Insulin action on glucose transporters through molecular switches, tracks and tethers

Abstract

Glucose entry into muscle cells is precisely regulated by insulin, through recruitment of GLUT4 (glucose transporter-4) to the membrane of muscle and fat cells. Work done over more than two decades has contributed to mapping the insulin signalling and GLUT4 vesicle trafficking events underpinning this response. In spite of this intensive scientific research, there are outstanding questions that continue to challenge us today. The present review summarizes the knowledge in the field, with emphasis on the latest breakthroughs in insulin signalling at the level of AS160 (Akt substrate of 160 kDa), TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) and their target Rab proteins; in vesicle trafficking at the level of vesicle mobilization, tethering, docking and fusion with the membrane; and in the participation of the cytoskeleton to achieve optimal temporal and spatial location of insulin-derived signals and GLUT4 vesicles.

Full text

Biochem. J. (2008) 413, 201–215 (Printed in Great Britain) doi:10.1042/BJ20080723 201
REVIEW ARTICLE
Insulin action on glucose transporters through molecular switches,
tracks and tethers
Hilal ZAID*, Costin N. ANTONESCU*†, Varinder K. RANDHAWA*†and Amira KLIP*†1
*Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada, and †Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
Glucose entry into muscle cells is precisely regulated by insulin,
through recruitment of GLUT4 (glucose transporter-4) to the
membrane of muscle and fat cells. Work done over more than
two decades has contributed to mapping the insulin signalling and
GLUT4 vesicle trafficking events underpinning this response. In
spite of this intensive scientific research, there are outstanding
questions that continue to challenge us today. The present review
summarizes the knowledge in the field, with emphasis on the
latest breakthroughs in insulin signalling at the level of AS160
(Akt substrate of 160 kDa), TBC1D1 (tre-2/USP6, BUB2, cdc16
domain family member 1) and their target Rab proteins; in vesicle
trafficking at the level of vesicle mobilization, tethering, docking
and fusion with the membrane; and in the participation of the
cytoskeleton to achieve optimal temporal and spatial location of
insulin-derived signals and GLUT4 vesicles.
Key words: actin dynamics, glucose transporter 4 (GLUT4),
insulin signalling, muscle cells, phosphoinositide, vesicle
traffic.
INTRODUCTION
Glucose is the major fuel of most mammalian cells, and insulin
is its principal regulator. A key function of insulin is to allow
glucose entry into muscle and fat cells. Skeletal muscle is the
largest site for disposal of dietary glucose, and GLUT4 (glucose
transporter-4) is the port of entry for glucose into this tissue.
One of the most fascinating biological discoveries of the past
two decades is the mechanism whereby insulin regulates GLUT4
availability at the surface of muscle and fat cells. It is noteworthy
that the original ‘translocation hypothesis’, whereby insulin would
rapidly increase the number of glucose transporters at the fat
cell membrane at the expense of an intracellular pool, was
postulated long before the cloning of GLUT4 or the availability
of dynamic assays of vesicle movement within cells. To date, the
concept of GLUT4 translocation is highlighted in textbooks and
pharmaceutical pamphlets, and in the scientific literature the trans-
porter has been the subject of almost 4000 publications. The
dynamic nature of this process has been further elucidated through
elegant and creative imaging methodologies, implemented by
many groups around the world. Understandably, the regulation
of GLUT4 by insulin has been the subject of scholarly reviews in
previous years [1–8]. In the present review we attempt to cover the
fundamental concepts that have been experimentally tested and
to highlight some ideas that are currently being discussed on the
dynamic nature of GLUT4 trafficking and how insulin-derived
signals regulate it. We also review the dynamic regulation of
the cytoskeleton by the hormone as a potential mechanism for
integration of insulin signalling and GLUT4 vesicle trafficking.
Particular attention is given to the current debate of whether
insulin signals impinge on one or numerous steps along the
intracellular itinerary of GLUT4.
Finally, we focus the present review on studies in both muscle
and fat cells. Of note, human and rodent skeletal muscle or
muscle cells respond to insulin with a 2- to 3-fold gain in surface
GLUT4 units; human adipocytes mount a 2- to 5-fold gain and
their rodent counterparts typically respond with a 10- to 20-fold
gain. Overall, the basic principles of GLUT4 translocation are
similar in all of these systems, and the quantitative differences
may be related to the fidelity of each step and, in some cases, to
the reliance on distinct isoforms of the individual insulin signals
and vesicle trafficking machinery.
GLUT4 CYCLING AND CELLULAR COMPARTMENTS
Kinetic models of GLUT4 cycling
In unstimulated muscle or adipose cells, 4–10 %of GLUT4
is located at the cell surface and >90 %at intracellular
compartments. This steady-state distribution of GLUT4 is the
balance of its fast endocytosis and slow recycling, which in muscle
cells have t1/2values of 3.5 and 120 min respectively [9,10]. The
cycling properties of GLUT4 in muscle cells are very similar
to those in adipose cells, although its recycling is even slower
in the latter (t1/2is 230 min) [7,11,12]. Insulin shifts the steady-
state distribution of GLUT4 from intracellular pools towards the
PM (plasma membrane) largely by elevating its exocytic rate. In
muscle cells, insulin does not alter its endocytic rate [9,10,12a],
but it modestly reduces it in adipocytes [13,14]. Within 10–
15 min of hormonal stimulation of muscle cells, surface GLUT4
Abbreviations used: ACTN4, α-actinin 4; aPKC, atypical protein kinase C; AS160, Akt substrate of 160 kDa; GAP, GTPase-activating protein; GFP, green
fluorescent protein; GLUT4, glucose transporter 4; GSV, GLUT4-storage vesicle; IGF, insulin-like growth factor; IRAP, insulin-regulated aminopeptidase;
IRS, insulin receptor substrate; NSF,
N
-ethylmaleimide-sensitive factor; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PIKfyve, FYVE-domain
containing PtdIns3
P
5-kinase; PKB, protein kinase B; PKC, protein kinase C; PM, plasma membrane; PTEN, phosphatase and tensin homologue deleted
on chromosome 10; RE, recycling endosome; SC, specialized or storage compartment; SHIP2, Src homology 2-containing inositol phosphatase-2; siRNA,
small interfering RNA; SM, Sec1p/Munc18; SNAP, soluble NSF-attachment protein; TBC1D1, tre-2/USP6, BUB2, cdc16 domain family member 1; TfR,
transferrin receptor; TGN,
trans
-Golgi network; TIRF, total internal reflection fluorescence; t-SNARE, target SNARE; TUG, tether containing UBX domain
for GLUT4; VAMP2, vesicle-associated membrane protein 2; v-SNARE, vesicular soluble
N
-ethylmaleimide-sensitive factor-attachment protein receptor;
WASP, Wiskott–Aldrich syndrome protein.
1To whom correspondence should be addressed (email amira@sickkids.ca).
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202 H. Zaid and others
Figure 1 GLUT4 concentrates in perinuclear compartments and translocates to the PM upon insulin stimulation
Adhered L6 myoblasts (A), rounded-up L6 myoblasts (B) or L6 myotubes (C) stably expressing GLUT4myc with or without insulin stimulation (100 nM insulin for 20 min) were subsequently fixed
and processed for immunofluorescence using an anti-myc antibody as described previously [10,96,153]. (i) Permeabilized cells showing total cellular GLUT4myc. (ii) Non-permeabilized cells
showing GLUT4myc exclusively at the cell surface. (D) 3T3-L1 adipocytes with or without insulin stimulation (100 nM insulin for 20 min) were fixed, permeabilized and labelled with anti-GLUT4
antibodies to detect total cellular GLUT4. Scale bars: (Aand C), 20 μm; (Band D), 5 μm.
levels double and remain stable for at least 30 min. Figure 1
illustrates the gain in surface GLUT4 in muscle and adipose cells
in culture. GLUT4 is retrieved from the PM by clathrin-dependent
and -independent routes of endocytosis [12a,14–16]. Internalized
GLUT4 is routed through a poorly understood series of endosomal
compartments, which ultimately concentrate GLUT4 into SCs
(‘specialized’ or ‘storage’ compartments ). The SC is also known
as the insulin-responsive compartment or GSVs (GLUT4-storage
vesicles), described as discrete endosomes and/or regions of the
TGN (trans-Golgi network) [4,17].
The complex GLUT4 itinerary has made it difficult to char-
acterize SC biochemically or spatially. Nonetheless, we and others
have provided evidence of partial segregation of GLUT4 from the
REs (recycling endosomes), as follows: (i) by velocity gradient
centrifugation of adipose cells, GLUT4 is found in two endo-
membrane fractions, only one containing the transferrin receptor,
TfR, a marker of the RE [18]; (ii) by ablation of the TfR-containing
compartment in intact 3T3-L1 adipocytes, only 40%of GLUT4
is affected [19,20]; (iii) by immunoprecipitation of GLUT4-rich
endomembranes from L6 muscle cells, recycling proteins are
partly excluded [21]. These and related studies have lent support
for the existence of two pools of GLUT4, differentiated by their
content of RE markers. The SC is considered to be the non-RE
pool of GLUT4, and it has been debated whether it is static in
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Insulin action on glucose transporters 203
Figure 2 Static and dynamic models of GLUT4 cycling
(A) In the static-retention model, GLUT4 in the SC does not recycle to the PM, but may engage
in an idle cycle with the TGN. Insulin (ins) redistributes GLUT4 through exit from the SC directly
to the PM. (B) In the dynamic model(s), GLUT4 recycles to the PM through the RE, but an
idle cycle between the RE and SC reduces the efficiency of recycling. Insulin (ins) redistributes
GLUT4 to the PM through exit from the SC directly to the PM, but potentially also through the
RE. See text for details.
the basal state or whether it slowly recycles (either to the PM
or back to the RE). In L6 muscle cells, the entire intracellular
complement of GLUT4 recycles to the PM within 6 h [9,10,22],
whereas such behaviour is controversial in 3T3-L1 adipocytes.
McGraw and co-workers [11] suggested the dynamic-exchange
model, whereby GLUT4 continuously cycles between the SC and
RE, and can slowly escape this dynamic exchange to reach the
PM. Insulin would shift the exocytic and endocytic rates, thereby
elevating GLUT4 at the PM [11]. An alternative model, termed
static retention, was proposed by James and co-workers [23],
suggesting that GLUT4 in the SC/GSV never reaches the PM
under basal conditions. Instead, GLUT4 would either be unable
to cycle out of the SC/GSV in the basal state, or engage in an
unproductive idle cycle between the TGN and/or RE and the SC/
GSV [23]. Insulin would either shift the balance towards the RE
or trigger GLUT4 exit directly from the SC/GSV to the PM, and
would further stimulate the fusion of RE-derived vesicles with
the PM, effectively drawing GLUT4 away from idle intracellular
cycling [23]. Both models propose intracellular idle cycling of
GLUT4 (whether or not including the TGN), but critically differ
in the proposed ability or inability of GLUT4 to slowly recycle to
the PM in the basal state. Figure 2 depicts the main differences
between these models. Interestingly, a recent report suggests that
the different results that support each model may have arisen
from the culture conditions employed [24]. In confluent cultures,
as used to propose the static model, approx. 80 %of GLUT4
is retained in non-recycling compartments. However, re-plating
of the cells, as used to propose the dynamic model, shifts 80 %of
GLUT4 to the recycling mode.
Contrary to the case of 3T3-L1 cells, in L6 myoblasts, GLUT4
recycles to the PM continuously, if slowly, in either confluent or
sparse cultures [9,10,22]. As only 50 %of intracellular GLUT4
coincides with the TfR in REs at steady state measured by a
‘contents mixing approach’ (V.K. Randhawa and A. Klip, unpub-
lished work), it is conceivable that, in myoblasts, GLUT4
undergoes idle cycling between the SC and RE, and this retards,
but does not preclude, its availability to recycle to the PM.
GLUT4 compartments
As described above, in unstimulated cells, nearly half of the
intracellular GLUT4 complement fails to share the lumen
of intracellular compartments containing markers of the RE
[25,26]. Important questions remaining to be answered are
whether GLUT4 vesicles reaching the PM in the basal and insulin-
stimulated states emanate from the same or different compart-
ments (RE or SC), and whether insulin-derived signals regulate
intracellular idle cycling, exit from these compartments and/or
their interaction and fusion with the PM. Our current understand-
ing of these events is summarized next.
One way to analyse the provenance of GLUT4 vesicles reaching
the PM in the basal or insulin-stimulated states is to compare
their biochemical characteristics. Although this has not been
feasible through standard biochemical approaches, use of live-
cell imaging and TIRF (total internal reflection fluorescence)
microscopy should be more revealing. In addition, functional
assays have aided in this distinction. Fusion of GLUT4 with the
PM requires the v-SNARE (vesicular soluble N-ethylmaleimide-
sensitive factor-attachment protein receptor) VAMP2 (vesicle-
associated membrane protein 2), whereas fusion in the basal state
is independent of this protein ([27,28] and references therein).
Moreover, in the basal state, GLUT4 insertion into the membrane
depends on the v-SNARE VAMP7/TI-VAMP (tetanus neurotoxin
insensitive VAMP) [29]. Neither basal-state recycling nor insulin-
dependent gain in surface GLUT4 depend on VAMP3 [27–29].
Collectively, these studies suggest that GLUT4 vesicles exiting the
RE and SC may be differentiated from each other and from other
recycling vesicles by virtue of their VAMP isoform. Moreover,
these findings raise the hypothesis that VAMP7 is present in
GLUT4 vesicles emanating from the RE and VAMP2 in GLUT4
vesicles emanating from the SC.
The differential presence of VAMP2 or VAMP7 could poten-
tially be used to isolate and characterize the SC, and efforts to
this effect are underway. To date, characterization of GLUT4-
containing intracellular compartments separated by differential
centrifugation and relatively devoid of cargo-loaded TfR [18]
revealed enrichment in IRAP (insulin-regulated aminopeptidase),
IGF (insulin-like growth factor) II/mannose 6-phosphate receptor,
sortilin, VAMP2 and VAMP3 [30–32], yet it is not clear whether
all of these are specific markers of the SC. Through a separate
approach, immunopurified GLUT4-containing compartments
examined by MS showed enrichment in a cohort of proteins,
among which IRAP and VAMP2 stood out [33]. More discerning
purifications are needed to identify bona fide markers of the SC.
Towards this goal, some studies have investigated the proteins that
bind directly to GLUT4. A detailed analysis of these proteins
[including TUG (tether containing UBX domain for GLUT4),
aldolase and Ubc9] has been presented in [6] and is not further
discussed in the present review.
Strikingly, it is not known where the SC is located within the
adipose or muscle cells. By immunofluorescence, the majority of
GLUT4 is found in a perinuclear region in both 3T3-L1 adipocytes
and L6 myoblasts and myotubes. Figure 1 illustrates this
perinuclear GLUT4 localization in these cell types. Perinuclear
GLUT4 exhibits partial co-localization with RE and TGN
markers, and significant co-localization with VAMP2 [29,34,34a].
However, such co-localization is limited by the resolution of
light microscopy, and does not prove spatial co-incidence.
Selective compartment ablation and high-resolution fluorescence
imaging techniques are needed for quantitative co-localization of
the SC. Moreover, it has also been proposed that the functional SC
is distributed throughout the cytosol and near the PM, particularly
in mature adipocytes [35]. Once incorporated into the cell surface,
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204 H. Zaid and others
the distribution of GLUT4 is also not homogeneous: in mature
skeletal muscle, GLUT4 is preferentially directed towards the
transverse tubules [36,37]. Two interesting recent studies suggest
that local submembrane depots furnish the transporter to the
transverse tubules and that tubular GLUT4 is a major determinant
of insulin-dependent glucose uptake [38,39]. In adipose cells,
some reports assign transitory deposition of surface GLUT4 in
areas of caveolae [40], and the functional significance of this
distribution merits further investigation.
Regulated steps in GLUT4 exocytosis
This question has been the subject of recent, elegant reviews
[1,3,7], reflecting the burgeoning information and continuously
revised models that should in the future be reconciled into a mol-
ecular and temporal map of the participating events. Here we
offer a bird’s eye view of the current knowledge of these
phenomena.
Vectorial transfer
There is current debate whether: (i) insulin induces net vectorial
transfer of GLUT4-containing vesicles towards the cell peri-
phery where additional signals accelerate their fusion; or,
(ii) GLUT4-containing vesicles continuously travel to and from
the cell periphery, and insulin-derived signals only promote their
interaction and fusion with the PM. The debate arose from the
difficulty in detecting net vectorial transfer, and from the com-
paratively well-documented regulation of events near/at the PM.
The observation that purified PM fractions from insulin-stimul-
ated rat adipocytes retain the molecular information to promote
fusion with intracellular GLUT4-containing vesicles derived
from unstimulated cells suggested that a major contribution to
regulation occurs at the PM [41]; however, this extrapolation from
in vitro studies does not rule out that there can be important
regulation in order to make the vesicles available to those
membrane events in intact cells. Even when the quantitative
contribution of PM-proximal events in adipocytes is large, there
is support for regulation of earlier steps in the GLUT4 itinerary,
as follows: by labelling GLUT4 at the surface of muscle cells
and tracking their intracellular itinerary, it was observed that
the transporters rapidly internalize (t1/23.5 min) from the cell
surface, reach the RE within 20 min and exit this compartment
20 min later [10]. As recycling back to the PM is very slow
(t1/2120 min), these results suggest that GLUT4 exiting the RE
is probably routed to the SC. Insulin significantly accelerated
the transit of GLUT4 in and out of the RE [10], and such
acceleration required input of PI3K (phosphoinositide 3-kinase)
and PKB (protein kinase B) (see section ‘Signals regulating
GLUT4 traffic’ below), suggesting that refilling the SC from the
RE is under insulin regulation. Additional observations supporting
the concept that insulin-derived signals regulate GLUT4 exit
from intracellular compartments are: (i) a reorganization of
GLUT4 in the perinuclear region that is not the result of
initial changes at the muscle cell surface [34a]; (ii) in vitro
budding of GLUT4-containing small vesicles (that do not sedi-
ment at 16 000 gfor 20 min) from purified intracellular mem-
branes, and increased abundance of these vesicles in isolates from
insulin-stimulated cells [42]; and (iii) increased abundance of GFP
(green fluorescent protein)–GLUT4 moving along linear tracks in
live cells [43].
Following release from idle cycling, GLUT4-containing
vesicles translocate towards the cell membrane where they tether,
dock and fuse. Although different definitions have been proposed
for tethering and docking, it can be assumed that tethering is a
cortical immobilization that primes or directs vesicles towards
docking sites. Docking is the high-energy, long-lasting but non-
covalent binding of GLUT4 vesicles to the PM, probably mediated
by SNARE proteins, and fusion is the integration of the vesicular
bilayer into the PM, resulting in exposure of the lumen of
the GLUT4 vesicle to the extracellular medium. There is sug-
gestive evidence that each of these three distinct steps is regulated
by insulin.
Vesicle tethering
The exocyst, a tethering machinery conserved from yeast to
mammals, is a stable complex of proteins Sec3p, Sec5p, Sec6p,
Sec8p, Sec10p, Sec15p, Exo70p and Exo84p [44]. Among them,
Exo70 is thought to play a central role as it interacts with almost all
of the other subunits [45,46]. Interestingly, in 3T3-L1 adipocytes,
Exo70 translocates to the PM upon insulin stimulation, through
activation of the Rho GTPase TC10 [47]. The C-terminal of
Snapin binds to the coiled-coil domain in Exo70, and Snapin
expression knockdown somewhat inhibited glucose uptake in
3T3-L1 adipocytes [48]. It is still unknown, however, whether
GLUT4 vesicles physically dock through the exocyst, and the
participation of Exo70 in GLUT4 translocation was recently
challenged [48a]. Additional or alternative components of the
functional peripheral tether of GLUT4 vesicles could include
elements of the cortical actin that remodels in response to insulin
(see the section ‘Cytoskeleton input on GLUT4 translocation’
below).
Vesicle docking and fusion
Owing to insufficient sensitivity of light microscopy techniques,
it had been difficult to distinguish between docking and fusion
events in a quantitative fashion, and to define their regulation by
insulin. Use of TIRF and fluorescent probes sensitive to the extra-
cellular pH have resulted in breakthroughs in this regard. As
TIRF only analyses events within 200 nm of the surface, these
studies do not rule out possible regulation of earlier steps such
as vesicle budding or vectorial traffic. By quantitative analysis
of TIRF images in live 3T3-L1 adipocytes expressing GFP–
GLUT4 chimaeras, three groups reported that GLUT4 vesicles
are mobile in the vicinity of the membrane, and insulin increases
the number of vesicles immobilized in that zone [35,49]. This
has been interpreted as reflecting increased docking, although it
could also result from tethering. Moreover, a study challenged
this tenet, proposing instead that immobilization occurs post-
fusion and reflects GLUT4 molecules in clathrin lattices [43].
Nonetheless, because interfering with PI3K or AS160 (Akt/PKB
substrate of 160 kDa) signalling reduced the insulin-induced
peripheral immobilization of GLUT4 and these signals contribute
to net GLUT4 exocytosis, it is likely that cortical immobilization
of GLUT4 is related to tethering/docking rather than its
endocytosis.
Analysis of the dwell time of each vesicle at the membrane also
suggested that docking is an obligatory step prior to vesicle fusion
with the membrane. Using a GLUT4 surrogate (IRAP) linked to
a GFP sensitive to the pH of the extracellular medium (pHluorin),
Jiang et al. [50] recently showed that IRAP-containing vesicles
‘dock’ and fuse in response to insulin, and that AS160 regulates
the docking step.
SNARE proteins are thought to mediate both docking and
fusion events, and hence it will be important to use this IRAP–
pHlouorin reporter to differentiate the step(s) that they mediate in
the GLUT4 interaction with the PM. The evidence that SNARE
proteins ultimately mediate exposure of GLUT4 at the cell surface
is vast. As mentioned earlier, the SC contains the v-SNARE
VAMP2, which binds to its cognate t-SNARE (target SNARE)
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Insulin action on glucose transporters 205
receptors syntaxin-4 and SNAP (soluble NSF-attachment protein)
23 at the PM [28,51]. Whether insulin promotes this interaction
or simply increases the proportion of GLUT4 vesicles available
for SNARE complex formation is not known. It is well docu-
mented that VAMP2, syntaxin4 and SNAP23 are the elements of
the SNARE complex involved in docking/fusion of insulin-sens-
itive GLUT4 vesicles with the PM. This has been determined by
interfering with SNARE complex formation by either selective
toxins (cleaving VAMP2), gene knockdown (targeting VAMP2),
neutralizing antibodies (binding to SNAP23) or peptides mim-
icking binding regions (of all three proteins) [28]. The SNARE
complex consists of four parallel α-helices formed from the
coiled-coil segments of SNARE proteins in two opposite mem-
branes [52]. Prior to docking/fusion, cis-SNARE complexes exist
on vesicles and must be disassembled to allow trans-SNARE
complex formation. cis-SNARE complex disassembly is driven by
the ATPase activity of NSF (N-ethylmaleimide-sensitive factor).
NSF recruitment to the cis-SNARE complex requires SNAP
[53]. NSF is believed to associate with GLUT4 vesicles and
cell membranes, but this interaction itself does not impact on
the formation of fusion complexes [54]. ATPase-deficient NSF
(that binds SNAREs but cannot disassemble them) predominantly
affected intracellular membrane fusion events involved in GLUT4
cycling from the endosomal system to the SC, but not to the
PM [55] in rat adipocytes. The same mutant precluded basal
state and insulin-dependent gain in surface GLUT4 in muscle
cells [22].
The regulation of SNARE complex formation is believed
to be exerted via SM (Sec1p/Munc18) [56]. Among the three
mammalian SM genes, Munc18c is involved in GLUT4 traffick-
ing, stabilizing syntaxin in the closed inactive conformation in the
basal state. Although there is good evidence from fixed cells or
in vitro analysis that Munc18c dissociates from syntaxin4 upon
insulin stimulation [57,58], by fluorescence correlation spectro-
scopy of live cells it was determined that Munc18c instead
switches to a different binding site on the t-SNARE, thereby
allowing VAMP2 and GLUT4 vesicle docking at the PM
[59].
The basic parameters of vesicle vectorial transfer, tethering,
docking and fusion are illustrated in Figure 3(A), along with the
insulin-derived signals that may control them (see below).
INSULIN SIGNALS REGULATING GLUT4 CYCLING
There are a number of excellent reviews of insulin signalling
[8,60], and we have previously reviewed signalling events leading
to glucose uptake [61]. However, as new elements are identified
and old controversies are reconciled, we have revisited and
updated these events in a concise way, focusing on GLUT4 trans-
location as the specific outcome. Figure 3(B) presents a working
model of insulin signals that impact on GLUT4 trafficking, which
summarizes most evidence to date.
IRSs (insulin receptor substrates)
It is well known that insulin binding elicits rapid autophosphory-
lation of its receptor, followed by binding and tyrosine
phosphorylation of IRS-1/2. Using siRNA (small interfering
RNA)-mediated knockdown of IRS proteins, it was found that
IRS-1, rather than IRS-2, is required for GLUT4 translocation to
the muscle cell surface and glucose uptake [62,63]. This finding
is consistent with the decrease of GLUT4 translocation observed
upon expression of a fragment encoding the PH (pleckstrin
homology) domain of IRS-1 that blocked signalling through
IRS-1 [64]. A number of studies have also reported differential
contributions of IRS-1 and IRS-2 to glucose metabolism. This
is outside of the scope of this review, but is summarized in
[65].
PI3K
IRS-1 binds the regulatory p85 subunit of class I PI3K activating
its catalytic p110 subunit. The PI3K family enzymes phosphoryl-
ate the third hydroxyl position of the inositol ring of phospho-
inositides. The PI3K family is subdivided into three classes,
the heterodimeric class I and III composed each of a catalytic
and regulatory subunit, and class II composed of only a cata-
lytic subunit [66].
Early in 1990, two groups showed that insulin stimulates
PI3K activity [67,68]. Those pioneering results were followed
by numerous studies geared to identify the precise mechanism
of PI3K impact on insulin action. Essential to this quest were
wortmannin and LY294002, unrelated inhibitors that block the
catalytic activity of the enzyme [69,70], as well as a dominant-
negative, class I PI3K mutant (delta-p85) [71,72]. There is a wide
consensus that the two chemical inhibitors (at concentrations
that inhibit classes I and III but not class II PI3K) or delta-
p85 overexpression vastly reduce the insulin-dependent gains
in glucose uptake and surface GLUT4 levels in adipose and
muscle cells (reviewed in [61]). It also goes unchallenged that
overexpression of constitutively active class I PI3K elevates
surface GLUT4 levels in adipocytes [71]. These results suggest
that one or more of the products of class I PI3K are essential for
this function.
In vivo, the major product of class I PI3K is PtdIns(3,4,5)P3,
although PtdIns(3,4)P2and PtdIns3Palso rise in muscle and
fat cells in response to insulin [69,73,74], and may originate
from either activation of class II [producing PtdIns(3,4)P2and
PtdIns(3)P] or class III (producing PtdIns3P), or from further
metabolism of PtdIns(3,4,5)P3. It is therefore interesting that
PtdIns(3,4,5)P3introduced into L6 muscle cells was not sufficient
to stimulate glucose uptake, although it effectively evoked a gain
in surface GLUT4 levels [75,76]. In contrast, PtdIns3Padmin-
istration induced GLUT4 arrival near the PM that did not fuse
with the membrane, and PtdIns(3,4)P2had no consequence on
GLUT4. From those studies a model arose whereby insulin-
induced activation of class I PI3K produces PtdIns(3,4,5)P3that
contributes, along with PtdIns3P, to mobilize GLUT4 vesicles to-
wards the surface, whereas only PtdIns(3,4,5)P3mediates vesicle
fusion with the membrane [76]. Consistent with this prediction,
PtdIns3Pwas insufficient to allow vesicle fusion in adipocytes,
until removal of a fusion block at the level of Munc18 allowed
fusion to proceed [57]. Also, overexpression of the PtdIns(3,5)P2
phosphatase ‘72-5ptase’ (72-kDa inositol polyphosphate 5-phos-
phatase) enhanced the production of PtdIns3P, and promoted
GLUT4 vesicle mobilization [77]. Conversely, overexpression of
aPtdIns3Pphosphatase, myotubularin, reduced insulin-depend-
ent GFP–GLUT4 movement to the cell periphery [78], further
substantiating the participation of this phosphoinositide in insulin
action. PtdIns3Pis a substrate of the enzyme PIKfyve (FYVE-
domain containing PtdIns3P5-kinase), an enzyme also involved
in insulin signalling (see below).
The model of dual input of PtdIns(3,4,5)P3and PtdIns3Pwas
further supported and expanded by the finding that PtdIns3P
is produced by other members of the PI3K family. Indeed,
PI3K-II C2αis activated in response to insulin [79,80] and
generates PtdIns3Pin L6 muscle cells upon insulin stimulation
[74]. The small GTPase TC10 appeared to be needed for such
PI3K-II activation based on overexpression of dominant negative
or constitutively active TC10 mutants. PI3K-II knockdown
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206 H. Zaid and others
Figure 3 Consolidated insulin signalling pathways regulating GLUT4 traffic
(A) Stages of GLUT4 exocytosis regulated by insulin.
Mobilization
: GLUT4 vesicles are transported to the cell periphery, possibly along microtubules.
Tethering
: GLUT4 vesicles are retained near the
cell periphery by the remodelled actin cytoskeleton through ACTN4, and/or by the exocyst complex.
Docking
: GLUT4 vesicles bind to the PM via SNARE complexes.
Fusion
: irreversible incorporation
of GLUT4 vesicles on to the PM is enhanced by insulin through action of Munc18c on SNARE proteins. Also shown are the possible steps of input by PtdIns3
P
and PtdIns(3,4,5)
P
3.Ananimated
version of this Figure is available at http://www.BiochemJ.org/bj/413/0201/bj4130201add.htm (B) Consolidated figure summarizing all signalling pathways proposed to lead to GLUT4 translocation
to the PM. The insulin receptor phosphorylates IRS1/2, which activates PI3K. The IRS-2 pathway is not involved in the GLUT4 trafficking. Downstream of IRS-1/PI3K, signalling bifurcates into two
arms. One arm is characterized by PKB/Akt, which in turn phosphorylates AS160 thus shutting of its GAP activity towards target Rabs. The other arm may involve aPKC and is characterized by
Rac and actin remodelling. ACTN4 possibly links GLUT4 vesicles to actin filaments. In addition, insulin can activate the CAP/Cbl pathway leading to TC10 activation, which has an impact on actin
remodelling. Several phosphoinositide phosphatases and kinases shown are predicted to regulate these pathways.
attenuated GLUT4 translocation and glucose uptake in the insulin-
stimulated state [74], supporting the participation of its products
in GLUT4 trafficking.
The dual participation of PtdIns(3,4,5)P3and PtdIns3Pto
increase surface GLUT4 does not, however, lead to productive
glucose uptake [76]. Hence it has been hypothesized that the gain
in glucose may involve activation of the PM-inserted transporters.
Moreover, a PtdIns(4,5)P2-binding peptide (PBP10) introduced
into adipocytes caused GLUT4 mobilization and insertion in the
membrane, without any accompanying gain in glucose uptake
[81]. Although the relationship of this peptide to PtdIns(4,5)P2
availability in the cells remains to be determined, the results
contribute to the diverse literature supporting separate inputs for
GLUT4 translocation and activation. Covering this topic is beyond
the scope of the present review, but further discussion can be found
in [82].
Downstream of class I PI3K lie three major signalling axes,
typified respectively by their initiating signals PKB/Akt, aPKC
(atypical protein kinase C) and Rac. We next discuss the involve-
ment of PKB and its targets in GLUT4 trafficking, followed by
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Insulin action on glucose transporters 207
analysis of the participation of aPKC, and Rac is discussed later
in the section on ‘Cytoskeleton input on GLUT4 translocation’
below.
PKB/Akt
A PKB requirement for GLUT4 trafficking in muscle cells was
first shown by Wang et al. [83], a result that was further confirmed
by several lines of evidence. First, mice lacking PKBβdisplayed
insulin resistance of glucose uptake [84]. Secondly, siRNA knock-
down in 3T3-L1 adipocytes impaired insulin-dependent 2-deoxy-
glucose uptake and PM-directed GLUT4 trafficking [85]. From
these studies it emerged that PKBβis more likely to control
GLUT4 trafficking than either PKBαor PKBγ. Thirdly, cons-
titutively active membrane-targeted PKB provoked GLUT4 trans-
location in the absence of insulin [86–88]. Finally, expression of
dominant-negative mutants or microinjection of blocking anti-
bodies against PKB in L6 muscle cells and 3T3-L1 adipocytes in-
hibited insulin-induced GLUT4 translocation [83,87,89]. Surpris-
ingly, however, a chemical inhibitor of Akt that fully precludes
its activation reduced insulin-dependent GLUT4 translocation by
only 50 %[90].
AS160, TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member
1) and target Rabs
Recently, there has been an intensive search for the PKB targets
that lead to GLUT4 trafficking. Although this enzyme can phos-
phorylate a number of proteins [91], AS160 (also known as
TBC1D4 [92]), was recently identified as a regulator of GLUT4
trafficking in 3T3-L1 adipocytes [33,93–95] and muscle cells
[96], as well as of glucose uptake in skeletal muscle [97]. In
all of these systems, insulin increases AS160 phosphorylation,
and this response was found to be impaired in skeletal muscle
obtained from insulin-resistant patients [98,99]. The latter finding
correlates with diminished insulin-mediated glucose uptake and
GLUT4 translocation to the PM observed in muscle and adipose
cells of diabetic animals and humans [99,100]. AS160 harbours
a GAP (GTPase-activating protein) domain that is thought to
maintain its target Rab(s) in an inactive, GDP-bound form. The
current understanding is that upon insulin stimulation, phos-
phorylation of AS160 shuts off its GAP activity, shifting the
equilibrium of its target Rab(s) to an active GTP-bound form,
enabling it to mediate GLUT4 trafficking by releasing GLUT4
from intracellular retention mechanisms [33,93–95]. This mode
of action is proposed based on results using AS160 mutants. An
AS160 mutant lacking four of the PKB phosphorylation sites
(termed ‘4P’), but not a mutant with a critical arginine/lysine
residue substitution in the GAP domain, prevented the insulin-
dependent GLUT4 translocation in 3T3-L1 adipocytes [94] and
muscle cells [96], and reduced insulin-dependent glucose uptake
in skeletal muscle [97]. Most telling, a mutant encoding both the
‘4P’ mutation and the arginine/lysine mutation in the GAP domain
was no longer inhibitory, suggesting that the phosphorylation
site is functionally linked to the GAP activity of AS160. Hence
AS160 can be viewed as a brake that is removed upon phosphoryl-
ation by PKB.
TBC1D1 is also a PKB substrate, highly homologous with
AS160. However, whereas AS160 harbours six phosphorylation
sites for PKB, TBC1D1 displays only two (Thr590 and Ser501 in
mouse TBC1D1 [101,102]). Differences were also found when
comparing the effects of mutants of each protein on GLUT4
translocation. Thus, whereas overexpression of wild-type AS160
had no effect, wild-type TBC1D1 diminished insulin-dependent
GLUT4 translocation in 3T3-L1 adipocytes [94,102]. However,
on closer scrutiny, basal state levels of surface GLUT4 were also
reduced by approx. 40 %, complicating the analysis of this result
with overexpressed protein. As in the case of AS160 mutants,
overexpression of TBC1D1 mutated in the PKB target sites largely
reduced insulin-dependent GLUT4 translocation, and TBC1D1
with a mutation in its GAP domain had no effect on GLUT4
translocation [102].
In addition to their GAP activity, AS160 and TBC1D1 can bind
14-3-3 proteins, which interact with phosphorylated serine or
threonine residues on numerous polypeptides, often in response
to growth factor stimulation. 14-3-3 binding to phosphorylated
AS160 is essential for GLUT4 translocation [103], yet the
exact mechanism remains to be elucidated. AS160 and possibly
TBC1D1 act as a convergence point for signalling from several
protein kinases in addition to PKB/Akt, such as AMPK (AMP-
activated protein kinase) and PKC [104].
The in vitro GAP activity of the TBC domains of AS160
and TBC1D1 is selective towards Rabs 2A, 8A, 8B, 10 and 14
[102,105]. In isolated GLUT4-containing intracellular mem-
branes from muscle cells and adipocytes, Rabs 2, 4, 8, 10, 11
and 14 were detected, suggesting that they may contribute in
GLUT4 trafficking [33,105,106]. Indeed, in L6 muscle cells,
overexpression of Rab8A and Rab14 (but not Rab10) rescued the
inhibition of GLUT4 translocation caused by the constitutively
active, i.e. inhibitory ‘4P’ mutant of, AS160. This suggested that
Rabs 8A and 14 are the targets of AS160 leading to GLUT4
translocation [107]. This conclusion was buttressed by the finding
that Rab8A or Rab14 knockdown, but not Rab10 knockdown,
reduced insulin-dependent GLUT4 translocation in muscle cells
(S. Ishikura and A. Klip, unpublished work). Furthermore,
knockdown of Rab8A or Rab14 rescued the rise in surface GLUT4
levels elicited by AS160 or TBC1D1 knockdown, suggesting
that these Rabs mediate the release of GLUT4 intracellular
retention exerted by the Rab-GAP proteins. On the other hand,
Rab10 was found to be the predominant AS160 target in 3T3-L1
adipocytes, where Rab10 knockdown lowered insulin-induced
GLUT4 translocation to the PM, an effect restored by its re-
expression. Moreover, Rab10 silencing partially overcame the
rise in surface basal GLUT4 caused by AS160 knockdown. These
effects were specific for Rab10 as they were not observed upon
silencing Rab8A or Rab14 [108,109]. The contrasting results
reported for L6 muscle cells and 3T3-L1 adipocytes could have
arisen from cell-specific reliance on distinct Rabs.
Rabs are typically considered to be molecular switches, linking
a signal transduction cascade to molecular effectors. As shown
above, some of these effectors appear to be molecular motors.
Other members of the Rab family, although not being targets
of AS160 or TBC1D1, also participate in GLUT4 trafficking,
particularly Rabs 4, 5, 11 and 31. Other reviews cover this topic
in detail [110,111]. It will be important to unravel the molecular
effectors of such Rabs and the specific steps in GLUT4 traffic that
they mediate.
aPKC
Of the four aPKC isoforms, PKCλand PKCζhave been implic-
ated in GLUT4 translocation. Diverse approaches suggest that
aPKC is both necessary and sufficient to elicit this major action
of insulin, although studies to the contrary have also emerged.
Overall the studies can be summarized as follows. RNAi (RNA
interference)-mediated PKCλsilencing in 3T3-L1 adipocytes
or dominant-negative PKCλoverexpression in muscle and fat
cells partially impaired insulin-elicited GLUT4 translocation and
stimulation of glucose uptake [89,112–115]. On the other hand,
two studies failed to see any effects on these outcomes upon
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208 H. Zaid and others
expression of dominant-negative aPKC mutants [116] or PKCλ/ζ
silencing [117] in the same cells. Overexpression of wild-type
PKCλ/ζin rat adipocytes mimicked insulin action on glucose
uptake [118] and provoked a 2-fold increase in GLUT4 transloc-
ation [119]. Compellingly, a mouse model with a muscle-targeted
PKCλgene deletion displayed whole-body insulin resistance,
impaired insulin-stimulated glucose uptake into muscle in vivo
and ex-vivo, and reduced GLUT4 translocation [120]. As a cor-
relative observation, human subjects with Type 2 diabetes and/or
obesity display diminished levels or poorly active aPKC in skeletal
muscle [121,122].
One of the further inconsistencies in the literature is that the
studies supporting a requirement for aPKC in GLUT4 transloc-
ation have also negated a required input by PKB, which is other-
wise widely supported. This controversy remains one of the most
fascinating roadblocks in our understanding of insulin action and
one that is worth re-addressing, perhaps through the concerted
action of several groups using diverse biological systems.
How could aPKC signal to GLUT4 vesicles? The connection
appears to involve the actin cytoskeleton (see the section on
‘Cytoskeleton input on GLUT4 translocation’ below), as PKCλ/ζ
can impinge on Rac and actin dynamics [119,123]. These find-
ings create the attractive hypothetical scenario that PKCλ/ζ
may converge with the Rac-dependent arm of insulin signalling
governing actin remodelling, that would act in parallel with PKB
leading to GLUT4 redistribution (see Figure 3B).
3- and 5-phosphoinositide regulation by PTEN (phosphatase and
tensin homologue deleted on chromosome 10), SHIP2 (Src
homology 2-containing inositol phosphatase-2) and PIKfyve
As seen in the preceding sections, phosphoinositides are crucial
elements in insulin action, and their levels are precisely regulated
by kinases and phosphatases. PTEN dephosphorylates the third
hydroxyl position of the inositol ring of phosphoinositides, mainly
PtdIns(3,4,5)P3. Hence its activity would be expected to be of a
major consequence on insulin signalling. Indeed, microinjection
of a neutralizing anti-PTEN antibody elevated basal and insulin-
stimulated PtdIns(3,4,5)P3levels, thereby increasing GLUT4
translocation to the PM [124]. Conversely, overexpression of wild-
type PTEN inhibited the metabolic actions of insulin, dependent
on PI3K, although a confounding result with overexpression of
dominant-negative PTEN questioned the extent to which PTEN
may modulate insulin action [125]. More recently, however, it
was established that selective PTEN deletion in skeletal muscle
protects against the development of insulin resistance in vivo
and enhances insulin-dependent Akt phosphorylation and glucose
uptake [126]. Furthermore, PTEN knockdown via siRNA elevated
glucose uptake into adipocytes [127]. Hence the majority of the
studies in vivo and in vitro support the view that PTEN is a negative
regulator of insulin action. PTEN then could potentially be a
key point of modulation, toning insulin signalling down (when
activated/up-regulated) or up (when inhibited/down-regulated) in
diverse metabolic conditions.
The levels of PtdIns(3,4,5)P3are regulated not only by dephos-
phorylation of the 3-position in the inositol ring, but also
by dephosphorylation of the 5-position. In this regard, SHIP2
dephosphorylates PtdIns(3,4,5)P3to produce PtdIns(3,4)P2[128],
and is hence an interesting molecule that would shift the levels of
these two phosphoinositides. Although PtdIns(3,4)P2is abundant
in cell membranes, there is growing evidence that its levels can be
locally regulated and impact on actin dynamics, vesicle traffic and
Akt activation [129]. Contrary to the case of PTEN, there is no
consensus as to how relevant SHIP2 may be in insulin action. In
some studies, PTEN but not SHIP2 knockdown enhanced insulin
signalling in 3T3-L1 adipocytes [117,127]. In others, SHIP2-
deficient embryonic fibroblasts showed elevated PtdIns(3,4,5)P3
and Akt activation in response to serum but not to IGF stimulation
[130]. Interestingly, SHIP2 expression is elevated in quadricep
muscle and epididymal fat tissue, but not in the liver of diabetic
db/db mice [131]; more studies are required to elucidate the
relevance of this phosphatase to insulin action in vivo.
As mentioned earlier, exogenous delivery of PtdIns(4,5)P2to
muscle cells does not suffice to mobilize GLUT4 [76]; however,
this does not rule out the direct participation of this phosphoino-
sitide in insulin action, beyond serving as the major source
for PtdIns(3,4,5)P3generation. Supporting such a requirement,
exogenous PtdIns(4,5)P2delivery reversed the insulin resistance
in adipocytes induced by endothelin-1 and other experimental
insults [132]. A compelling body of evidence shows that
PtdIns(4,5)P2is a key regulator of actin dynamics and membrane
trafficking [133], and indeed the restoration of insulin sensitivity
by PtdIns(4,5)P2delivery was linked to actin repolymerization.
This cellular function is addressed further below.
Among the kinases regulating 3-and5
- phosphoinositide
levels, PIKfyve stands out. PIKfyve produces PtdIns5Pand
PtdIns(3,5)P2from phosphatidylinositol and PtdIns3Prespect-
ively [134]. This enzyme is essential for cargo sorting from
late endosomes to lysosomes [135]. Although PIKfyve activity
is not altered by insulin, it is reportedly inactivated by autophos-
phorylation [136] or activated in response to Akt [137]. Moreover,
expression of a non-phosphorylatable PIKfyve mutant enhanced
insulin stimulated GLUT4 translocation [137].
In principle, PIKfyve could contribute to insulin action by
changing the levels of its substrates (phosphatidylinositol acid and
PtdIns3P) or of its products [PtdIns5Pand PtdIns(3,5)P2]. Indeed,
PtdIns5Plevels increase transiently upon insulin stimulation of
3T3-L1 adipocytes [138], as do PtdIns(3,5)P2levels in intracellu-
lar membranes [139]. Accordingly, microinjection of PtdIns5P
into 3T3-L1 adipocytes mobilized GLUT4 towards the cell peri-
phery, and intracellular sequestration of intracellular PtdIns5P
reduced insulin-induced GLUT4 translocation [138]. Whether
this is due to the action of PtdIns5Pitself or to changes in other
phosphoinositides is not known, as the same group observed
that PIKfyve knockdown reduced PtdIns(3,5)P2levels, insulin-
induced Akt phosphorylation and glucose uptake [139]. It will
be important to dissect out in greater detail the role of the two
products of PIKfyve and their respective targets, as well as to
reconcile the divergent results on whether the enzyme exerts
positive or negative influence on GLUT4 translocation.
Finally, for all experiments analysing phosphoinositide action,
it remains to be determined whether exogenous delivery or forced
changes in abundance through overexpression of kinases or phos-
phatases can faithfully reproduce the actions of the endogenous
counterparts with regards to levels and location. This caveat must
be considered when analysing all results described above, while
understanding that experimental deviation from equilibrium is a
scientific approach to explore physiological phenomena.
CYTOSKELETON INPUT ON GLUT4 TRANSLOCATION
Microtubules
Although both microfilaments and microtubules are responsible
for maintaining the integrity of perinuclear GLUT4 compart-
ments, the relevance of microtubules to GLUT4 trafficking
has been controversial. Some studies show that microtubule-
depolymerizing agents such as nocodazole, taxol or colchicine
attenuate the insulin-stimulated PM fusion of GLUT4 [140–142].
Conversely, others report GLUT4 trafficking to be independent of
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Insulin action on glucose transporters 209
microtubule integrity, since low concentrations of nocodazole that
disrupted microtubules did not prevent GLUT4 trafficking [143–
145]. Moreover, some of the agents used to alter microtubule
stability affected PKB activation [141]. In light of these variable
findings, several studies revisited the role of microtubules.
Therein, microtubules were shown to contribute to the regulation
of GLUT4 vesicle transport to the cell periphery and docking at the
PM, as nocodazole pre-treatment reduced GLUT4 build-up within
the TIRF zone [42,101,146]. Given that several studies report
long-distance linear movement of GLUT4 vesicles that coincide
with the position of tubulin, it seems reasonable to propose that
microtubules could serve as tracks for the long-range trafficking
of vesicles to and from the PM. Such mobilization would also
require the association of GLUT4 with microtubule-based motor
proteins. In fact, expression of dominant-negative dynein or
kinesin motors (KIF3 and KIF5B) disrupts the internalization
or exocytosis of GLUT4 respectively [147–149]. An additional
possibility is that microtubules co-ordinate their function with the
actin cytoskeleton (see next), potentially handing on vesicles to
cortical actin filamints.
Actin filament remodelling
Actin molecules are in equilibrium between monomeric G-actin
and filamentous F-actin forms. In unstimulated muscle cells, F-
actin is present as stress fibres running longitudinally in myotubes
or radially in myoblasts, whereas, in suspended myoblasts or in
adipocytes, actin filaments are located in the cortical zone beneath
the PM. Insulin rapidly promotes the formation of cortical actin
projections in cultured and primary muscle and fat cells [150–
156]. This manifests as cortical membrane ruffling in muscle and
fat cells. Actin filaments that reorganize in this fashion are com-
posed of β-actin, to be differentiated from αor sarcomeric
actin that in mature skeletal muscle constitutes the contractile
apparatus. The intense actin branching and repolymerization that
occurs at the cell cortex occurs in part at the expense of depolymer-
ization of actin filaments, and indeed it is expected that a large
turnover of actin filaments occurs in response to the hormone.
The contribution of the actin cytoskeleton to the subcellular local-
ization of insulin-derived signals has been reviewed earlier [157].
In the present review we focus on the impact of actin dynamics
on GLUT4 trafficking and highlight recent developments in this
area.
There is considerable evidence for the participation of filamen-
tous actin in insulin-induced GLUT4 exocytosis in muscle and
fat cells in culture, as well as in primary muscle tissue and fat
cells. Agents that disrupt actin dynamics abrogate insulin-induced
GLUT4 translocation. This has been observed upon interfering
with the dynamic equilibrium between G- and F-actin by cyto-
chalasin D [140,158–160], latrunculin B [152,161,162], swin-
holide A or jasplakinolide [150,155,160]. However, these observ-
ations do not differentiate between the need for actin cables for
the purpose of processive vesicular motion, and the participation
of insulin-induced actin remodelling. The analysis of signals and
molecules involved in such remodelling has aided in answering
this question.
Rac and TC10
In myoblasts and myotubes, actin remodelling requires the activity
of class I PI3K. Indeed, expression of the dominant inhibitory
mutant delta-p85, pre-treatment with inhibitors of its enzymatic
activity (wortmannin or LY294002), or high overexpression of the
PH domain of GRP (general receptor for phosphoinositides) that
binds and mops up PI3K lipid products, prevents actin filament
reorganization in muscle [154] or fat [163,164] cells. Actin re-
modelling downstream of PI3K is not, however, mediated by
PKB, suggesting signalling bifurcation at this juncture (see Fig-
ure 3B). As is well established, all aspects of actin remodelling
are under the control of small G-proteins of the Rho family
(reviewed in [165]). The type of actin-dependent ruffles formed
in muscle cells are reminiscent of the action of one Rho family of
small GTPase, Rac. Accordingly, insulin stimulation led to Rac
activation, revealed by enhanced GTP-loading of this protein;
and this response was reduced when PI3K activity was inhibited
[166]. Moreover, expression of a dominant-negative Rac1 mutant
or silencing Rac1 expression via siRNA in muscle cells precluded
insulin-induced actin remodelling [153,167]. This occurred in the
absence of the disruption of stress fibres, allowing one to test
the specific consequence of the remodelling on GLUT4
translocation. Importantly, both expression of the Rac mutant and
silencing Rac expression largely prevented GLUT4 translocation
in muscle cells [153,167]. Since Rac1 silencing did not prevent
PKB activation, the PKB signalling arm alone is insufficient
to evoke GLUT4 translocation. Conversely, overexpression of
dominant-negative PKB or acute PKB inhibition via the chemical
AktI1/2 failed to prevent actin remodelling while reducing
GLUT4 translocation ([83] and A. Koshkina, V.K. Randhawa
and A. Klip, unpublished work). These results prove that each
signalling arm, defined by Rac/actin and PKB, is necessary but
insufficient to regulate GLUT4 trafficking.
In addition to Rac1, other Rho-family GTPases have also been
implicated in GLUT4 translocation. In some cellular systems,
Rho-family GTPases provide co-ordinated regulatory input to
actin dynamics, and Cdc42 can regulate Rac. In muscle L6
cells, Cdc42 was rapidly activated (i.e. GTP-loaded) following
insulin stimulation (A. Koshkina and A. Klip, unpublished
work). The functional significance of this activation is currently
under investigation. In 3T3-L1 adipocytes, there is controversial
evidence for the participation of either Cdc42 and/or Rho activity
in insulin-dependent GLUT4 trafficking [168–170]. In fact, in
those cells yet another different GTPase, TC10, has been
highlighted to govern cortical actin polymerization and contribute
to GLUT4 exocytosis [169,171,172]. Conversely, the contribution
of Rac to adipocyte actin remodelling is less explored, despite
the activation of both TC10 and Rac1 within minutes of insulin
treatment [166]. In adipocytes, TC10 becomes activated in
response to insulin and, in contrast with Rac, this activation
appears to be independent of PI3K or PKB. Instead, TC10
activation is linked to the upstream GEF (guanine nucleotide
exchange factor), CGD, itself the consequence of clustering of the
CAP–Cbl–CrkII complex within caveolae. However, the reliance
on the CAP–Cbl–CrkII–C3G relay for GLUT4 translocation has
been questioned. Moreover, the inhibitory effect of the TC10
mutants occurred independently of its GTP-binding domain
[169,172]. Efforts to explore this pathway further have also
yielded opposing results, as silencing CAP or Cbl expression did
not affect insulin-stimulated glucose uptake [173], but silencing
selective TC10 isoforms precluded GLUT4 translocation [171].
More work is required to investigate the mode of action of
TC10 in this function, and the recent implication of TC10 in
PtdIns3Pproduction via Rab5 [173a] and PI3K-C2α[74] may
provide the connecting link. In addition, TC10 activation may
also contribute to positioning aPKC near the cell surface [123].
In contrast with these extensive, if divergent, studies implicating
TC10 in GLUT4 trafficking in adipocytes, there is less support
for the participation of the Cbl–CAP–TC10 pathway in muscle
cells. Hence, in myoblasts, TC10 activation could occur in the
absence of CAP expression, and overexpression of a GDP-locked
TC10 dominant-negative mutant was inconsequential on GLUT4
translocation [166]. More studies are required to investigate the
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210 H. Zaid and others
relevance of this alternative pathway in skeletal muscle and its
impact on whole body glucose homoeostasis.
Connection to aPKC?
As mentioned above, PI3K activates PKB, aPKC and Rac, and
PKB activation does not lead to Rac activation or actin remodell-
ing. This poses the question as to whether aPKC is related to
Rac activation/actin remodelling, or whether there is a three-way
split of insulin signals downstream of PI3K. The former scenario
appears likely, as it has been reported that PKCζactivity lies
downstream of Rac1 [174]. Moreover, overexpression of PKCζ
was adequate to elicit a type of actin reorganization and, as dis-
cussed above, it also enhanced GLUT4 translocation [119,161].
There is contrasting information on whether or not aPKCs co-
localize with cortical actin structures near the PM [119,154]. How
aPKC is involved in GLUT4 trafficking remains to be elucidated,
but in neurons, aPKC binds proteins that contribute to cellular
polarity and protein compartmentation [175]. Interestingly, the
kinase was also linked to serine phosphorylation of VAMP2
[176], a component of specialized insulin-responsive GLUT4
vesicles (discussed in the section on ‘Vesicle docking and fusion’).
It is plausible that there are hotspots of actin remodelling
and subsequent PM insertion where insulin-responsive GLUT4
vesicles accumulate as a result of aPKC input. Consistent with
this possibility, GLUT4 appears to insert preferentially into ruffled
areas of the PM supported by the remodelled actin mesh [155].
Rac effectors governing actin dynamics
It is reasonable to rationalize that the requirement for Rac activ-
ation in insulin-dependent GLUT4 translocation is a direct
consequence of the action of Rac on actin remodelling. However,
it is conceivable that Rac has an independent, parallel input on
both functions. Hence it is important to map the events unleashed
by Rac activation that control actin dynamics and to examine
their requirement for GLUT4 traffic. These include actin filament
capping and severing by gelsolin, filament severing by dephos-
phorylated cofilin and filament branching effected by WASP/
WAVE (Wiskott–Aldrich syndrome protein/WASP verprolin
homologous) acting on Arp2/3. There is no information on
whether these pathways participate in insulin-induced actin re-
modelling. Interestingly, insulin elicits cofilin dephosphorylation
in muscle cells, and cofilin knockdown via siRNA partly inhibited
GLUT4 externalization [176a]. These results suggest that the
dynamic remodelling of actin (cycles of severing and polymer-
ization) helps to furnish GLUT4 to the cell surface. In adipocytes,
dominant-negative N-WASP mutants reduced GLUT4 traffic to
the PM [169,177].
GLUT4-associated cytoskeletal proteins: ACTN4 (α-actinin 4)
How do GLUT4 vesicles interact with the cytoskeleton? In
insulin-stimulated muscle cells, GLUT4 is found within the area
of cortical actin remodelling by both immunofluorescence and
electron microscopy [157]. In an effort to identify molecular
linkers between GLUT4 and the cytoskeleton, we embarked
on a differential screen of insulin-dependent changes in protein
association to immunoprecipitated GLUT4. Through stable
isotope labelling of amino acids and MS analysis, the protein that
most markedly increased its association with GLUT4 was ACTN4
[178]. Silencing ACTN4 protein expression by siRNA specifically
diminished the increase in surface GLUT4 content by insulin
[178a]. ACTN4 knockdown allowed normal PKB activation
and cortical actin remodelling but precluded the localization of
GLUT4 within the cortical actin mesh. ACTN4 may potentially
tether the insulin-responsive GLUT4 vesicle to the actin cyto-
skeleton for progression towards docking and fusion. Interesting
in this context is the protein TUG, which, in contrast with
ACTN4, binds to GLUT4 in the absence of insulin and hormonal
stimulation, causing its dissociation from the transporter in both
adipose [179] and muscle (J. D. Schertzer, X. Huang and A.
Klip, unpublished work) cells. It will be interesting to investigate
whether TUG release might allow GLUT4 binding to ACTN4.
Alternatively, TUG may be a GLUT4 tether that releases vesicles
from the SC for progression towards the cell surface.
Tracks or tethering scaffold?
It is plausible that the requirement for actin dynamics inherent to
the insulin-induced GLUT4 translocation is due to participation of
actin filament tracks along which GLUT4 vesicles would move
near the cell surface. Disrupting these tracks could potentially
prevent GLUT4 exit from the SC. Consistent with this scenario
would be a need for actin-based motor proteins to allow the PM-
directed transport of GLUT4 vesicles. As per this prediction,
several actin-based motor proteins have been implicated in
GLUT4 trafficking. Partial knockdown of myosin 1c expression
reduced the gain in surface GLUT4 in 3T3-L1 adipocytes [180].
Also, myosin Va is phosphorylated by Akt2, enhancing its asso-
ciation with actin, and myosin Va knockdown reduced GLUT4
translocation [181]. This led Yoshizaki et al. [181] to suggest
that myosin Va is the motor that propels vesicles along actin
tracks to the cell surface. Finally, in L6 muscle cells, a fragment
of the processive myosin Vb that interferes with its interaction
with Rab8 precluded insulin-dependent GLUT4 translocation
(S. Ishikura and A. Klip, unpublished work). All of these results
suggest that actin-based motors are required for the final gain in
surface GLUT4 elicited by the hormone, but they do not directly
prove that there is processive movement of the vesicles on actin
tracks mediated by these motors.
On the other hand, it is also possible that insulin-induced actin
remodelling into a highly branched cortical filamentous mesh
serves to position vesicles and signals, effectively priming them
for docking and fusion. Such actin platforms would presumably
allow the insulin signalling molecules to come into close prox-
imity with the intracellular GLUT4 vesicles. Interestingly, along
with the remodelled cortical actin mesh co-localize several insulin
signalling molecules (e.g. IRS-1/2, PI3K subunits, phospho-Akt,
and possibly PKCζ) together with PtdIns(3,4,5)P3,VAMP2and
GLUT4 [153,154]. How GLUT4 vesicles are tethered to the actin
cytoskeleton remains to be elucidated. In this regard, ACTN4
may be the missing link, as this protein is found only within
remodelled actin but not along filaments in unstimulated cells,
and insulin promotes cortical co-localization of actin filaments,
ACTN4 and GLUT4, as well as the physical association of
GLUT4 with ACTN4 [178]. The functional significance of Rabs
should be revisited in this context. Rab proteins bind molecular
tethers, and can regulate the actin cytoskeleton in certain cell
systems [111,156,182,183]. Alternatively, the actin cytoskeleton
may serve as a barrier to PM-directed vesicle release. This latter
phenomenon has been well described for dense core granule
release or synaptic vesicle trafficking [184]. In this final scenario,
one would envision the dissolution of the actin mesh specifically
about the PM by insulin to subsequently allow for vesicle
fusion.
The cytoskeleton in insulin-resistant states
The importance of identifying the molecular mechanisms by
which cytoskeletal elements contribute to GLUT4 translocation
is underscored by recent studies that highlight defects in actin
dynamics in conditions of insulin resistance. In muscle cells
c
The Authors Journal compilation c
2008 Biochemical Society

Insulin action on glucose transporters 211
incubated for prolonged times with high glucose or insulin, or
for short times in the presence of ceramide or oxidative-radical-
generating enzymes, Rac activation and actin remodelling by
insulin were compromised [167]. All of those conditions emulate
aspects of the cellular environment in Type 2 diabetes, which
is accompanied by insulin resistance. Moreover, the loss of
cortical actin filaments observed in insulin-resistant conditions
was relieved by regeneration of PtdIns(3,4)P2, concomitantly
relieving the insulin resistance of GLUT4 trafficking [185]. With
the rapid advances in imaging, it should become possible to tease
out the specific steps at which actin and microtubules regulate
GLUT4 vesicle trafficking to better understand how their dysregu-
lation may contribute to insulin resistance.
CONCLUDING REMARKS
GLUT4 trafficking and in particular its regulation by insulin have
become the paramount example of regulated vesicle trafficking.
The segregation of a large portion of GLUT4 away from the conti-
nuously recycling pathway is a feature that evolutionarily was
developed both in the structural characteristics of this transporter
isoform, as well as of proteins that define a ‘specialized storage
compartment’, SC. The molecular markers of such definers are
still to be unravelled, but they appear to be exclusively character-
istic of muscle and fat cells. Impressive progress has been made
in mapping the signals that, in series and in parallel, collude to
relay information to GLUT4 vesicles and target membranes. Yet,
further work is required to elucidate the final steps in the insulin-
signalling cascade downstream of molecular switches (Rabs) and
molecular motors (myosins) that convey molecular information
to the SC. In this regard, progress has been made in mapping bio-
chemical (e.g. VAMP2 phosphorylation), structural (dispersion
and generation of small vesicles) and organizational (association
or dissociation of TUG, ACTN4) changes in the vesicles
themselves. Additional changes at the PM are essential for actual
docking/fusion of the vesicles (SNARE complex availability
through Munc18c and probably other regulators). Finally, the
entropic input of the cytoskeleton, potentially to position vesicles
and signals, provide tethers or allow final access of vesicles to the
PM, has emerged as an essential constituent for effective GLUT4
translocation. As we approach the 30th anniversary of the first
glimpse of this phenomenon, we look back and marvel at the intri-
cate series of intracellular events governing it. The next years
should add the fine spatial and temporal resolution of this funda-
mental biological process.
We apologize to all of the colleagues whose work could not be covered in the present
review. We thank Dr Philip J. Bilan and Dr Shuhei Ishikura for insightful comments on
this manuscript. The work from our laboratory that is described in the present review
was supported by grants MOP-7307 and MOP-1202 to A.K. from the Canadian Institutes
of Health Research and from the Canadian Diabetes Association. H.Z. was supported
by a postdoctoral fellowship from the Hospital for Sick Children (Toronto, ON, Canada).
V.K. R. was supported by a doctoral studentship from the Canadian Institutes of Health
Research, and C. N.A. was supported by a doctoral studentship from the Canadian Diabetes
Association.
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Received 8 April 2008/1 May 2008; accepted 2 May 2008
Published on the Internet 26 June 2008, doi:10.1042/BJ20080723
c
The Authors Journal compilation c
2008 Biochemical Society