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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This is the first article of three in the “Insulin-stimulated Glucose Transport Minireview Series.”
Insulin, the major hormonal regulator of glucose transport in humans, has served as a prototypic molecule for understanding cell signaling pathways since its discovery in 1922. It was among the first proteins for which primary amino acid sequence and three-dimensional structure were determined. The insulin receptor was likewise among the first peptide receptors to be identified by ligand binding, and its subunit structure was deduced in the earliest days of modern receptor biology (
). Despite these advances, our understanding of how insulin and the related proteins, insulin-like growth factors I and II, stimulate glucose transport (via the translocation of glucose transporter proteins from intracellular to plasma membranes) is fragmentary. One reason for this is that the major target for insulin signaling to glucose transport is a complex membrane trafficking pathway that is likely to contain many unknown components.
As summarized in the preceding introductory comments (
), glucose uptake via the GLUT4 isoform of mammalian hexose transporters accounts for most of the stimulatory effect of insulin on this process in muscle and fat cells. GLUT4 rapidly recycles through the plasma membrane/endosomal membrane system in the presence of insulin. However, in the basal state this transporter protein is directed to and retained within specific intracellular membranes through the action of distinct elements within the GLUT4 structure (
). Insulin causes movement of GLUT4 out of this sequestered localization, leading to an increase in its steady state concentration on the cell surface membrane where it can catalyze glucose uptake. However, to accomplish this, insulin signaling is likely to regulate GLUT4 trafficking at multiple steps, consistent with data showing insulin also enhances cell surface display of proteins such as transferrin receptor and GLUT1 that recycle to a large extent independently of GLUT4. To further complicate our understanding of this system, different signaling elements may be required to modulate each of these different putative regulated steps in the GLUT4 trafficking pathway.
Insulin Receptor Signaling Circuits
Substrate phosphorylation by the insulin receptor tyrosine kinase appears to involve the binding of phosphorylated receptor tyrosine 960 to phosphotyrosine-binding (PTB)1 domains of substrate proteins (
). Adjacent Pleckstrin homology (PH) domains on some substrate proteins also appear critical for receptor binding and phosphorylation. Tyrosine kinase signaling is often initiated by the recruitment of signaling proteins through their Src homology 2 (SH2) or PTB domains to phosphotyrosine sites. In the case of insulin receptor, tyrosine phosphorylation of four related substrate (IRS) proteins (
) causes many candidate signaling proteins to be recruited, including: 1) the p110-type phosphatidylinositol 3-kinase (PI 3-kinase) through the SH2 domains of p85 regulatory subunits; 2) Grb2 and the protein tyrosine phosphatase SH-PTP2, which appear to be necessary for p21ras activation (
), which may modulate processes such as actin assembly and mitogenesis under control of the small GTPase Rho. Insulin receptor signaling can also engage p21ras through tyrosine phosphorylation of Shc and its subsequent binding to complexes of Grb2 and Sos (
As illustrated in Fig. 1, p21rasand the p85/p110-type PI 3-kinases represent two major initial switch elements for insulin receptor signaling. There is also evidence that the p21ras-related GTP-binding proteins Rap (
) are engaged by insulin receptor signal transduction. A central paradigm of insulin signaling is the involvement of multiple protein serine/threonine kinases downstream of both the p21ras and PI 3-kinase elements (Fig. 1). More recently, a group of non-receptor tyrosine kinases denoted Btk/Itk/Tec has been found in hemopoietic cells to respond to the generation of 3′-polyphosphoinositide, apparently by recruitment through their PH domains to membranes where they can be tyrosine-phosphorylated and activated (
). It is not yet known whether insulin or insulin-like growth factor-1 receptor signaling can act through 3′-phosphoinositides on these or other tyrosine kinases, and they are therefore not included in Fig. 1. Another recently discovered class of likely downstream effectors of 3′-phosphoinositides includes proteins that regulate membrane-related functions (Fig. 1) such as actin assembly (Rac GTPase), early endosome fusion (EEA1), and guanine nucleotide exchange (GRP1, cytohesin-1, and ARNO) or possibly GTPase activation (α-centaurin) of ARF proteins. It is certain that the list of potential downstream effectors of insulin receptor signaling will rapidly expand, as additional protein targets of 3′-phosphoinositides are discovered.
Much effort has been directed toward identifying which of the above insulin receptor signaling elements are actually linked to GLUT4 translocation. Although some evidence suggested p21rasactivation might influence glucose transport in fat (
). Rather, strong indications that p85/p110-type PI 3-kinase activity is necessary for this insulin response have accumulated. Such evidence includes complete inhibition of insulin action on glucose transport by specific inhibitors of PI 3-kinase such as wortmannin (
), suggesting PI 3-kinase activity is also sufficient for glucose transporter translocation.
Although the above results strongly support the hypothesis that p85/p110-type PI 3-kinase activation through its recruitment to phosphotyrosine sites on insulin receptor substrate proteins mediates GLUT4 translocation, it is instructive to review the limitations related to the current supporting data. First, the inhibitors wortmannin and LY29004 are not fully specific for PI 3-kinase activity and in any case are known to block PI 3-kinase isoforms lacking known regulatory subunits that contain SH2 or other domains that can be recruited to phosphotyrosine sites. Such other PI 3-kinase isoforms (
) rather than, or in addition to, the type p110 PI 3-kinase may be necessary for exocytosis of GLUT4. The use of the truncated p85 subunit containing SH2 domains as a dominant inhibitory reagent to inhibit native p85/p110 PI 3-kinase function also has the potential limitation of inadequate specificity. When expressed at high concentrations, SH2 domains can be promiscuous in binding phosphotyrosines within various amino acid sequences and thus may not be restricted to binding sites specific for endogenous p85/p110-type PI 3-kinase. Thus, although a key role for this PI 3-kinase in GLUT4 regulation by insulin seems almost certain, there are potential gaps in our understanding of even this point.
Multiple Signaling Pathways Regulate Glucose Transport
Recent results call into question the hypothesis that PI 3-kinase activation by insulin is sufficient for GLUT4 translocation. For example, it is well established that recruitment of PI 3-kinase to phosphotyrosines on the platelet-derived growth factor (PDGF) receptor in response to PDGF, which occurs in 3T3-L1 adipocytes to the same extent as recruitment of PI 3-kinase to protein phosphotyrosines in response to insulin, has virtually no effect on GLUT4 translocation (
) also fails to enhance glucose transport in the absence or presence of submaximal concentrations of insulin. Conversely, severe inhibition of insulin-mediated IRS protein tyrosine phosphorylation and recruitment of PI 3-kinase in response to incubation of 3T3-L1 adipocytes with PDGF failed to diminish glucose transport stimulation by insulin at all concentrations along the dose-response relationship (
The lack of correlation between PI 3-kinase activation and GLUT4 translocation in the studies cited above may reflect an additional insulin-specific signaling pathway or pathways required to operate in conjunction with PI 3-kinase activation to effect this biological response. According to this model, agents such as interleukin-4 and anti-integrin antibody fail to enhance glucose transport because they are unable to mimic the effect of insulin on this other signaling pathway. Recent surprising results support this hypothesis. In these experiments, a cell-permeable analog of the PI 3-kinase product PI(3,4,5)P3, thought to be a downstream effector for insulin action through PI 3-kinase, was unable to cause GLUT4 translocation when added alone to cells (
). These results suggest that insulin may uniquely act to initiate PI 3-kinase-independent signaling events in the presence of wortmannin that collaborate with the PI 3-kinase signaling pathway to effect GLUT4 translocation.
Substantial recent information suggests that PI 3-kinase-independent mechanisms also regulate GLUT4 translocation in skeletal muscle during contraction. For example, the stimulations of glucose transport in skeletal muscle caused by insulin and contraction are additive, but only the effect of the former is blocked by wortmannin (
). Taken together, these recent findings suggest the hypothesis that AMPK may mediate, at least in part, exercise-stimulated glucose uptake in skeletal muscle.
Another cell signaling pathway that appears to markedly stimulate glucose uptake in muscle involves nitric oxide, which stimulates guanylate cyclase to produce cyclic GMP. The NO donor sodium nitroprusside and dibutyryl-cGMP both accelerated epitrochlearis (
), studies differ on whether NO is involved in glucose transport regulation by contraction. NO synthase inhibitors failed to block increased glucose uptake in response to contraction in rat epitrochlearis muscle (
Phorbol ester-sensitive protein kinase C isoforms may also be significant regulators of muscle glucose uptake. Thus, the protein kinase activator 12-deoxyphorbol 13-phenylacetate 20-acetate causes a 3–4-fold stimulation of glucose transport in isolated rat epitrochlearis muscle (
). These effects of catecholamines and bradykinin are not blocked by wortmannin, suggesting independence of the actions of most known isoforms of PI 3-kinase. Particularly interesting is the reported effect of adenosine to potentiate the stimulation by insulin of glucose uptake in brown fat cells from 15- to 30-fold (
). Thus it is not clear whether some of these insulin-independent modulations of glucose transport involve other glucose transporter isoforms. It appears, for example, that cell surface GLUT1 transporter activity can be regulated, in some cases potentially independent of its recruitment to the cell surface (
). This may be particularly relevant in tissues that express significant levels of this glucose transporter isoform, such as brown fat. Table I summarizes many of the cellular regulators reported to stimulate glucose transport, and the isoform likely to be involved.
Table IRegulators and signaling pathways reported to stimulate glucose transport
Role of Docking Proteins and PI 3-Kinase Localization
Of the regulatory mechanisms for glucose transport described above, research on the PI 3-kinase pathways has been the most extensive. Whether IRS proteins are required for PI 3-kinase signaling to act on the GLUT4 system is unclear at present. Microinjection or expression of the dominant inhibitory PTB or SAIN domains of IRS-1 in insulin-sensitive cultured adipocytes is able to block the mitogenic and membrane-ruffling effects of insulin but not the stimulation of glucose transport (
). These dominant inhibitory constructs are expected to block binding of all IRS protein isoforms to the insulin receptor through binding to the insulin receptor juxtamembrane sequence surrounding Tyr-960, the docking site for IRS and Shc protein PTB domains. Indeed, nearly complete inhibition of IRS-1 tyrosine phosphorylation by insulin was caused by expression of its PTB domain in these studies (
). Furthermore, studies in animals in which the IRS-1 or theIRS-2 gene has been ablated provide support for a role of these proteins in glucose transport regulation. Mice lacking IRS-1 exhibit some insulin resistance although no diabetes (
), which is tyrosine-phosphorylated in response to insulin. Such redundancy may account in part for some of these apparently conflicting results. The information gained from IRS knockout mice provide compelling support for a role of these proteins in glucose transport regulation by insulin, with the caveat that unrelated changes in response to IRS loss during development may contribute to the phenotypes observed. Taken together, the data reported to date related to the role of IRS proteins in GLUT4 translocation are difficult to reconcile unless we again invoke the hypothesis that PI 3-kinase·IRS protein complexes are necessary but not sufficient for GLUT4 translocation by insulin.
An important question about the role of PI 3-kinase docking proteins in regulating GLUT4 trafficking is whether they function to localize the enzyme to specific cellular sites required for regulation of GLUT4 trafficking. IRS-1 (
) are present in cells as both cytosolic and membrane-bound proteins, both in basal and insulin-stimulated conditions. Membrane binding of PI 3-kinase, through its binding to docking proteins, is expected to be necessary for the enzyme to come into contact with its phospholipid substrate. The exact cellular location of membrane-bound PI 3-kinase·IRS complexes is not known, but endosomes and GLUT4-containing vesicles, potentially through binding AP3 adaptin complexes (
), have been suggested. Only a very small fraction of the insulin-stimulated, IRS-1-bound PI 3-kinase activity co-purifies with GLUT4-containing vesicles, but the downstream protein kinase Akt appears to be recruited as well (
). This latter result is not inconsistent with the hypothesis that a second signaling pathway is necessary for GLUT4 translocation, however.
Downstream Targets of 3′-Phosphoinositides
The insulin-sensitive p85/p110 PI 3-kinases can catalyze phosphorylation of PI, PI(4)P, or PI(4,5)P2 at the D-3 position of the inositol ring to produce the PI(3)P, PI(3,4)P2, or PI(3,4,5)P3. Recent results indicate PI(3,4,5)P3 is necessary for insulin action on glucose transport, based on the inhibitory effect of microinjection of a phosphatase specific for this species into 3T3-L1 adipocytes (
). It is not yet clear whether the other two PI 3-kinase products are also necessary, but the recent identification of a protein target, EEA1, that binds PI(3)P through its RING finger and functions in the context of Rab5 in early endosome fusion (
) denoted GRP1, ARNO, and cytohesin-1 containing PH domains that bind PI(3,4,5)P3with high affinity and a Sec7 domain that catalyzes guanine nucleotide exchange of ARF1, -5, and -6 proteins has been shown to be recruited to plasma membranes in response to insulin (
), but future studies on these and similar proteins in relation to GLUT4 translocation are warranted. The potential connection between PI 3-kinase signaling in insulin action and ARF function is highlighted by the discovery of polyphosphoinositide-binding protein α-centaurin, which contains some sequence similarity to ARF GTPase-activating protein (
). Taken together, these discoveries of proteins that bind directly to PI 3-kinase products and regulate known membrane modulators such as ARF proteins highlight a potentially rich interface between signaling and membrane trafficking.
Intense research has focused on protein serine/threonine kinases downstream of PI(3,4)P2 and PI(3,4,5)P3 that may regulate GLUT4. For example, recent reports (
) have shown that protein kinase Cζ (Fig. 1) is activated by polyphosphoinositides, is stimulated in intact cells by insulin in a wortmannin-sensitive manner, and may contribute to glucose transport regulation. Another prime candidate has been Akt/protein kinase B, which is activated in conjunction with its binding to these lipids by the 3′-polyphosphoinositide-dependent protein kinase PDK1 and another protein kinase (
). However, in well controlled experiments using a mutant Akt/protein kinase B with alanines substituted at phosphorylation sites threonine 308 and serine 473 as a dominant inhibitory construct in both Chinese hamster ovary cells and 3T3-L1 adipocytes, protein synthesis but not insulin-stimulated glucose transport was inhibited (
) to suggest that protein kinase Cλ is directly involved in GLUT4 translocation. Taken together, there is not yet a clear consensus of data derived from multiple laboratories that any of the known protein kinases downstream of PI 3-kinase directly mediates insulin action on glucose transport.
Conclusions and Future Directions
Over the past few years many new components of the insulin receptor signaling network have been discovered and their cDNA clones isolated. Particularly exciting has been the identification of downstream targets of the insulin-regulated PI 3-kinase lipid products, including protein kinase C isoforms ζ and λ, regulatory protein kinases of the Akt/protein kinase B system, tyrosine kinases Itk/Btk/Tec, the early endosome regulator EEA1, and ARF exchange factors GRP1, ARNO, and cytohesin-1. Because PI 3-kinase appears to be required for glucose transport regulation by insulin, future work is likely to connect one or more of these proteins (or additional ones to be discovered) to specific membrane trafficking components involved in GLUT4 translocation. We have also learned that additional cellular signaling elements such as trimeric G proteins, NO, and cGMP can regulate glucose transport and that unknown signaling events may be required in conjunction with PI 3-kinase for insulin to act. In the meantime, progress is being made on understanding the components of membrane systems through which GLUT4 traverses in insulin-sensitive cells, as will be discussed in the accompanying minireviews on this topic (
). Merging these two fertile research fields, signaling and membrane trafficking, will be the natural result of future exciting work on the mechanisms that underlie GLUT4 regulation.
We thank the members of the Czech and Corvera laboratories for their contributions and creative discussions, and Drs. Jerry Olefsky, Jeffrey Pessin, and Maureen Charron for helpful comments on this review. We thank Jane Erickson for excellent assistance in preparation of this manuscript. We regret the number of references had to be restricted due to space limitations.
Proc. Natl. Acad. Sci. U. S. A.1980; 77: 7137-7141