the knockdown of Rab10 and Rab14 on GLUT4
translocation [42]. Both Rab10 and Rab14 have been found to
interact with myosin Va, a molecular motor implicated in the
final phases of GLUT4 translocation [42]. Other Rab proteins
that are implicated in intracellular GLUT4 traffic but that act
most likely at other places than the plasma membrane include
Rab31, found to play a negative role in GLUT4 translocation,
possibly via regulating the fusion of GSVs with endosomes at
the expense of the fusion of GSVs with the plasma membrane
[128], Rab5, acting on GLUT4 internalization or on endosomal
GLUT4 traffic [129], Rab11, proposed to play a role in the sor-
ting of GLUT4 into GSVs [20], possibly by regulating the traffic
of GLUT4 from recycling endosomes to the TGN, from where
it can be processed into GSVs, Rab4 [130] and Rab8A [42].
2.8. SNARE proteins
The docking and fusion of GLUT4-containing vesicles with
the plasma membrane are regulated by insulin. Especially the
fusion event is highly insulin-sensitive [70]. The entire process
of tethering, docking and fusion of vesicles with an acceptor
membrane is a highly coordinated system. The key players
that form the basis of this process are the so-called SNAREs
(soluble N-ethylmaleimide-sensitive-factor attachment protein
receptors) together with the proteins that regulate SNARE
function [131]. The SNARE proteins involved in the fusion
of GLUT4 vesicles with the plasma membrane are syntaxin
4, SNAP23, and VAMP2 [132,133]. The target SNAREs (t-
SNAREs) syntaxin 4 and SNAP23 are located on the plasma
membrane. It is generally accepted that VAMP2, also known
as synaptobrevin, is the vesicle SNARE (v-SNARE) present on
GSVs, though v-SNAREs VAMP3 and VAMP8 have also been
suggested to be present on GLUT4 vesicles and to be possibly
implicated in GLUT4 translocation [134–136]. VAMP2 specif-
ically displays affinity for the syntaxin 4/SNAP23 complex and
hence this affinity aids in the regulation of the specificity (i.e.
selectivity) of the fusion process. Together, syntaxin 4, SNAP23
and VAMP2 form a tight four-helix bundle, enabling the docking
and fusion of the GLUT4 vesicles with the plasma membrane.
Additional SNARE proteins are involved as well in intracel-
lular GLUT4 traffic as they are implicated in the other GLUT4
trafficking steps. One example concerns a TGN t-SNARE-
complex consisting of syntaxins 6 and 16 [137]. These SNAREs
are present in the TGN as well as on GLUT4-containing vesi-
cles and substantially redistribute to the plasma membrane upon
insulin stimulation [58]. Functionally, syntaxins 6 and 16 have
been suggested to be implicated in the sequestration of GLUT4
from endosomes into GSVs [137,138]. Given their usual role
in protein traffic between endosomes and TGN [139], these t-
SNAREs are likely to mediate the traffic of GLUT4 to the TGN,
from where it can be further sorted into GSVs. Also the syntaxin
16 regulator mVps45, member of the SM (Sec1/Munc18) family
of proteins, has been demonstrated to play a role in this pathway
[140].
At present, about ten proteins have been identified that are
implicated in the regulation of the SNAREs that mediate the
docking and fusion of GLUT4 vesicles with the plasma mem-
brane. One of them is SM family member munc18c. This protein
is an inhibitory regulator that binds syntaxin 4, thereby reduc-
ing the interaction of syntaxin 4 with SNAP23 and VAMP2
[141,142]. Upon insulin stimulation, the activated insulin recep-
tor phosphorylates munc18c [143] which reduces its affinity for
syntaxin 4, leading to a decrease in the interaction between
munc18c and syntaxin 4 [144], enabling the formation of the
trimeric SNARE-complex, required for the fusion of GLUT4
vesicles with the plasma membrane. In accordance, Munc18c
ablation results in an increased GLUT4 translocation [145],
while munc18c overexpression causes insulin resistance [146].
Another protein that binds and regulates syntaxin 4 is Doc2b
[147]. Insulin increases the interaction between Doc2b and syn-
taxin 4, thereby enhancing the docking and/or fusion of GSVs
with the plasma membrane [147]. Accordingly, GLUT4 translo-
cation is enhanced by Doc2b overexpression and inhibited by
Doc2b knockdown [147]. Recently, Doc2b has been suggested
to enhance the SNARE-mediated fusion process by inducing a
local curvature of the target membrane [136].
3. Concluding remarks
T2D is taking epidemiological proportions and is associated
with insulin resistance, in which muscle and adipose tissue take
up less glucose. Since GLUT4 is the main transporter that is
responsible for this uptake, GLUT4 is considered to play an
essential role in insulin resistance and T2D. While the intracellu-
lar GLUT4 traffic and its sensitivity to insulin have proven to be
 R. Govers / Diabetes & Metabolism 40 (2014) 400–410
regulated in a complex manner, our understanding of its itinerary
and of the molecules implicated in the various trafficking steps
has been largely improved over the years. However, how GLUT4
is efficiently retained intracellularly and how insulin exactly
impinges on this retention is still largely unknown and remains to
be clarified. Unveiling these mechanisms remains a future goal
and may aid in the development of novel therapeutic avenues
for the treatment of T2D.
Disclosure of interest
The author declares that he has no conflicts of interest con-
cerning this article.
Acknowledgements
I apologize to the research groups in this field whose excel-
lent work was not cited due to restricted space limitations. I
am grateful to Teresa Gonzalez for assistance in preparing the
French abstract. This work is supported by an Inserm Avenir
grant and by an Alfediam/SFD-Roche Diagnostics award.
Appendix A. Supplementary data
Supplementary data (French abstract) associated with this
article can be found, in the online version, at http://dx.doi.
org/10.1016/j.diabet.2014.01.005.
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