Departments of Medicine and of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Lipid rafts are domains within the plasma membrane that are enriched in cholesterol and lipids with saturated acyl chains. Specific proteins, including many signaling proteins, segregate into lipid rafts, and this process is important for certain signal transduction events in a variety of cell types. Within the past decade, data have emerged from many laboratories that implicate lipid rafts as critical for proper compartmentalization of insulin signaling in adipocytes. A subset of lipid rafts, caveolae, are coated with membrane proteins of the caveolin family. Direct interactions between resident raft proteins (caveolins and flotillin-1) and insulin-signaling molecules may organize these molecules in space and time to ensure faithful transduction of the insulin signal, at least with respect to the glucose-dependent actions of insulin in adipocytes. The in vivo relevance of this model remains to be determined.
adipocyte; caveolin; flotillin; glucose transport; Cbl-associated protein
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INTRODUCTION |
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THE BINDING OF INSULIN to its receptor
has multiple tissue-specific effects on cells, including both metabolic
and mitogenic actions. Insulin is the primary hormone responsible for
maintenance of plasma glucose within a narrow physiological range,
which it does by inhibiting hepatic glucose output and promoting
glucose disposal into muscle and adipose tissues. Disruption of glucose homeostasis by target organ resistance or by pancreatic -cell failure results in significant morbidity and excess mortality. For
these reasons, discovery of the intracellular molecules and pathways
that mediate insulin action has preoccupied many investigators for many
years, but controversy and confusion remain. For example, substantial
evidence has accumulated that activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin-stimulated glucose transport in adipocytes, but the relative contribution of kinases downstream of PI 3-kinase is uncertain, and data suggest that activation of PI 3-kinase alone is not sufficient (40).
Further difficulty is posed by the promiscuity of the signaling
molecules that are involved in transducing insulin's signal. Despite
the fact that many of these signaling molecules are common to the signaling pathways of other receptors, insulin has specific metabolic effects on cells that are not common to the other receptors
(29).
An emerging concept to explain such signaling specificity in the face of molecular promiscuity is that specialized scaffold, anchor, and adapter proteins segregate signaling molecules into specific cellular compartments via protein-protein interactions (38). One level of spatial organization proposed for the plasma membrane is that of the liquid-ordered phase. This phase is enriched in cholesterol and lipids with saturated acyl chains, such as sphingolipids and glycosphingolipids, but is relatively depleted of phospholipids (5). According to a model proposed by Simons and Ikonen (54), lipids in the liquid-ordered phase pack together to form dynamic "rafts" in the plasma membrane, and these lipid rafts either recruit or exclude specific molecules, including signaling proteins. The biophysical and biochemical microenvironment of the rafts (e.g., membrane fluidity) may influence the function of the raft proteins. Also, the physical segregation of proteins into such "microdomains" may regulate the accessibility of those proteins to regulatory or effector molecules. The structural bases suggested for protein association with rafts include glycophosphatidylinositol (GPI) anchors (e.g., folate receptor), dual fatty acylation by myristylation and palmitoylation (e.g., Src family tyrosine kinases), transmembrane domain structure (e.g., influenza hemagglutinin polypeptide), cholesterol binding (e.g., caveolins), and protein-protein interactions (e.g., caveolin-interacting proteins). On the basis of their ability to sort proteins, rafts have been implicated in a number of cell functions, including membrane targeting in polarized cells, endocytosis, uptake of small molecules via potocytosis, and signal transduction. Recent, detailed reviews of lipid rafts and their functions are available (1, 19, 50, 55, 56); this review will focus on proposed roles lipid rafts may play in insulin signaling, particularly in adipocytes from which most of the data derive.
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CAVEOLAE |
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A morphological correlate of rafts is caveolae ("little caves"), which were first described over 40 years ago in electron micrographs of endothelial cells as 50- to 100-nm flask-shaped invaginations of the plasma membrane (50). These structures are distinct from clathrin-coated pits and have their own characteristic striated coat, which consists largely of oligomerized caveolin proteins. Caveolae are enriched in cholesterol, and the presence of cholesterol in caveolae is necessary for their proper morphology and function (44). The extent to which lipid rafts and caveolae overlap in composition and function is not known, but rafts that do not contain caveolins likely exist in some mammalian cells.
Caveolae may participate in intracellular trafficking of lipids and proteins. Caveolae are dynamic structures that assume different shapes on the basis of their functional state (1). At the plasma membrane, they may exist as invaginations open to the extracellular space, as closed invaginations, and as flattened patches. Purified caveolae contain the protein components of vesicular transport, including SNARE proteins (47). Plasma membrane caveolae bud intracellularly in a GTP- and dynamin-dependent process (18, 36). Such budding and subsequent trafficking of caveolae vesicles could regulate signaling events that occur in caveolae.
Caveolae are present in most cell types but are especially abundant in adipocytes, smooth muscle cells, endothelial cells, and fibroblasts. As 3T3-L1 fibroblasts differentiate into adipocytes, plasma membrane caveolae identified morphologically by electron microscopy (EM) increase dramatically in number (9). The highest expression of caveolin messenger RNA and protein is in adipose tissue, and caveolin expression increases significantly during 3T3-L1 adipocyte differentiation (21, 46). These data suggest that caveolin and caveolae have important functions in adipocytes. One clue to such a function was suggested by the ultrastructural data of Goldberg et al. (13), who found that ferritin- or gold-labeled insulin concentrated in "micropinocytotic invaginations" of the adipocyte plasma membrane that were distinct from coated pits. More recently, several laboratories have localized important elements of the insulin signal transduction machinery to caveolae or rafts, including the insulin receptor itself. Baumann et al. (3) and Chiang et al. (7) have reported the discovery of a novel insulin-signaling pathway in adipocytes that relies on proper localization of signaling molecules to lipid rafts and that is required for insulin-stimulated glucose uptake. This review is organized according to the lipid raft-associated proteins that have been implicated in insulin signaling in adipocytes. Investigations of these pathways in skeletal or cardiac muscle or in cultured myoblasts or myotubes have not been reported.
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THE CAVEOLAE UNCERTAINTY PRINCIPLE |
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Before embarking on a discussion of how lipid rafts and caveolae may be involved in insulin signaling, it is important to recognize that there has been considerable controversy about the protein constituents and even existence of lipid rafts. The process of isolating rafts and measuring them may change their nature or perhaps even create them. That lipid rafts exist as functional units in the plasma membrane in vivo has received support from multiple, independent methods, and this topic has been reviewed elsewhere (19, 20, 55). Membrane domains that are highly enriched in the components of rafts and caveolae, including cholesterol, glycosphingolipids, and raft proteins, can be isolated on the basis of their insolubility in nonionic detergent (Triton X-100) at 4°C and their low density in sucrose density gradients. There is no unified terminology for these biochemically purified membrane domains. They have been called DIG (detergent-insoluble glycolipid-rich membranes), DRM (detergent-resistant membranes), TIFF (Triton-insoluble floating fractions), and CMD (caveolin-enriched membrane domains). In lung tissue, the DIG fraction excludes >98% of cellular protein but contains >85% of immunoreactive caveolin-1 (27).
Several investigators have emphasized that proteins in the DIG fraction cannot be assumed to be resident proteins of caveolae or even of rafts (24). For example, some DIG proteins, including GPI-linked proteins, may cluster in caveolae only after cross-linking with antibodies or upon detergent solubilization (32, 33). Recent work has used chemical cross-linking (10) and fluorescence resonance energy transfer (58) to show that GPI-linked proteins are organized in cholesterol-dependent, submicron domains of the plasma membrane, which likely correspond to rafts. Alternative methods of isolating caveolin-enriched membranes that do not rely on detergent solubilization have been developed. These methods differ from each other in which proteins cofractionate with caveolins. For instance, a detergent-free method developed by Smart et al. (57) includes GPI-linked proteins in the caveolin-enriched fraction, but another method, based on the shearing of caveolae vesicles from plasma membranes immobilized on silica beads, does not (48). All methods are susceptible to contamination of raft fractions with nonraft proteins (19), and at this time no one method can be considered the "gold standard." As we shall see, even the same method used in different laboratories can yield conflicting results.
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CAVEOLAE, CAVEOLINS, AND INSULIN SIGNALING |
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The caveolins are a family of ~21- to 25-kDa integral membrane
proteins that form the coat structure of caveolae (56). An unusual hairpin membrane domain results in both the amino and carboxy
termini facing the cytoplasm. Caveolins avidly bind cholesterol, which
may be the basis for their association with the liquid-ordered phase of
the plasma membrane. Caveolin-1 and -2 have a widespread, overlapping
tissue distribution; in contrast, caveolin-3 expression has been
detected only in muscle and astrocytes. Caveolin-1 and -2 form
heterooligomeric complexes with each other and colocalize by
immunofluorescence. Caveolin-1 is expressed as two isoforms, (22 kDa) and
(24 kDa), which differ at the amino terminus because of
alternative translation initiation sites. Caveolins have been
evolutionarily conserved with orthologs, being detected in organisms as
remote from humans as Caenorhabditis elegans.
Caveolin initially was identified as a major substrate for tyrosine
phosphorylation in Rous sarcoma virus-transformed fibroblasts (12). A possible role for caveolae and caveolins in
insulin signaling was suggested when Saltiel and colleagues [Mastick
et al. (30)] found that insulin stimulation of 3T3-L1
adipocytes was associated with tyrosine phosphorylation of caveolin-1,
with maximal phosphorylation being detected at 5 min of the insulin pulse. Tyrosine phosphorylation of caveolin did not occur in adipocytes after stimulation with either platelet-derived growth factor (PDGF) or
epidermal growth factor (EGF). In the case of PDGF, this may have been
due to low expression of the PDGF receptor in the majority of 3T3-L1
adipocytes (53). Insulin-stimulated tyrosine
phosphorylation of caveolin did not occur in undifferentiated
preadipocytes (31), despite the fact that the insulin
receptor and caveolin are expressed in preadipocytes. Lee et al.
(25a) investigated insulin-stimulated phosphorylation of caveolin-1 with a monoclonal antibody specific for
caveolin-1 phosphorylated on tyrosine-14. Again, insulin stimulation of
3T3-L1 adipocytes resulted in a rapid increase in caveolin-1 tyrosine
phosphorylation. No effect was seen with PDGF, EGF, bovine fibroblast
growth factor (bFGF), tumor necrosis factor- (TNF-
), or
interleukin-6 (IL-6). In contrast to the results obtained by Mastick et
al. (30), 3T3-L1 fibroblasts showed similar
insulin-stimulated tyrosine phosphorylation of caveolin-1. This
discrepancy may result from the different methods
(immunopreciptation-immunoblotting vs. immunoblotting) and reagents
(antiphosphotyrosine vs. antiphosphocaveolin-1) used by the
investigators. In any event, the physiological significance of
insulin-mediated caveolin phosphorylation in adipocytes has not been determined.
Using a detergent-based method of lipid raft preparation, Mastick et
al. (30) found only trace levels of insulin receptor in
the CMD of 3T3-L1 adipocytes. Recently, Müller et al.
(34) characterized the composition of CMD from isolated
rat adipocytes. The CMD and non-CMD were prepared by both detergent and
detergent-free methods from total cell lysates and from plasma
membranes, respectively. Representative fractions were analyzed by
immunoblotting for known raft and nonraft proteins. Despite the
methodological differences, there was good agreement between the
proteins enriched in CMD an or in the non-CMD by the two procedures. By
both methods, the -subunit of the insulin receptor was relatively
depleted from the CMD and relatively enriched in the non-CMD. However,
Gustavsson et al. (14) reached the opposite result by
double immunogold transmission EM and detergent-free isolation of CMD.
In these experiments, caveolin and both subunits of the insulin
receptor colocalized in ~50-nm-round structures that corresponded to
individual caveolae and in clusters of these structures. This
colocalization was confirmed by immunofluorescence microscopy in
paraformaldehyde-fixed 3T3-L1 adipocyte plasma membrane sheets. The
immunofluorescence labeling pattern appeared as doughnut-like spots of
0.3-0.5 µm diameter, a size that was noted to be similar to that
of the caveolae clusters visualized by EM. Biochemically, the insulin
receptor cofractionated with caveolin in detergent-free isolation of
CMD from rat adipocyte plasma membranes. The addition of insulin was not associated with a significant change in localization of the insulin
receptor, as assessed biochemically or by immunofluorescence. Even low
concentrations of Triton X-100 (0.1%) completely solubilized the
-subunit of the insulin receptor from the caveolin-enriched fractions. This observation may explain why Mastick et al. found only
trace amounts of the insulin receptor in CMD prepared on the basis of
detergent insolubility.
The presence of the insulin receptor in caveolae likely explains the
finding of labeled insulin in caveolae-like invaginations of adipocyte
plasma membranes previously seen by EM. The localization of proximal
components of insulin receptor signaling to CMD has also been reported
in a hepatoma cell line (22). Also, caveolae-like membranes isolated from neuronal plasma membranes were enriched in
receptor tyrosine kinases, including the insulin receptor -subunit (61). At this point the weight of the published data
supports localization of a significant subset of plasma membrane
insulin receptors to caveolae in adipocytes, but the issue is not settled.
Does localization of the insulin receptor to caveolae have any
significance for insulin signaling? If yes, then one would predict that
disruption of caveolae would inhibit insulin receptor function. As
noted, caveolae are highly enriched in cholesterol, and depletion of
cholesterol in the plasma membrane disrupts caveolae structures and
functions (6, 16, 45). Gustavsson et al. (15)
incubated 3T3-L1 adipocytes for 50 min with -cyclodextrin, which
binds cholesterol and extracts it from the plasma membrane. This
treatment resulted in dose-dependent effects, including depletion of
plasma membrane cholesterol and flattening of caveolar invaginations. With respect to insulin action,
-cyclodextrin treatment inhibited insulin-stimulated glucose uptake, downstream tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and protein kinase B
/Akt, and insulin-dependent binding of IRS-1 to the insulin receptor. Treatment with
-cyclodextrin had no significant effect on insulin binding to intact adipocytes or on levels of plasma membrane insulin receptor and caveolin. Nor did
-cyclodextrin affect
insulin-stimulated tyrosine-specific insulin receptor
autophosphorylation, IRS-1 serine/threonine phosphorylation, or
activation of the mitogen-activating protein (MAP) kinase pathway.
These investigators noted the similarities between these cell culture
results and the findings in many cases of clinical insulin resistance
(59), thereby suggesting a potential role for caveolae in
the pathogenesis of this disorder.
On what basis might the insulin receptor preferentially segregate into
caveolae as opposed to the rest of the plasma membrane? Some signaling
proteins interact with caveolin by direct protein-protein interactions.
These interactions involve the "caveolin scaffolding domain," a
short amino-terminal cytosolic domain of caveolin near the plasma
membrane (26), and hydrophobic motifs in the
caveolin-interacting proteins (X
XXXX
or
XXXX
XX
, where
is any aromatic amino acid) (8). The insulin receptor
contains this motif. Yamamoto et al. (62) prepared CMD
from Chinese hamster ovary (CHO) cells that overexpressed the insulin
receptor and IRS-1 by a detergent-free method. The insulin receptor,
but not IRS-1, cofractionated with caveolin-1. Expression of
caveolin-3, the muscle- and astrocyte-specific isoform, in HEK293T
cells enhanced insulin-stimulated tyrosine phosphorylation of IRS-1 at
1 and 5 min. This enhancement occurred without any effect of caveolin-3
expression on insulin receptor autophosphorylation. These authors
demonstrated a direct effect of caveolin scaffolding domains on insulin
receptor tyrosine kinase activity by use of in vitro assays. Briefly,
the cytoplasmic tyrosine kinase domain of the insulin receptor (BIRK)
and full-length IRS-1 were expressed in insect cells and subsequently
purified. These polypeptides were then incubated with peptides
corresponding to the scaffolding domains of caveolin-1, -2, and -3. There was a dose-dependent augmentation of IRS-1 tyrosine
phosphorylation by BIRK in the presence of caveolin-1 and -3 peptides
but not the caveolin-2 peptide. BIRK autophosphorylation was unaffected by caveolin peptides. Direct interaction between the insulin receptor and caveolins was further supported by the ability of immobilized caveolin-1 and -3 scaffolding peptides to bind BIRK in vitro. This
binding was eliminated specifically by coincubation with the potential
caveolin-binding domain of the insulin receptor (TTSSDMWSFGVVIWEITS, amino acids
1175-1192).
The ability of caveolins to modulate insulin receptor signaling in transfected cells was confirmed by Nystrom et al. (35). Although it was not shown in the article, these authors were able to coimmunoprecipitate endogenous insulin receptors with endogenous caveolin-1 in freshly isolated rat adipocytes. Overexpression in Cos-7 cells of wild-type caveolin-1 enhanced insulin-induced Elk-1 phosphorylation and inhibited extracellular signal-regulated kinase (ERK)2 phosphorylation, but mutant caveolin-1 with a disrupted scaffolding domain had no effect on insulin signaling. In rat adipocytes, on the other hand, an intact caveolin-1 scaffolding domain was not necessary to inhibit ERK2 phosphorylation or recruitment of epitope-tagged GLUT-4 to the cell surface. Thus it appears that some effects of caveolin on insulin signaling in some cell types may not depend on direct protein-protein interactions between these two molecules, at least via the caveolin scaffolding domain. The precise role that caveolins play in the regulation of insulin signaling requires further investigation.
An insulin-independent pathway has been described that mimics the metabolic actions of insulin in adipocytes (34). This pathway can be stimulated in adipocytes by exogenous phosphoinositolglycans (PIG), cleavage products of the glycosyl-phophatidylinositol anchor of GPI-linked proteins. PIG treatment of isolated rat adipocytes leads to dose-dependent increases in tyrosine phosphorylation of IRS-1, IRS-1-associated PI 3-kinase activity, and glucose uptake in an insulin-independent fashion. A critical mechanism in this pathway may involve the dissociation of the nonreceptor tyrosine kinases Lyn and Fak from caveolin-1, their activation by tyrosine phosphorylation, and their redistribution from rafts to nonraft membranes. Incubation of adipocytes with PIG, as with insulin, is followed by tyrosine phosphorylation of caveolin-1. The physiological role of this pathway and its potential cross talk with the insulin-signaling pathway in caveolae remain to be determined.
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LIPID RAFTS, FLOTILLIN-1, AND INSULIN SIGNALING |
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Independent of any direct role for caveolin proteins in insulin signaling, recent reports have suggested that lipid rafts and perhaps caveolae provide a necessary organizing principle for insulin-stimulated glucose transport in adipocytes. Baumann et al. (3) reported that resident lipid raft protein flotillin-1 recruits a complex of tyrosine-phosphorylated Cbl and Cbl-associated protein (CAP) to rafts, and that this recruitment is required for GLUT-4 translocation in response to insulin.
Flotillin-1 originally was identified as an integral membrane protein resident in CMD (4). White adipose tissue expresses more flotillin-1 RNA per microgram poly(A)+ than any other mouse tissue, followed by heart, brain, diaphragm, and lung (4). Flotillin-1 protein levels increase as 3T3-L1 cells progress through the adipocyte differentiation program, whereas levels of the related protein flotillin-2 (47% amino acid identity) do not change during adipogenesis. Like the caveolins, flotillin-1 and -2 are ancient genes with orthologs in mouse, rat, human, goldfish, and drosophila (11, 17, 25, 49). Whether the flotillins are resident in caveolae, in noncaveolar lipid rafts, or in both has not been determined by immunogold EM. Nevertheless, the weight of published data provides compelling evidence that flotillin-1 is a resident caveolar and lipid raft protein (4). First, by both detergent and detergent-free methods, flotillin-1 and -2 cofractionate with caveolin-1. Second, caveolae vesicles purified from silica-coated plasma membranes of rat lung endothelial cells contain almost all of the plasma membrane flotillins and caveolin-1. Third, in paraformaldehyde-fixed, Triton-permeabilized 3T3-L1 adipocytes, flotillin-1 has a largely overlapping distribution with caveolin-2 (unpublished data). Also, immunofluorescent staining of adipocyte plasma membrane sheets for flotillin-1 and caveolin-2 results in an overlapping pattern of doughnut-like structures (3) similar to those observed by Strålfors and colleagues (Gustavsson and co-workers, Refs. 14 and 15) by use of insulin receptor and caveolin-1 antibodies. Fourth, Volonté et al. (60a) have reported that, in A-498 kidney carcinoma cells, flotillin proteins form a stable hetero-oligomeric complex with caveolin-1 and that heterologous expression of flotillin-1 in insect cells was sufficient to lead to formation of caveolae-like intracellular vesicles. Because flotillin-1 is also present in the Triton-insoluble, buoyant membrane (DIG) fraction of brain gray matter and differentiated PC-12 cells, which lack classic caveolae, flotillin-1 also likely resides in noncaveolar lipid rafts (4).
In 3T3-L1 adipocytes, but not preadipocytes, Cbl is a substrate for
tyrosine phosphorylation in response to insulin and, once phosphorylated, associates with the adapter protein Crk and the Src
family kinase Fyn, as assessed by coimmunoprecipitation
(43). The significance of the insulin-dependent
interaction of Cbl with Fyn is unknown but may be important for
caveolin phosphorylation (30). Reasoning that the
adipocyte-specific, insulin-mediated phophorylation of Cbl might
involve an adipocyte-expressed adapter protein, Ribon et al.
(42) screened an adipocyte yeast two-hybrid library with
full-length Cbl as bait and identified Cbl-associated protein, or CAP,
as this protein. CAP exists as several different isoforms because of
alternative splicing. CAP RNA and protein are induced during 3T3-L1
adipocyte differentiation. Treatment of adipocytes with peroxisome
proliferator-activated receptor- (PPAR
) agonists induced
transcription of CAP RNA and resulted in increased CAP protein
(41). The predicted structure of CAP contains 3 SH3
domains in its carboxy-terminal half and a domain homologous to sorbin
(a porcine peptide hormone) in its amino-terminal half. CAP interacts
with Cbl via the most carboxy-terminal SH3 domain, and this association
is independent of insulin. One CAP isoform also interacts with the
insulin receptor, but this association diminishes with time upon
receptor activation by ligand. By 5 min into an insulin pulse, little
CAP coimmunoprecipitates with the insulin receptor.
What is the fate of the CAP-Cbl complex after it dissociates from the activated insulin receptor? Mastick and Saltiel (31) reported that, after insulin stimulation, tyrosine-phoshorylated Cbl (PY-Cbl) is present in both the Triton-soluble supernatant and Triton-insoluble pellet of 3T3-L1 adipocytes. Although the method used did not distinguish between enrichment of PY-Cbl in lipid rafts and some other detergent-insoluble subcellular compartment (e.g., cytoskeleton), the results were suggestive that lipid rafts were a possible destination. This possibility has now been confirmed. Again, by yeast two-hybrid screening, Baumann et al. (3) identified the lipid raft protein flotillin-1 as a CAP-interacting protein. In vitro experiments confirmed that immobilized flotillin-1 was able to specifically bind to epitope-tagged CAP in transfected HEK293T cells, as well as coprecipitate endogenous Cbl. By a nondetergent method to separate raft from nonraft proteins, almost all Cbl protein is present in nonraft fractions in the basal state. However, after insulin stimulation, Cbl rapidly accumulates in caveolin- and flotillin-enriched membrane fractions (caveolae/rafts). By immunofluorescence staining of adipocyte plasma membrane sheets, colocalization of flotillin-1 and Cbl was detectable by 2 min, maximal at 5 min, and undetectable by 15 min. A mutant CAP that lacked all SH3 domains was able to bind flotillin-1 but not Cbl. Overexpression of this mutant in 3T3-L1 adipocytes significantly inhibited insulin-stimulated enrichment of PY-Cbl in the Triton-insoluble pellet. Most significantly, insulin-stimulated glucose uptake and GLUT-4 translocation to the plasma membrane were also inhibited. Overexpression of the mutant CAP did not affect insulin-stimulated GLUT-1 translocation, insulin receptor autophosphorylation, or phosphorylation of the downstream targets IRS-1, Akt, or MAP kinase. Not all metabolic actions of insulin involve the flotillin-1/CAP/Cbl pathway. Only the glucose-dependent, but not the glucose-independent, pathways that regulate insulin-stimulated activation of glycogen synthase are inhibited by overexpression of the CAP dominant-negative mutant (2).
As noted above, the PI 3-kinase pathway is required for insulin-stimulated glucose transport in adipocytes but may not be sufficient. The data reported by Baumann et al. (3) point to the existence of another pathway that involves recruitment to lipid rafts of the CAP/PY-Cbl binary complex by flotillin-1. Because disruption of this recruitment inhibits insulin-stimulated glucose transport but not downstream effectors of PI 3-kinase, the flotillin-1/CAP/Cbl pathway appears to be a second independent and necessary pathway for this metabolic action of insulin.
But what is the role of PY-Cbl in the lipid raft, and why must it be there for proper insulin-regulated GLUT-4 translocation? Clues may lie in the proteins that are known to interact with PY-Cbl in other contexts. First, in macrophages, integrin-mediated cell adhesion promotes tyrosine phosphorylation of Cbl and complex formation between PY-Cbl and both Src and PI 3-kinase (37). Adhesion of macrophages to fibronectin results in translocation of Cbl to the plasma membrane and relative enrichment of PY-Cbl at that location. Whether this translocation is to lipid rafts is unknown. Might PY-Cbl associate with PI 3-kinase in insulin-stimulated adipocytes and thereby influence its activity? Prevention of PY-Cbl recruitment to rafts does not affect Akt phosphorylation, but it is not known whether it affects other downstream targets, such as atypical protein kinase C isoforms. Second, Cbl has ubiquitin ligase activity in some cell types. In rat basophilic leukemia cells, activation of IgE receptors results in recruitment of these receptors and Cbl into rafts (51). Cbl-mediated ubiquitination may target the receptor and other membrane proteins for endocytosis, and with subsequent recycling to endosomes or degradation in proteosomes. It is not known whether the ubiquitin ligase activity of Cbl modulates the insulin signal in adipocytes. In 3T3-L1 adipocytes, recruitment of PY-Cbl to lipid rafts after insulin receptor activation is accompanied by its association with Fyn, a Src-family tyrosine kinase of which caveolin is a potential substrate (31). The significance of this recruitment, like that of insulin-stimulated caveolin phosphorylation, is not known. Insulin also induces association of Cbl with CrkII in these cells (43), and this association is likely the critical one, as described below.
The best evidence for the function of Cbl in insulin-stimulated GLUT-4 translocation has come from parallel studies of the role of small GTP-binding proteins in this process. As molecular switches that have been shown to function in both signal transduction and vesicular trafficking, this class of proteins has received considerable attention in the quest for mediators that may connect insulin signaling to the GLUT-4 translocation machinery. Chiang et al. (7) surveyed several different classes of small GTPases for the ability of wild-type, dominant-negative, or constitutively active mutants to modulate insulin-stimulated glucose uptake and GLUT-4 translocation. Expression of wild-type TC10, a Rho-family GTPase, or its dominant-negative mutant TC10/T31N, in 3T3-L1 adipocytes inhibited this process but did not affect insulin-stimulated GLUT-1 translocation. Insulin stimulation of these cells resulted in activation (i.e., GTP loading) of TC10, with maximal activation by 5 min, and this activation was not blocked after inhibition of PI 3-kinase by wortmannin. Because TC10 is a constitutive lipid raft protein, these data suggested that TC10 may be activated downstream of the flotillin-1/CAP/Cbl pathway. This hypothesis was supported by demonstration that overexpression of the same dominant-negative CAP mutant that prevents PY-Cbl recruitment to rafts also inhibits TC10 activation in response to insulin.
How does PY-Cbl in rafts lead to TC10 activation? Here we return to a common theme in signal tranduction: protein phosphorylation generates docking sites for other signaling molecules. Just as insulin-stimulated tyrosine phosphorylation of Cbl creates a binding site for Fyn, so too a binding site for the SH2 domain of the adapter protein CrkII is formed. An SH3 domain of CrkII, in turn, binds C3G, a guanyl nucleotide exchange factor for Rap1. Following this clue, Chiang et al. (7) found that insulin treatment of 3T3-L1 adipocytes leads to the time-dependent recruitment of CrkII and C3G to caveolin- and flotillin-enriched membranes. This recruitment was blocked by expression of the dominant-negative CAP mutant. The authors also found that C3G functions as an exchange factor for TC10 in 3T3-L1 adipocytes. The relevance of these observations to insulin-stimulated glucose transport was supported by the finding that overexpression of C3G in adipocytes led to a four- to fivefold left shift in the insulin dose-response curve. Furthermore, whereas expression of the constitutively active p110 subunit of PI 3-kinase (p110CAAX) partially promoted translocation of GLUT-4 tagged with green fluorescent protein in the absence of insulin, coexpression of p110CAAX and C3G promoted approximately the same amount of translocation as that stimulated by insulin. Overexpression of C3G alone had no effect on basal glucose uptake or GLUT-4 localization to the plasma membrane. These data suggest that activation by insulin of the PI 3-kinase pathway and the flotillin-1/CAP/Cbl/CrkII/C3G/TC10 pathway may be sufficient for full insulin-stimulated GLUT-4 translocation in adipocytes.
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LIPID RAFTS AND GLUT-4 |
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If lipid rafts and caveolae are sites of insulin signaling, do these structures also participate in the downstream actions of insulin? Several laboratories have investigated whether there is overlap between membrane compartments that contain caveolin and GLUT-4. Using biochemical isolation techniques, Scherer et al. (46) detected a transient increase in GLUT-4 immunoreactivity in CMD after insulin stimulation of 3T3-L1 adipocytes. Also, insulin stimulation led to an increase in plasma membrane caveolin and a corresponding decrease in caveolin detected in low-density microsomes. These investigators detected caveolin in GLUT-4 vesicles immunoisolated from rat adipocytes. Kandror et al. (21) conducted similar studies but arrived at a different conclusion. They found that intracellular caveolin and GLUT-4 were detectable on vesicles of similar size and buoyant density and that these vesicles translocated to the plasma membrane in response to insulin. However, in contrast to the findings of Scherer et al., only limited amounts of caveolin protein were present in immunoabsorbed GLUT-4 vesicles, which suggested that the intracellular pools of these proteins, although biophysically similar, were distinct. Gustavsson et al. (15) isolated subcellular membrane fractions from rat adipocytes and performed glucose uptake assays on intact adipocytes at various times during an insulin pulse. As expected, after the addition of insulin, GLUT-4 increased in the plasma membrane (PM) fraction rapidly, so that 50% of maximum GLUT-4 in the PM was present by ~3 min. Intriguingly, insulin also led to a slower increase (half-time ~7 min) of GLUT-4 in the DIG (caveolin-enriched) membrane fraction that had been prepared from the PM fraction. The kinetics of glucose uptake more closely followed the time course of GLUT-4 appearance in the DIG fraction than in the PM fraction. The authors proposed a model such that, upon insulin stimulation, GLUT-4 moves first to the PM and then to detergent-resistant PM microdomains (caveolae?), where GLUT-4-mediated glucose transport occurs.
Immunoelectron microscopy data have not supported insulin-stimulated movement of GLUT-4 to caveolae. Voldstedlund et al. (60) measured the density of Na+-K+-ATPase subunits and of GLUT-1 and GLUT-4 in isolated rat adipocyte plasma membrane sheets. Immunogold labeling for GLUT-4 confirmed an increase in GLUT-4 density in the PM with insulin, but the GLUT-4 labeling was confined to the planar membrane and was not seen in morphologically defined caveolae. More recently, Malide et al. (28) labeled ultrathin cryosections of embedded white and brown rat adipocytes with anti-GLUT-4 immunogold particles. Insulin stimulation was associated with increased gold particles at the plasma membrane but not in caveolae specifically. The authors estimated that 2% of the GLUT-4 label was in caveolae but that 17% of the rat adipocyte PM consisted of caveolae.
How do we reconcile the biochemical data, some of which support movement of GLUT-4 to caveolae upon insulin stimulation, and the ultrastructural data, which do not? First, we must be clear as to terminology. The biochemical data are based on isolation of DIG fractions from either whole cells or plasma membrane, which cannot discriminate between caveolae and noncaveolar lipid rafts. The ultrastructural data can only distinguish between morphologically identified caveolae and the rest of the plasma membrane, which would include noncaveolar lipid rafts. So one possible explanation is that insulin leads to translocation of GLUT-4 from its intracellular storage depot to noncaveolar lipid rafts in the plasma membrane. Second, the discrepancy may be methodological; the two approaches are each subject to artifact. As a twelve-membrane-spanning protein, GLUT-4 may be intrinsically insoluble in detergent, thereby leading to spurious cofractionation with DIG membranes. If this is true, then one must posit that insulin stimulation promotes this insolubility by an unknown mechanism. Although immunoelectron microscopy is regarded by some experts as the "gold" standard for definitive localization of proteins to caveolae (55), cell fixation may lead to redistribution of proteins. Thus, it remains an open question whether insulin stimulation results in movement of GLUT-4 to lipid rafts, in general, to caveolae specifically, or simply to nonraft plasma membranes.
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SMOKE FROM THE CAVES![]() |
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Where there's smoke, there's fire. Perhaps not always, but the
data reviewed in this article support the notion that the smoke emerging from the "little caves" is generated by a physiological function for caveolae and rafts in insulin signaling, at least in
adipocytes, where these structures are abundant. As noted, many
discrepancies remain among data reported by different laboratories, and
many more are likely to emerge as more investigators enter this dynamic
field. It will be critical for all investigators to describe their
methods of raft/caveolae isolation and detection in meticulous detail
and to use positive and negative controls to establish the quality of
their preparations. For example, the literature contains examples in
which modifications are made to well characterized protocols for raft
isolation but putative isolated "raft" membranes are not shown to
be depleted of nonraft proteins. Also, whenever possible, more than one
method for determining localization to rafts or caveolae should be used
to eliminate technique-specific artifact. The strongest data combine
careful localization with physiological function. Does either
increasing or decreasing raft/caveolae localization of a specific
molecule or set of molecules by some experimental method perturb a cell function? One problem with the frequently used cholesterol depletion method (e.g., by -cyclodextrin) is that it is such a blunt
instrument: cholesterol depletion may have effects on other cellular
membrane compartments that are not specific to caveolae/raft
disruption. Finally, if the conclusion is drawn that two or more
potential raft proteins directly interact on the basis of their
coimmunoprecipitation with one another, then it is critical to
completely solubilize raft/caveolae structures (e.g., by addition of
octyl-glucoside to the lysis buffer) during cell lysis. If this is not
done, then coimmunoprecipitation may result merely from two proteins
being Triton insoluble and may have nothing to do with protein-protein interactions or even raft localization.
At present, the evidence favors the conclusion that at least a subset of plasma membrane insulin receptors reside in lipid rafts/caveolae. Furthermore, molecular components of insulin signaling also reside in or are recruited into these structures, and this recruitment is required for at least some insulin actions in adipocytes (insulin-stimulated glucose transport). The recent discovery of the flotillin-1/CAP/Cbl/CrkII/C3G/TC10 pathway has opened up a new area of investigation and has raised many questions. The data so far suggest that activation of TC10 in response to insulin is a major outcome of this pathway and that the role of caveolae/rafts in this pathway is to ensure the proper temporal-spatial arrangement of the molecular components of the pathway. But what happens after TC10 activation? What are the TC10 effector molecules in the context of GLUT-4 translocation? Because TC10 belongs to the Rho-family of GTPases, it is attractive to speculate along with Chiang et al. (7) that insulin-mediated TC10 activation is involved in cytoskeletal rearrangements necessary for GLUT-4 vesicle translocation, docking, or fusion. Another possibility proposed by Chiang et al. is that activated TC10 interacts with components of the SNARE machinery that participate specifically in GLUT-4 vesicle docking and fusion. Clearly, much remains to be discovered about the increasingly complex pathways that lead from insulin binding to insulin action.
This minireview has attempted to synthesize a growing literature about
the role of lipid rafts/caveolae in insulin signaling. Controversies
and agreements have been highlighted. The critical theme to emerge is
the importance of proper subcellular localization of signaling
molecules for their proper function. In many circumstances it may not
be enough to measure the effects of a treatment on expression or
even phosphorylation of a given target molecule. Rather, one must
also determine the effects of the treatment on where the target
molecule is in the cell, as well as when and how long it is there. Not
discussed in this minireview are potential ways in which rafts and
caveolae may also function in the negative regulation of insulin
signaling. For example, TNF--mediated apoptosis of U937
cells, a monoblastoid cell line, may be initiated in caveolae-like domains via the generation of ceramide from sphingomyelin
(23). A mechanism by which TNF-
inhibits insulin
signaling is by stimulation of sphingomyelinase activity
(39). A product of sphingomyelinase activity, ceramide, is
increased in the muscle of diabetic mice, and exogenous ceramide
inhibits insulin-stimulated GLUT-4 translocation and Akt
phosphorylation in adipocytes (52). Because of the
proximity of insulin signaling molecules in adipocyte caveolae/rafts,
perhaps the local production of ceramide in those structures regulates insulin's signal.
In conclusion, it must be acknowledged that the data summarized in this review are from either cultured cells or isolated adipocytes. It is not known whether lipid rafts/caveolae are involved in insulin-stimulated glucose uptake or exercise-induced glucose uptake in skeletal muscle, the major site of glucose disposal after a meal. Validation of the role of caveolae/rafts in vivo awaits the generation and study of animal models, such as gene knockouts of the key players. Also, whether variations in the expression, function, or localization of raft proteins such as caveolins and flotillins are associated with insulin resistance in animal models or in humans is not known. As of the writing of this review, no mutations in the genes that encode these molecules have been reported in insulin-resistant states.
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NOTE ADDED IN PROOF |
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Two independent groups, led by Lisanti and Kurzchalia, respectively, have reported the creation of caveolin-1 knockout mice, which are viable but have significant vascular dysfunction and hyperproliferation of endothelial cells and fibrosis in lung alveolar septae (Razani et al. J Biol Chem 276: 38121-38138, 2001, and Drab et al. Science 293: 2449-2452, 2001). Characterization of the metabolic phenotype of these mice promises to be instructive. Also, Gould and colleagues recently reported that flotillin-1 and caveolin-1 are overexpressed in the skeletal muscle of the hypertensive, insulin-resistant SHRSP rat strain vs. the normotensive, insulin-sensitive WKY strain (James et al. Diabetes 50: 2148-2156, 2001). It will be important to determine whether these differences in expression are accompanied by differences in the subcellular localization of these proteins and whether altered expression of flotillin-1 and caveolin-1 contributes to the insulin-resistant phenotype or is a compensatory response.
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ACKNOWLEDGEMENTS |
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I want to recognize the generous research support provided by the American Diabetes Association, the Monsanto-Pharmacia/Washington University Biomedical Research Program, the Washington University Diabetes Research and Training Center [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant 5P60 DK-20579], and the Washington University Clinical Nutrition Research Unit (NIDDK Grant 5P30 DK-56341).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. E. Bickel, Depts. of Medicine and of Cell Biology and Physiology, 660 S. Euclid Ave., Campus Box 8127, St. Louis, MO 63110 (E-mail: pbickel{at}im.wustl.edu).
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