Insulin-induced glucose transport
The major glucose transporter responsible for insulin-regulated glucose uptake in muscle and adipose tissue is GLUT4, discovered by David James in 1988. In the absence of insulin, GLUT4 is mainly found in intracellular vesicles termed GLUT4 storage vesicles. Activation of Akt/PKB following insulin binding to the insulin receptor leads to the translocation of GLUT4 glucose transporter vesicles to the plasma membrane. Fusion of GLUT4 storage vesicles with the plasma membrane in response to insulin results in an increase in the amount of GLUT4 on the cell surface, thereby increasing the transport of glucose into the cell. Insulin signaling also inhibits GLUT4 endocytosis ensuring a longer duration of GLUT4 residence in the plasma membrane.
Insulin-stimulated GLUT4 translocation involves homologues of many of the proteins involved in nerve cell transmission and other types of intracellular transport. Such transport is executed by a conserved and universal fusion machinery composed of two components: SNARE and SM proteins. Fusion results from the thermodynamic coupling of protein folding (assembly of v-SNAREs with t-SNAREs, spatially and temporally organized by SM proteins) to bilayer perturbation.
Energy made available from folding is productively channeled into the bilayer so that, on balance, fusion is the favored, spontaneous reaction. Nonetheless, fusion is tightly regulated in a spatial and temporal manner, the degree and nature of regulation varying with cell type and signaling response time.While numerous proteins and compounds have been identified as potential regulators of synaptic and other fusion processes in different tissues the underlying principles of vesicle mediated signaling are likely the same, driven by the universal mechanism summarized above.
A glossary of proteins involved in membrane fusion is provided to assist the reader.** Details about the structures of glucose transporters, including GLUT4, can be found in the section Glucose transport.
The universal docking and fusion machinery
SNAREs, NSF and SNAP: The proteins involved in intracellular membrane fusion include NSF (N-ethylmaleimide–sensitive factor), SNAP (soluble NSF attachment protein) and the SNARE protein complex which is the receptor for SNAP and NSF (hence the name SNARE, derived from SNAP receptor). The SNARE complex consists of three membrane proteins proposed to bridge the exocytic vesicle to the plasma membrane: the t-SNAREs syntaxin-1 and synaptosomal-associated protein 25 (SNAP-25) in the plasma membrane, and the v-SNARE, vesicle-associated membrane protein-1 (VAMP- 1; also called synaptobrevin-1), located in the synaptic vesicle. The synaptic SNARE proteins are the targets for botulinum and tetanus toxins, exquisitely specific proteases that block synaptic vesicle fusion.
Individual SNARE proteins spontaneously assemble (zipper up) into a remarkably stable, four-helix bundle (‘trans-SNARE complex’ or ‘SNAREpin’) that pulls the two membranes tightly together to exert the force required for fusion (Figure 1). The force required to rupture trans-SNARE complexes is estimated to be in the range of 100–300 pN, and each SNAREpin releases about 35 kBT of energy (equivalent to about 20 kcal/mole) as it zippers up. The activation energy for lipid bilayer fusion is in the range of 50–100 kBT) and so three or more individual SNAREpins suitably arranged will provide enough energy to drive fusion, in line with current estimates).
Figure 1. The zippering model for SNARE-catalyzed membrane fusion. (A) Three helices anchored in one membrane (the t-SNARE) assemble with the fourth helix anchored in the other membrane (v-SNARE) to form trans-SNARE complexes, or SNAREpins. Assembly proceeds progressively from the membrane-distal N-termini towards the membrane-proximal C-termini of the SNAREs. This generates an inward force vector (F) that pulls the bilayers together, forcing them to fuse. Complete zippering is sterically prevented until fusion occurs, so that fusion and the completion of zippering are thermodynamically coupled. (B) Therefore, when fusion has occurred, the force vanishes and the SNAREs are in the low energy cis-SNARE complex. Reproduced from Südhof & Rothman, 2009 with permission. [Click to enlarge]In the post-fusion state, the fully-zippered SNARE complex (emanating from the fused membrane) is termed the ‘cis-SNARE complex’(Figure 1). Once assembled, the cis-SNARE complexes are recycled by the ATPase NSF and its adaptor protein, SNAP, the latter binding directly to the SNARE complex. NSF is a hexamer that presumably uses 3–6 ATP’s with each catalytic cycle (totaling about 20–40 kcal/mole to disrupt the SNARE complex. Thus overall, fusion is driven by an ATP-dependent cycle of SNARE association and dissociation. In this cycle, the bilayer merger is thermodynamically coupled to exergonic folding of SNARE proteins, followed by their endergonic unfolding by a specialized ATPase (NSF) that binds to the SNARE complex via its adapter protein SNAP and by ATP hydrolysis returns the SNARE proteins to their initial state for another round.
Sec/Munc proteins: While Sec/Munc (SM) proteins can be dispensed with in vitro at high SNARE concentrations, the system in vivo universally requires an SM protein as a subunit of the t-SNARE complex to clasp the assembling SNARE complexes. The synaptic SM protein is Munc 18-1 (Munc 18a), a ~600 residue protein that folds into an arch-shaped “clasp” structure with an N-terminal lobe and a C-terminal lobe which can bind four helix bundles. As summarized in Figure 2, Munc 18-1 associates with SNARE proteins in two ways. Initially it binds to a four helix bundle located solely within the t-SNARE syntaxin-1 in its ‘closed’ conformation. This four helix bundle is composed of the N-terminal three helix “Habc” domain of syntaxin-1 folded back onto part of the long helical SNARE motif (which provides the fourth helix). This blocking/sequestration of part of the syntaxin-1 SNARE motif prevents the formation of syntaxin-1/SNAP-25/Vamp-1 SNARE complexes suggesting Munc 18-1 acts as a negative regulator of membrane fusion.
Figure 2. SM proteins are designed to bind four helix bundles. (A) The “closed” conformation of Syntaxin-1A, in which the SM protein Munc18-1 binds the four helix bundle composed of syntaxin’s own Habc domain (three helices, in brown) and its own SNARE motif helix (fourth helix, in red). This closed state has so far only been found with syntaxins involved in exocytosis. Inset: SM proteins are universally attached to Habc domains by a specialized sequence at the N-terminus of Habc (labeled as N-peptide). (B) The “open” conformation of a t-SNARE complex, consisting of a t-SNARE and its cognate SM protein bound to the N-peptide of its syntaxin’s Habc domain. This is believed to be the universal state in which t-SNAREs are open (i.e. reactive) with cognate v-SNAREs to form trans-SNARE complexes (C) resulting in fusion. Positioning of the protein domains in B and C are arbitrary. Panel C illustrates SNAREs and SM proteins, the universal fusion machinery. Reproduced from Südhof & Rothman, 2009 with permission. [Click to enlarge]However SM proteins are now known to be positively required in all membrane fusion reactions. SM proteins promote membrane fusion when syntaxin-1 adopts an ‘open’ conformation (with the SM protein anchored by its N-terminal lobe to a specific N-terminal peptide of syntaxin-1), exposing the SNARE motif of syntaxin-1 (single helix) to interaction with the other t-SNARE SNAP-25 (double helix) to form the t-SNARE complex, a three helix bundle which then docks with the v-SNARE, VAMP-1(single helix). This conformational change leaves the ‘arch-shaped’ body of Munc 18-1 free to fold back and clasp the zippering syntaxin-1/Snap-25/VAMP-1 four-helix bundle near the membrane as shown in Figure 2.
Exactly how SM proteins cooperate with SNARE complexes for fusion is not yet known but a kinetic role in which SM proteins co-operate with SNAREs by helping them assemble into productive topological arrangements at the interface of two membranes has been suggested. Thus, SM proteins likely act as catalysts for SNAREs which in turn are catalysts for membrane fusion.
In summary, the universal fusion machinery consists of a v-SNARE protein and a t-SNARE complex, the latter comprised of a syntaxin ‘heavy chain’ with one or two associated non-syntaxin SNARE ‘light chains’, and a cognate SM protein bound to the N-terminus of the syntaxin. The t-SNARE complex engages the cognate v-SNARE in the opposing membrane, and as these two SNAREs zipper-up towards the membrane, the SM protein cooperates in fusion at least in part by circumferentially clasping the assembling trans-SNARE complex.
GLUT4 vesicle docking and fusion machinery in muscle and fat
Insulin-stimulated GLUT4 translocation involves a similar process of membrane fusion mediated by homologues of the SM protein and v- and t-SNARES found in synaptic transmission. The v-SNARE found on GLUT4-containing vesicles is VAMP-2, first identified by immunological detection in GLUT4-enriched vesicles from rat adipocytes and subsequently confirmed in muscle and adipose cells by PCR, Northern blots and immunological methodologies (see ref ). Similar approaches revealed that the t-SNARES in insulin responsive tissues are syntaxin-4 and SNAP23.
The SM protein isoform involved in insulin-stimulated GLUT4 vesicle fusion is Munc 18c which binds to the trans-SNARE complex and strongly accelerates the fusion rate. Munc 18c recognizes both vesicle-rooted SNARE and target membrane-associated SNAREs, and promotes trans-SNARE zippering at the post-docking stage of the fusion reaction. The stimulation of fusion by Munc 18c is specific to its cognate SNARE isoforms. Munc 18c also binds the syntaxin-4 monomer but unlike Munc 18-1 does not block target membrane-associated SNARE assembly. Thus, the inhibitory "closed" syntaxin binding mode demonstrated for Munc 18-1 is not conserved in Munc 18c. Unexpectedly, it was found that Munc 18c recognizes the N-terminal region of the vesicle-rooted SNARE, whereas Munc 18-1 requires the C-terminal sequences, suggesting that the architecture of the SNARE/SM complex likely differs across fusion pathways.
Different physiological events require different degrees of regulation
Different intracellular fusion reactions have distinct regulatory processes that adapt the universal fusion machinery to specific situations. The regulatory processes associated with neurotransmitter release where the response time is less than a millisecond would be expected to differ from those controlling insulin-induce glucose transport which has a half time of 2-5 minutes. In neurons the activated SNAREs (those whose v-SNAREs have already formed partially zippered trans-SNARE complexes with the plasma membrane t-SNAREs) are clamped by a protein called complexin which binds via its central α-helix to the membrane-proximal portion of the t-SNARE thus preventing the v-SNARE from completing its zippering and triggering fusion. This inhibition is released when Ca2+ enters in response to an arriving action potential and binds to the protein kinase C-like C2 domains of a second regulatory protein, synaptotagmin. Calcium-activated synaptotagmin competes with complexin for binding to the assembled SNARE complex and displaces it, allowing the zippering to be completed and fusion to occur.
Such exquisite regulation is not required for insulin-stimulated glucose transport given its much slower response times where GLUT4-containing vesicles translocate from their storage positions inside the cell to the plasma membrane.
Insulin-stimulated translocation of GLUT4 vesicles to the plasma membrane
As Larance, Ramm and James point out in their 2008 review insulin-stimulated glucose transport is a complex process reflecting the convergence of two convoluted biological systems – vesicular transport and signal transduction. More than 60 proteins have been implicated in this orchestrated process and the challenge is to distinguish between the mainly passive players on the one hand and the molecules that are clearly driving the process on the other. The maze-like nature of the endosomal system adds to the difficulty of unravelling what appears to be a medley of many overlapping and rapidly changing transitions. GLUT4 trafficking involves six discrete steps: i) Biogenesis of GLUT4 storage vesicles (GSVs), small 50-nm vesicles that contain the majority of intracellular GLUT4; ii) Transport of GSVs to the cell cortex via elements of the cytoskeleton including microtubules and actin; iii) Tethering, the low-affinity interaction between GSVs and the plasma membrane mediated by a tethering complex; iv) Docking, the assembly of the trans SNARE complex as described above; v) Fusion of the lipid bi-layers of the GSV and the PM as described above; vi) Endocytosis, where after incorporation into the surface membrane, GLUT4 can be efficiently retrieved via endocytosis, involving, at least in part, a clathrin-dependent mechanism.
Figure 3. A model for GLUT4 trafficking. In the absence of insulin (basal), a pool of GLUT4 is targeted to GSVs, which are derived from the TGN and/or endosomes. In the presence of insulin, these GSVs fuse directly with the plasma membrane in an initial burst (insulin burst). GLUT4 subsequently recycles through endosomes, the TGN and back to the plasma membrane through generic recycling compartments in the presence of insulin (‘continuous insulin’). Following insulin withdrawal, GLUT4 traffics through the TGN to re-form GSVs. Arrows represent different trafficking rates: low (light grey), medium (dark grey), high (black). Endo, endosomes. Reproduced from Stockli J et al 2011 with permission. [Click to enlarge]As summarized in Figure 3, in the absence of insulin, GLUT4 is sequestered at a perinuclear location in small vesicles and tubules termed GLUT4 storage vesicles (GSVs) that are derived from the transgolgi network (TGN) and/or endosomes. Sorting of GLUT4 into its insulin-sensitive store requires the GGA [Golgi-localized, gamma-ear-containing, ADP ribosylation factor (ARF)-binding] adaptor proteins and ubiquitination of GLUT4. A ubiquitin-resistant version of GLUT4 fails to translocate to the cell surface in response to insulin, supporting the model that ubiquitination acts as a signal for the trafficking of GLUT4 from the endosomal/trans-Golgi network (TGN) system into its intracellular storage compartment of GSVs. In the presence of insulin these GSVs translocate and fuse with the plasma membrane in an initial burst. Under prolonged insulin signaling GLUT4 recycles through endosomes, the TGN and then back to the plasma membrane, whereas on insulin withdrawl, GLUT4 traffics through the TGN to re-form GSVs.
Insulin activation of GLUT4 translocation involves the PI3K/Akt pathway where activated Akt phosphorylates the RabGAP protein AS160 (TBC1D4) which is associated with GLUT4 vesicles via its interaction with the IRAP cytosolic tail and which regulates GLUT4 trafficking in adipocytes. Nonphosphorylated AS160 binds to GLUT4 vesicles and through the activity of its GTPase-activating protein (GAP) domain inhibits GLUT4 translocation by maintaining Rab10 in an inactive GDP-bound state. On insulin stimulation AS160 is phosphorylated by Akt at six sites[14,16], which results in it being bound by a member of the 14-3-3 family of phosphoserine binding proteins which in turn inhibits its RabGAP activity leading to Rab activation (maintenance of the GTP bound state). Activated Rab10 on GLUT4 vesicles, in turn promotes the recruitment to and subsequent docking and fusion of GLUT4 vesicles with the plasma membrane via the formation of SNARE complexes as discussed above.
Both Rab10 and Rab14 have been shown to be key regulators of GLUT4 trafficking in adipocytes and to function at independent, sequential steps of GLUT4 translocation. Rab14 functions upstream of Rab10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane whereas Rab10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. AS160, which forms tether-like homo-oligomers and binds to GLUT4 vesicles and the plasma membrane, plays a crucial role during tethering by bridging the interaction between Rab proteins on GLUT4-containing vesicles, actin and the exocyst.
A proteomic survey of GLUT4 vesicles immuno-purified from adipocytes identified a number of Rabs including Rab8, Rab10, Rab11, and Rab14. It is not surprising that multiple Rabs have been implicated in GLUT4 trafficking in view of the large number of compartments that GLUT4 traffics through. This could call for the activity of multiple Rabs as well as multiple RabGAPs and GEFs in order to regulate the sequential movement of GLUT4 through its itinerant compartments. Consistent with this, Rab cascades whereby the activity of individual Rabs that act in series are coupled via the coordinated regulation of GAPs and GEFs. Thus far, the RabGAP TBC1D4/AS160 and DENND4C, a Rab GEF, have been implicated in GLUT4 trafficking in adipocytes and these are thought to control the activity of Rab10.
While Rab10 is a key regulator in of GLUT4 translocation in adipose tissue, it is not involved in GLUT4 translocation in muscle where the role is carried out by a different small GTPase Rab8A. Both Rab8A and Rab13 are targets of AS160-GAP activity in the context of GLUT4 traffic in muscle. Rab13 has a broader intracellular distribution compared with the perinuclear restriction of Rab8A, and insulin promotes Rab13 colocalization with GLUT4 at the cell periphery. Thus both Rab13 and Rab8A are Rab-GTPases activated by insulin, and that downstream of AS160 they regulate the traffic of GLUT4 vesicles, possibly acting at distinct steps and sites, paralleling the combined activities of Rab14 and Rab10 in adipose tissue.
Given the complexity of GLUT4 trafficking the question arises as to the involvement of additional RabGAPs and RabGEFs in this process. A qualitative screen identified the RabGAP TBC1D13 as a potent suppressor of GLUT4 translocation. TBC1D13 interacted with Rab10 and Rab1 in a GTP-dependent manner but it did not display GAP activity toward either of these Rabs. Rather, TBC1D13 stimulated Rab35 GTP hydrolysis in vitro and in vivo. Overexpression of constitutively active Rab35 but not Rab10 overcame the TBC1D13-mediated block in insulin-stimulated GLUT4 translocation suggesting an important role for Rab35 and TBC1D13 in GLUT4 trafficking in adipocytes.
Other proteins involved in GLUT4 trafficking
As Rothman and Sudhof point out in their review there are a plethora of proteins and compounds that fragmentary evidence suggests may regulate synaptic and other fusion processes—including the large families of Rab GTPases, tethering proteins, and phosphoinositides. In neurons, the SNARE-Munc 18-1 complex works in collaboration with C2-domain-containing proteins including synaptotagmin, double C2-like domain beta (DOC2b) and Munc 13. C2 domains bind Ca2+ and phospholipids providing a link to the regulation of synaptic vesicle exocytosis through Ca2+.
One candidate C2-domain-containing protein implicated in GLUT4 exocytosis is extended synaptotagmin-like protein 1 (ESYT1) which has five C2 domains, is targeted to the plasma membrane and is phosphorylated by CDK5 in response to insulin. Another is DOC2b, a protein that contains two C2 domains and translates from small punctuate structures to the plasma membrane and binds syntaxin-4 in response to insulin in a Ca2+–dependent manner. C2 domains insert into membranes and induce curvature which together with the energy provided by the assembling SNARE complex, may be sufficient to induce fusion.
Figure 4. Overview of the steps involved in GLUT4 trafficking at the plasma membrane. GSVs interact with a host of molecules as they are delivered to the cell periphery and tethered, docked and fused at the plasma membrane. GSVs approach the plasma membrane where they are tethered by actin, the exocyst or TBC1D4, or a combination of these in parallel or in series. The vesicle docks with the plasma membrane as the ternary complex is formed between VAMP2 on the GSV and syntaxin-4 and SNAP23 on the plasma membrane. This complex can then facilitate fusion of a GSV with the plasma membrane. Reproduced from Stockli J et al 2011 with permission. [Click to enlarge]As summarized in Figure 4, there are additional proteins involved in the events (approach and tethering) occurring upstream of the docking and fusion of GLUT4 vesicles with the plasma membrane. The approach is mediated by either the cytoskeleton or microtubules depending on the cell type with vesicles delivered by insulin-regulated kinensin motors. Tethering is more complex with the exocyst and actin being implicated along with the RabGAP protein AS160 (TBC1D4) discussed above. The exocyst is a ‘Y”-shaped complex comprised of eight subunits and interacts with many molecules: phospholipids, actin, actin nucleators, atypical protein kinase C, JNK, small molecular GTPases (RAL, CDC42, Rho, Rab8, Rab10, Rab11, Rab14, TC10, ARF6) as well as SNAREs and SNARE-associated proteins such as snapin. Each tip of the “Y”-shaped exocyst may interact with the plasma membrane and the transport vesicle through different small GTPases such as Rab10 and/or Rab11. Insulin causes actin rearrangements in fat and muscle cells and actin likely serves as a longer range tether than the exocyst. Myosin motors MYO5 and MYO1C have been identified on GLUT4 vesicles and at the plasma membrane, making them candidate molecules that link GLUT4-containing vesicles, actin and the plasma membrane. Both are phosphorylated by CamKII in response to insulin. MYO5 binds the small GTPases Rab8, Rab10 and Rab11 and MYO1C binds RAL, all of which have been implicated in GLUT4 trafficking and bind the exocyst.
Exercise-induced stimulation of glucose transport in muscle
Similarly to insulin, a single bout of exercise increases the rate of glucose uptake into the contracting skeletal muscles, a process that is regulated by the translocation of GLUT4 glucose transporters to the plasma membrane. Exercise and insulin utilize different signaling pathways, both of which lead to the activation of glucose transport. Exercise training in humans results in numerous beneficial adaptations in skeletal muscles, including an increase in GLUT4 expression. There is evidence that there are two distinct intracellular locations or pools of glucose transporters in skeletal muscle, one that responds to exercise and one that responds to insulin. However the exact intracellular locations of the putative exercise- and insulin-stimulated GLUT4 pools have not yet been elucidated.
The molecular signaling pathways that lead to the stimulation of glucose uptake in skeletal muscle or other cell types have not been completely elucidated but do not involve autophosphorylation of insulin receptors, IRS tyrosine phosphorylation, or PI 3-kinase activity or the mitogen-activated protein kinase signaling cascade, which is also increased in response to exercise.
There are several lines of evidence to suggest that the increase in intracellular calcium that leads to the interaction of actin and myosin filaments with muscle contraction is a critical mediator of contraction-stimulated glucose transport. Since cytoplasmic calcium concentrations are elevated for only a fraction of a second following each muscle contraction, these molecules probably do not directly activate the glucose transport system. Instead, the rise in cytosolic calcium may initiate or facilitate the activation of intracellular signaling molecules or cascades of signaling proteins that lead to both the immediate and prolonged effects of exercise on muscle glucose transport. Candidates include protein kinase C (PKC) a calcium-dependent signaling intermediary that has been shown to be activated by muscle contraction and may be involved in the regulation of contraction-stimulated glucose transport.
There is also evidence for an autocrine or paracrine component for the activation of contraction-stimulated glucose uptake, one important example being nitric oxide. Nitric oxide is released from skeletal muscle contracted in vitro, and inhibition of nitric oxide synthase has been demonstrated to decrease both basal and contraction-stimulated glucose transport. Another molecule that may be involved with contraction-stimulated glucose transport is kallikrein, which catalyzes the production of bradykinin and is a potential stimulator of nitric oxide synthase. Adenosine has also been shown to be secreted from contracting muscle fibers, and it has been suggested that the adenosine receptor mediates the signaling mechanism through which contraction results in the synergistic stimulation of glucose transport.
In addition to the activation of specific intracellular signaling molecules, the glycogenolytic process may be an important regulator of exercise-induced GLUT4 translocation in skeletal muscle. Although there is still no direct evidence, it has long been hypothesized that transporter molecules are associated with glycogen particles in the muscle, and that the contraction-stimulated hydrolysis of glycogen releases GLUT4, leading to translocation of these transporters to the cell surface.
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