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Spatial and temporal regulation of GLUT4 translocation by flotillin-1 and caveolin-3 in skeletal muscle cells

Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

³Correspondence: E-mail: feg5{at}pitt.edu

SPECIFIC AIMS

The aim of the present study is to investigate whether flotillin-1 and caveolin-3 coordinately modulate the insulin-stimulated signaling cascades that lead to the translocation of GLUT4 to the plasma membrane in skeletal muscle cells.

PRINCIPAL FINDINGS

1. GLUT4 moves together with flotillin-1 from intracellular stores to the plasma membrane upon insulin stimulation in skeletal muscle cells through PI3K- and PKC-dependent pathways
Differentiated skeletal muscle cells were subjected to immunofluorescent labeling using antibody probes against flotillin-1 and GLUT4. Figure 1 (upper panels) illustrates that the two proteins are colocalized to a perinuclear compartment in the absence of insulin. After insulin stimulation (160 nM for 10 min), flotillin-1 together with GLUT4 moved to the sarcolemma (Fig. 1 , lower panels). The translocation from a perinuclear compartment to the plasma membrane was PI3K- and PKC-dependent, as shown by the perinuclear localization of flotillin-1 and GLUT4 after insulin stimulation in the presence of wortmannin and protein kinase C pseudosubstrate inhibitor, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 1. Flotillin-1 and GLUT4 move to the sarcolemma in response to insulin in skeletal muscle cells. Skeletal muscle cells were differentiated for 5 days. Differentiated myotubes were left untreated or treated with insulin (160 nM for 10 min). Localization of flotillin-1 and GLUT4 was examined using anti-flotillin-1 and anti-GLUT4 IgGs, followed by incubation with fluorescent secondary antibodies.

2. Activation of the PI3K/Akt pathway occurs within 2 min after insulin stimulation and requires caveolin-3
To examine the time-course of activation of the PI3K/Akt pathway, we began to evaluate the phosphorylation of the insulin receptor after insulin stimulation. Phosphorylation of the insulin receptor occurred 2 min after insulin treatment. Five min after insulin stimulation, activation of the receptor remained elevated. Consistent with these data, stimulation with insulin for 2 min was sufficient to activate PI3K and Akt. Caveolin-3 expression was necessary to activate this pathway as demonstrated by the significant reduced activation of both PI3K and Akt after insulin stimulation in caveolin-3 null skeletal muscle cells.

3. Lack of caveolin-3 reduces glucose uptake by inhibiting the movement of GLUT4 to the sarcolemma
To further investigate the specific role of caveolin-3 in insulin signaling, we examined the effect of lack of caveolin-3 on insulin-stimulated GLUT4 translocation and glucose uptake by employing skeletal muscle cells derived from caveolin-3 null mice. We demonstrate that the ability of flotillin-1/GLUT4 domains to reach the plasma membrane after insulin stimulation was significantly compromised. In fact, while flotillin-1 and GLUT4 reached the sarcolemma in 68 ± 5% of control cells after insulin stimulation, flotillin-1/GLUT4-containing domains were found at the plasma membrane only in 18 ± 3% of caveolin-3 null cells. Both flotillin-1 and GLUT4 remained in a perinuclear region in the majority of caveolin-3 null cells (82±7%) upon insulin stimulation, in contrast to control cells (32±4%). In support to this finding, caveolin-3 null cells showed a drastic inhibition (68% vs. control cells) of insulin-mediated glucose uptake. In contrast, basal glucose uptake was not affected.

4. Caveolin-3 moves from the plasma membrane toward the cytoplasm upon insulin stimulation and temporarily interacts with flotillin-1/GLUT4-containing domains
We next asked whether the localization of caveolin-3 was affected by insulin in differentiated myotubes. We show in Fig. 2 that caveolin-3 moved from the sarcolemma (left panel) to the cytoplasm (right panel) upon insulin stimulation (160 nM for 10 min), as GLUT4 moved from perinuclear compartments (left panel) to the plasma membrane (right panel). Caveolin-3 and GLUT4 colocalized in the cytoplasm when the cells were treated with insulin for 5 min, suggesting that caveolin-3-containing domains, as they move away from the sarcolemma (arrowhead in Fig. 2 , middle panel), may temporarily interact with flotillin-1/GLUT4-containing domains, before they reach the sarcolemma (see arrows in Fig. 2 , middle panel).

View larger version (19K): [in this window] [in a new window]   Figure 2. Caveolin-3 moves away from the sarcolemma and temporarily co-localizes with GLUT4 after insulin stimulation. Skeletal muscle cells were differentiated for 5 days. Cells were left untreated (left panel), and treated with insulin (160 nM) for 5 (middle panel) and 10 (right panel) min. Cells were then incubated with antibody probes specific for caveolin-3 and GLUT4, followed by incubations with fluorescent secondary antibodies. Green: caveolin-3; red: GLUT4; yellow indicates colocalization between caveolin-3 and GLUT4.

Consistent with these findings, caveolin-3 and flotillin-1 did not interact in the absence of insulin and their interaction peaked 5 min after insulin stimulation. Their interaction returned to basal levels after 20 min.

5. Insulin promotes the movement of the insulin receptor from caveolin-3-containing domains to flotillin-1-containing domains after 5 min
The insulin receptor has been previously shown to coimmunoprecipitate with caveolin-3 in skeletal muscle tissue. As we observed interaction between caveolin-3-containing domains and flotillin-1/GLUT4-containing domains 5 min after insulin stimulation, we next asked whether the insulin receptor is part of the same protein complex with caveolin-3 before insulin stimulation, and can interact with flotillin-1 after insulin stimulation. Toward this end, cell lysates from differentiated myotubes were immunoprecipitated with anti-insulin receptor IgGs, and immunoprecipitates subjected to immunoblot analysis with antibody probes specific for caveolin-3 and flotillin-1. We demonstrated that the insulin receptor was mainly localized in caveolin-3-containing domains in the absence of insulin. After insulin stimulation, the insulin receptor moved from caveolin-3-containing domains to flotillin-1-containing domains.

6. The movement of Cbl and CrkII to flotillin-1-containing domains, as well as the activation of C3G, occurs 5 min after insulin stimulation
Because we have shown that the insulin receptor moved from caveolin-3-containing domains to flotillin-1-containing domains 5 min after insulin stimulation, and flotillin-1 is known to directly bind the Cbl-associated protein CAP in adipocytes, we next asked whether flotillin-1 mediates insulin-stimulated activation of the Cbl/C3G/TC10 pathway in skeletal muscle cells. Differentiated myotubes were immunoprecipitated using specific antibody probes directed against Cbl, CrkII, and C3G. Immunoprecipitates were then subjected to immunoblot analysis using anti-flotillin-1 IgGs. We found that both Cbl and CrkII temporarily moved into flotillin-1-containing domains 5 min after insulin stimulation. In contrast, the GDP/GTP exchange factor C3G was located in flotillin-1-containing domains before and after insulin stimulation. However, activation of C3G occurred 5 min after insulin treatment, which temporally matched the translocation of the insulin receptor, Cbl, and CrkII into flotillin-1-containing domains.

7. Disruption of flotillin-1-containing domains prevents the activation of C3G, movement of GLUT4 to the sarcolemma, and glucose uptake in response to insulin
To confirm a major role of flotillin-1 in activation of the Cbl/C3G/TC10 pathway and GLUT4 translocation after insulin stimulation, we disrupted flotillin-1-containing domains using the cholesterol-sequestering agent methyl-ß-cyclodextrin (MbetaCD). Treatment with MbetaCD resulted in the exclusion of both flotillin-1 and GLUT4 from DRMs in differentiated myotubes stimulated with insulin. Treatment with MbetaCD prevented also the interaction between the insulin receptor and flotillin-1 5 min after insulin stimulation.

Consistent with the idea that the insulin-promoted translocation of the insulin receptor to flotillin-1-containing domains is required for the consequent activation of the Cbl/C3G/TC10 pathway, we found that the insulin-stimulated activation of C3G is dramatically inhibited by the treatment with MbetaCD. Down-regulation of flotillin-1 expression by siRNA resulted in a virtually identical inhibition of the insulin-stimulated activation of C3G.

Disruption of flotillin-1-containing domains with MbetaCD prevented the translocation of both flotillin-1 and GLUT4 to the sarcolemma in response to insulin. In support to these data, insulin-stimulated glucose uptake was prevented by the treatment with MbetaCD.

CONCLUSIONS AND SIGNIFICANCE

The glucose transporter type-4 is found in intracellular stores before moving to the plasma membrane in response to insulin. However, the precise nature of these stores remains to be determined. We demonstrate here for the first time that GLUT4 localizes to flotillin-1-containing domains, which are concentrated in perinuclear regions in skeletal muscle cells under basal conditions. After insulin stimulation, GLUT4, together with flotillin-1, moves to the sarcolemma where glucose uptake takes place. Insulin fails to stimulate GLUT4 translocation to the plasma membrane and glucose uptake when flotillin-1-based domains are disrupted by a cholesterol-sequestering agent.

We also find that the insulin receptor, PI3K, and Akt are all activated within 2 min after insulin stimulation in skeletal muscle cells. Expression of caveolin-3 is necessary for activation of this pathway, as demonstrated by reduced activation of both PI3K and Akt in cells lacking caveolin-3 expression. We conclude that activation of the Ins-R/PI3K pathway occurs in caveolar membranes in skeletal muscle cells and is an early event. In support to these data, we show that flotillin-1/GLUT4-containing domains remain localized to a perinuclear compartment after insulin stimulation and insulin-stimulated glucose uptake is impaired in caveolin-3 null myotubes.

The role of the Cbl/C3G/TC10 pathway in the modulation of GLUT4 translocation in skeletal muscle cells remains to be fully understood. We demonstrate that c-Cbl and CrkII transiently move into flotillin-1-containing domains 5 min after insulin stimulation in differentiated myotubes. In contrast, the GDP/GTP exchange factor C3G, which activates the Rho family GTPase TC10, coimmunoprecipitates with flotillin-1 before and after insulin treatment. Phosphorylation has been previously shown to activate C3G. Phosphorylation of C3G transiently occurs 5 min after stimulation with insulin. Flotillin-1-containing domains are required for activation of this pathway as demonstrated by the dramatically reduced activation of C3G upon insulin stimulation when flotillin-1-based domains are disrupted by either a cholesterol-sequestering agent or flotillin-1 siRNA. Taken together, these results indicate that activation of the Cbl/C3G/TC10 pathway occurs in flotillin-1-based domains in skeletal muscle cells and is a late event (i.e., occurs 5 min after insulin stimulation).

How are the two pathways activated? It is believed that two separate pools of insulin receptor mediate activation of the PI3K/Akt and Cbl/C3G/TC10 pathways in adipocytes. Our results suggest for the first time that the same pool of insulin receptor may activate both pathways in a two-step event which is spatially and temporally regulated by caveolin-3 and flotillin-1 in skeletal muscle cells (Fig. 3 ). To our knowledge, this is the first example of functional cooperation between two different types of DRMs in the regulation of the same signaling pathway within the same cell type.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing the modulation of GLUT4 translocation by flotillin-1 and caveolin-3. Phase I (0–2 min). Upon insulin stimulation, the insulin receptor, which is localized into caveolar membranes, activates IRS1, which phosphorylates and activates PI3K, with the consequent production of PIP3. PIP3 serves as an allosteric regulator of PDK. PDK phosphorylates and activates Akt and PKC, which stimulate the movement of flotillin-1/GLUT4-containing domains from a perinuclear compartment toward the plasma membrane. Caveolin-3 expression is necessary for activation of both PI3K and Akt. Phase II (2–5 min). Caveolin-3-containing domains move from the sarcolemma to the cytoplasm, where they interact with flotillin-1/GLUT4-containing domains. The insulin receptor now moves from caveolin-3-containing domains to flotillin-1/GLUT4-containing domains, where it promotes recruitment of Cbl, and CrkII, as well as activation of C3G (indicated as C3G*). Flotillin-1 expression is necessary for activation of C3G. Activation of the flotillin-1/Cbl/C3G-dependent, but PI3K-independent, pathway finalizes the movement of flotillin-1/GLUT4-containing domains to the plasma membrane, where glucose uptake takes place. Caveolin-3 expression is also required for the insulin-dependent activation of p38 MAP kinase, which is necessary for glucose uptake.

FOOTNOTES

¹ These authors contributed equally to this work.

² Current address: Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4661fje;

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