From the Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242
Among all the diverse actions
of insulin, one of the most critical and intensively studied is its
regulation of glucose homeostasis. In the postabsorptive state, insulin
action in muscle and adipose tissue results in increased glucose uptake
from the circulation, thereby maintaining plasma euglycemia and
preventing hyperglycemia (1-3). Defects in this pathway result in
insulin resistance, a condition in which excessive concentrations of
insulin are required to reduce blood glucose levels and often precede
the development of frank Type II diabetes (3, 4). Although
it has been appreciated for almost two decades that this major action of insulin results from a redistribution of glucose transporter proteins from intracellular storage sites to the plasma membrane (5,
6), the cellular mechanisms responsible for these trafficking events
and the defects associated with insulin resistance have remained
largely enigmatic.
We now know that facilitative glucose uptake occurs through a
family of highly related integral membrane proteins that share significant sequence similarity. Although several lines of evidence suggest the presence of additional glucose transporters, to date only
four members of this gene family have been described and documented to
function as authentic glucose transporters. For example, although GLUT5
was originally thought to be a glucose transporter it was subsequently
identified as a facilitative fructose transporter (7). Of the four
established glucose transporter isoforms, the following GLUT4 is highly
expressed in adipose tissue and striated muscle with significantly
lower levels of the GLUT1 isoform (see accompanying minireview by
Charron et al. (8)). In the basal state, GLUT4 cycles slowly
between the plasma membrane and one or more intracellular compartments,
with the vast majority of the transporter residing in vesicular
compartments within the cell interior (9, 10). Activation of the
insulin receptor triggers a large increase in the rate of GLUT4 vesicle
exocytosis and a smaller decrease in the rate of internalization by
endocytosis (11-14). The stimulation of exocytosis by insulin is
probably the major step for GLUT4 translocation because complete
inhibition of GLUT4 endocytosis only modestly increases plasma
membrane-associated GLUT4 protein without affecting the extent of
insulin-stimulated GLUT4 translocation (15-17). In contrast to GLUT4,
GLUT1 is localized both to the plasma membrane and intracellular
storage sites in the basal state but only displays a modest
insulin-stimulated redistribution to the plasma membrane. Thus, the
overall insulin-dependent shift in the cellular dynamics of
GLUT4 vesicle trafficking results in a net increase of GLUT4 on the
cell surface, thereby increasing the rate of glucose uptake.
As detailed in the previous minireview by Czech and Corvera (18), it is
unclear if a single or multiple overlapping pathways can lead to GLUT4
translocation. At present, substantial evidence has documented that the
activation and/or appropriate targeting of the Type I
phosphatidylinositol (PI)1
3-kinase is necessary for insulin-stimulated GLUT4 translocation and
glucose uptake (19). However, the specific downstream targets for PI
3-kinase have remained questionable with evidence both for and against
protein kinase B, also known as Akt (20-22). Alternatively, the
atypical protein kinase C isoforms (protein kinase C In addition to PI 3-kinase-dependent signal transduction
pathways, several studies have demonstrated the presence of PI
3-kinase-independent mechanisms of GLUT4 translocation. For example,
osmotic shock, GTP It is well established that integral membrane proteins traffic to
and from the plasma membrane through an endosomal recycling membrane
system. Immunoelectron microscopy has demonstrated that the GLUT4
protein is predominantly localized in small vesicles and
tubulovesicular structures, with relatively smaller amounts found in
the trans-Golgi network, clathrin-coated vesicles, and endosomes
(31-35). Subcellular fractionation and immunoabsorption studies have
indicated a substantial co-distribution of GLUT4 with markers of
several intracellular membrane compartments including GLUT1 and the
transferrin and the insulin-like growth factor-2/mannose 6-phosphate
receptors (9, 10). However, several studies have observed the presence
of a separate subpopulation of GLUT4-containing vesicles different from
the constitutively recycling endosomal system. For example,
GLUT4-containing vesicles are enriched in the v-SNARE protein, VAMP2,
but not the related VAMP3/cellubrevin isoform, which is present in the
constitutively recycling endosome population (36-38). The presence of
distinct vesicle populations may also account for the ability of
insulin to stimulate 2-4-fold plasma membrane increases of several
recycling proteins (i.e. GLUT1, transferrin, and
insulin-like growth factor-2/mannose 6-phosphate receptors), whereas
plasma membrane GLUT4 content can increase 10-20-fold. Thus, it
appears that this unique subpopulation of GLUT4-containing vesicles is
primarily responsible for the majority of insulin-stimulated translocation.
Irrespective of which vesicle population is insulin-responsive, there
must be specific mechanisms in place to localize GLUT4 to its
appropriate intracellular storage sites. To address this issue, several
laboratories have examined the subcellular localization of GLUT4/GLUT1
and GLUT4/transferrin receptor chimeric proteins. Steady-state analysis
of the C-terminal region of GLUT4 identified a dileucine motif (SLL)
that when mutated resulted in the plasma membrane accumulation of GLUT4
(39-42). However, further mutational studies and kinetic analysis
revealed that this motif was, in fact, required for efficient
internalization (43, 44). Thus the apparent lack of intracellular
sequestration in these mutants resulted from an accumulation of GLUT4
at the plasma membrane because of a reduction in the rate of
endocytosis. Even though a contiguous GLUT4 amino acid sequence
responsible for intracellular sequestration or retention has not yet
been identified, kinetic and morphological data strongly suggest the
presence of an intracellular targeting signal within the GLUT4
cytoplasmic C-terminal domain.
There are two general models that can account for the
insulin-stimulated translocation of these intracellular
GLUT4-containing vesicles to the plasma membrane. Schematic
representations of the retention and synaptic models of GLUT4 vesicle
trafficking are illustrated in Figs. 1
and 2. The retention model predicts that
the specific sequences within GLUT4 or other co-localized vesicular
proteins (see below) specifically target this vesicle population away
from recycling endosomes (Fig. 1). Insulin stimulation allows these
sequestered vesicles to enter the constitutive recycling endosome
pathway and thereby results in default trafficking to the plasma
membrane. This model is consistent with the predominant localization of
GLUT4 to small vesicles and tubulovesicular structures adjacent to
endosomes typically underlying the plasma membrane (32, 34, 35). In
addition, expression of GLUT4 in neuroendocrine cells and fibroblast
cell lines results in the localization of GLUT4 to secretory granules
and to endosomes (42, 45-47). These data are consistent with the
presence of specific GLUT4 intracellular sequestration sequences that
can function in heterologous cell systems.
INTRODUCTION
Top
Introduction
References
Insulin Regulation of Glucose Uptake
and
) are
downstream targets of PI 3-kinase and have also been implicated in the
insulin stimulation of GLUT4 translocation (23, 24, 81).
S, and exercise/contraction in skeletal muscle
induce GLUT4 translocation in the complete absence of PI 3-kinase
activity (25-28). These treatments can result in increased AMP levels,
and it has been suggested that activation of the
AMP-dependent protein kinase stimulates glucose uptake
(29). G protein-coupled receptors linked to Gq have also
been observed to induce GLUT4 translocation (30). Future studies will
be necessary to ascertain the specific roles and relationships between
protein kinase B, protein kinase C
and
, and/or other PI
3-kinase-dependent as well as independent signaling
processes in GLUT4 translocation. In any case, whatever specific signal
transduction pathway(s) are involved, they must directly impact on the
GLUT4 intracellular storage sites and its subsequent trafficking to and
fusion with the plasma membrane.
Identification of Intracellular GLUT4 Storage Compartments
Retention versus Synaptic Vesicle Model of GLUT4 Translocation
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Fig. 1.
Schematic representation of the postulated
retention model of intracellular GLUT4 sequestration. In this
model, GLUT4-containing vesicles are sequestered away from the
constitutive endosome recycling system by the specific association with
a retention receptor (RR). Insulin stimulation results in
the release of these GLUT4-containing vesicles, which can then enter
the recycling endosome system and thereby accumulate at the cell
surface. IR, insulin receptor.
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Fig. 2.
Schematic representation of the hypothesized
synaptic model of GLUT4 vesicle trafficking. In this model GLUT4
vesicles are localized to both small synaptic-like vesicles as well as
larger tubulovesicular compartments, both enriched for the v-SNARE
protein VAMP2. Presumably the tubulovesicular structures are in
equilibrium with the smaller vesicles. In either case, insulin
stimulation results in the association of GLUT4-containing vesicles
with the plasma membrane through the interaction of VAMP2 with the
t-SNARE complex composed of syntaxin 4 and SNAP23. In addition, the
modulator proteins Munc18c and Synip are thought to provide an
inhibitory and stimulatory function, respectively. IR,
insulin receptor.
A functional regulatory property of the retention model can be proposed based upon the recent observation that expression of the GLUT4 C-terminal domain results in the translocation of the endogenous GLUT4 protein to the plasma membrane (48). Similarly, the insulin-responsive aminopeptidase (IRAP), also known as vp165, co-localizes to biochemically and morphologically identical GLUT4 vesicles (49, 50). The cytoplasmic N-terminal domain of IRAP/vp165 displays some homology with the GLUT4 C terminus, and introduction of the cytoplasmic domain of IRAP/vp165 also results in plasma membrane translocation of GLUT4 (51). This model also accounts for the apparent masking of the GLUT4 C-terminal domain, which becomes more immunoreactive following insulin stimulation (34, 35). The simplest interpretation of these data is that insulin-responsive cells contain a saturable retention receptor, which is responsible for the intracellular sequestration of the GLUT4-containing vesicles. Disruption of this interaction by either insulin stimulation or through competition with an appropriate peptide allows the GLUT4-containing vesicles to enter the recycling endosome system, thereby resulting in plasma membrane translocation.
Although several aspects of the retention model are quite appealing,
there are several lines of evidence inconsistent with this model.
First, a substantial proportion of the GLUT4-containing vesicles
appears to be segregated away from recycling endosome markers, having
characteristics analogous to small synaptic vesicles. This includes the
co-localization of GLUT4 with VAMP2 and the fact that a substantial
amount of the GLUT4 protein remains distinct from the recycling of the
transferrin receptor (36, 37). Furthermore, ablation of the transferrin
receptor-containing endosomes does not prevent insulin-stimulated GLUT4
translocation. These data demonstrate that insulin-stimulated GLUT4
trafficking can occur independent of the recycling endosome system, at
least the endosome population defined by the transferrin receptor. In
either case, it is important to recognize that both the retention and
synaptic vesicle models are not necessarily mutually exclusive. For
example, insulin could induce the trafficking of multiple GLUT4 vesicle populations through different mechanisms. Alternatively, GLUT4 may be
stored in synaptic-like vesicles that are sequestered because of GLUT4
and/or IRAP/vp165 retention signals.
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SNARE Model of GLUT4 Trafficking |
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In contrast to GLUT4 translocation, substantial progress has been
made in our understanding of synaptic vesicle trafficking in the
regulation of neurotransmitter release from the presynaptic membrane
(52-54). In this process, protein complexes in the vesicle compartment
(v-SNAREs, for vesicle SNAP
receptors) pair with their cognate receptor complexes at
the target membrane (t-SNAREs, for target membrane
SNAP receptors). Direct interactions between
the t-SNARE and v-SNARE proteins in combination with the association of
several accessory proteins (for example, -SNAP,
N-ethylmaleimide-sensitive factor, synaptophysin, and
synaptotagmin) are responsible for determining trafficking specificity
and formation of the core complex required for membrane fusion.
In adipocytes, the insulin-stimulated trafficking of GLUT4 vesicles has several features in common with the regulated exocytosis pathway of synaptic vesicles, including the presence of specialized vesicular proteins (Fig. 2). As described previously, the v-SNARE VAMP2 co-localizes with GLUT4 vesicles and, based on several criteria, appears to be essential for insulin-stimulated GLUT4 translocation. Although there are no specific endoproteases that cleave either syntaxin 4 or SNAP23/Syndet plasma membrane t-SNARE proteins, studies using blocking antibodies, dominant-interfering mutants, and/or peptide inhibitors have strongly implicated syntaxin 4 and SNAP23/Syndet as the t-SNARE proteins required for insulin-stimulated GLUT4 translocation (55-58).
In the case of synaptic transmission there are also several modulators of v- and t-SNARE interactions. The mammalian Munc18 proteins, homologues of Saccharomyces cerevisiae n-Sec1/rbSec1, Caenorhabditis elegans unc18, and Drosophila melanogaster Rop proteins, bind with high affinity to their cognate plasma membrane syntaxins. Null mutations in the n-Sec1, unc18, or Rop genes dramatically reduce vesicle exocytosis, suggesting that these proteins are essential for normal v- and t-SNARE function. The Munc18a isoform is predominantly expressed in neurons, where it inhibits the association of VAMP1 and SNAP25 with syntaxin 1 (59). In contrast to Munc18a, Munc18b and Munc18c are expressed ubiquitously, but only Munc18c binds syntaxin 4 (60, 61). In analogy to Munc18a, the binding of Munc18c to syntaxin 4 precludes the binding of VAMP2, and increased expression of Munc18c inhibits insulin-stimulated GLUT4 translocation (62, 63). Because insulin treatment apparently reduces the binding of Munc18c to syntaxin 4, this suggests that GLUT4 translocation is inhibited by Munc18c in the basal state and that insulin stimulation may result in a removal of the Munc18c repression.
More recently, we have identified a novel protein (Synip) that specifically binds to syntaxin 4 and is uniquely expressed in muscle and adipose tissue.2 Similar to Munc18c, the binding interaction of Synip with syntaxin 4 is regulated by insulin, which also induces a dissociation of the Synip-syntaxin 4 complex. However, in contrast to Munc18c, expression of the wild type Synip protein has no effect on insulin-stimulated GLUT4 or IRAP/vp165 translocation. Furthermore, expression of the C-terminal domain of Synip does not display an insulin-stimulated dissociation from syntaxin 4 but functions in a dominant-interfering manner by inhibiting both GLUT4 and IRAP/vp165 translocation. These data suggest that although Munc18c may have an inhibitory role, Synip functions as a positive regulator of insulin-stimulated GLUT4 translocation. Future studies will be necessary to determine the stoichiometry of these interactions and the relationship between Synip-syntaxin 4 and Munc18c-syntaxin 4 complexes.
In addition to the involvement of these SNARE proteins in GLUT4
translocation, the small GTP-binding protein Rab4 also appears to play
an important functional role. Based upon the relationship between Rab3A
function and synaptic transmission, insulin-responsive tissues were
found to express several Rab isoforms. In particular, Rab4 was found to
co-localize with the GLUT4-enriched microsome fraction (65).
Furthermore, insulin stimulation resulted in a redistribution of Rab4
into the cytosolic fraction. More recent studies have also demonstrated
that introduction of a Rab4 C-terminal peptide, expression of a
C-terminal Rab4 deletion mutant, or high level expression of wild type
Rab4 all result in an inhibition of insulin-stimulated GLUT4
translocation (66-68). These data have been interpreted to indicate
that Rab4 is an essential component in the GLUT4 translocation machinery.
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Regulation of GLUT4 Endocytosis |
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Accompanying the paucity of information available about GLUT4 exocytosis, even less is known about the mechanism of plasma membrane GLUT4 retrieval (endocytosis). Kinetic analysis has indicated that insulin increases the rate of GLUT4 exocytosis approximately 10-20-fold with a 2-3-fold decrease in the rate of endocytosis (10, 69, 70). Although insulin-stimulated exocytosis requires activation of the PI 3-kinase, inhibition of this activity has no effect on the rate of endocytosis (71). Electron microscopic analysis has demonstrated that in the basal state, the small amount of plasma membrane-associated GLUT4 is almost exclusively found in coated pit regions (32, 33, 72). However, following insulin stimulation only a fraction of the plasma membrane-localized GLUT4 is found in coated pits whereas the majority of GLUT4 is dispersed in non-coated plasma membrane regions (32, 72). Although the mechanism responsible for this observation is not known, the insulin-stimulated decrease in GLUT4 coated pit localization could result from either saturation of the targeting machinery or specific segregation of the GLUT4 protein. In any case, disruption of clathrin lattices by potassium depletion or acidification of the cytosol has clearly established a role for clathrin-mediated endocytosis in GLUT4 internalization (44, 73).
One essential molecule implicated in endocytic coated vesicle formation is the GTPase dynamin (Fig. 3). In addition to its highly conserved N-terminal GTPase domain, dynamin contains a central pleckstrin homology domain and a C-terminal proline-rich region that negatively and positively regulate the dynamin self-assembly and GTPase activities, respectively (74, 75). These domains can functionally interact with several signaling intermediates, including Grb2, inositol phospholipids, and amphiphysin (76, 77). In this regard, insulin has been observed to induce the association of a dynamin-Grb2 complex with tyrosine-phosphorylated Shc and IRS1, which in turn modulates the rate of dynamin GTPase activity (78, 79). Furthermore, insulin has been reported to induce the tyrosine phosphorylation of dynamin (79). Together, these data suggest that insulin may regulate the endocytic rate of constitutively recycling cell surface proteins such as GLUT4 through a dynamin-dependent mechanism.
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Consistent with this hypothesis, expression of a dominant-interfering
dynamin mutant (K44A/dynamin) completely prevented GLUT4 endocytosis
without affecting insulin-stimulated GLUT4 translocation (16, 17, 80).
In addition, microinjection of a dynamin peptide encompassing the
amphiphysin binding sites prevents GLUT4 endocytosis and results in the
accumulation of GLUT4 at the cell surface (64). These data provide
compelling evidence that the internalization of GLUT4 occurs through a
clathrin-coated pit-mediated dynamin-dependent endocytic
pathway. These findings underscore the importance of dynamin function
in GLUT4 endocytosis and suggest that the assembly and/or
dynamin-dependent formation of coated vesicles may be a target for the insulin regulation of GLUT4 endocytosis.
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Summary and Conclusions |
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Since the original observation nearly 20 years ago that insulin
stimulates the translocation of glucose transport proteins from
intracellular storage sites to the plasma membrane, we have considerably advanced our understanding of the insulin signal transduction pathway, the molecular/cellular regulation of glucose transport, and their relationships to whole body glucose metabolism. During this time, four functional facilitative glucose transporter isoforms have been identified, cloned, and characterized. We now know
that the GLUT4 isoform is primarily responsive to insulin and accounts
for the majority, if not all, of insulin-stimulated glucose transport
in muscle and adipose tissue under normal physiological circumstances.
In the basal state, this transporter is distributed between multiple
intracellular membrane compartments, some of which overlap with the
recycling endosome system and others which may be localized to unique
vesicular compartments. Future studies are needed to unambiguously
identify the intracellular retention signals present in GLUT4, the
mechanism by which GLUT4 is targeted to these intracellular storage
sites and the functional properties of these compartments. Although it
is evident from this brief review that precisely defining the GLUT4
intracellular trafficking pathways continues to be a daunting task,
substantial progress is being made on characterizing the specific v-
and t-SNARE proteins directly involved. Clearly, the next major advance
will require the identification of the downstream signal transduction
pathway that regulates the priming/docking and/or fusion of these GLUT4 vesicles with the plasma membrane. Progress on all these issues is
continuing at an accelerating pace, and the future looks bright for the
ultimate development of an integrated and uniform model that links
insulin action at the molecular level with the physiological control of
glucose homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Maureen Charron, Michael Czech, Jerry Olefsky, Paul Pilch, and Alan Saltiel for helpful comments and discussions.
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FOOTNOTES |
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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This is the second article of three in the "Insulin-stimulated Glucose Transport Minireview Series."
To whom correspondence should be addressed. Tel.:
319-335-7823; Fax: 319-335-7330; E-mail:
Jeffrey-Pessin{at}uiowa.edu.
The abbreviations used are:
PI, phosphatidylinositol; GTPS, guanosine
5'-3-O-(thio)triphosphate; IRAP, insulin-responsive
aminopeptidase; t-SNARE, target membrane SNAP receptor; v-SNARE, vesicle SNAP receptor; SNAP, soluble NSF
(N-ethylmaleimide-sensitive factor) attachment protein.
2 J. Min, S. Okada, M. Kanzoki, B. P. Ceresa, K. Coker, J. S. Elmendorf, L.-J. Syu, Y. Noda, A. R. Saltiel, and J. E. Pessin, submitted for publication.
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REFERENCES |
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