Insulin-stimulated Interaction between Insulin Receptor Substrate 1 and p85α and Activation of Protein Kinase B/Akt Require Rab5*

Binding of insulin to the insulin receptor initiates a cascade of protein phosphorylation and effector recruitment events leading to the activation of multiple distinct signaling pathways. Previous studies suggested that the diversity and specificity of insulin signal transduction are accomplished by both subcellular localization of receptor and the selective activation of downstream signaling molecules. The small GTPase Rab5 is a key regulator of endocytosis. Three Rab5 isoforms (Rab5a, -5b, and -5c) have been identified. Here we exploited the RNA interference technique to specifically knock down individual Rab5 isoforms to determine the cellular function of Rab5 in distinct insulin signaling pathways. Small interference RNA against a single Rab5 isoform had no effect on protein kinase B (PKB)/Akt or MAPK activation by insulin in NIH3T3 cells overexpressing human insulin receptor. However, simultaneous knockdown of all three Rab5 isoforms dramatically attenuated PKB/Akt activation by insulin without affecting MAPK activation. This inhibition of PKB/Akt activation was because of the impaired interaction between insulin receptor substrate 1 and the p85α subunit of phosphatidylinositol 3-kinase. These results indicate a requirement of Rab5 in presenting p85 to insulin receptor substrate 1. Additional evidence supporting a role for Rab5 was suggested by studies with GAPex-5, a vps9 domain containing exchange factor. Down-regulation of GAPex-5 impaired insulin-stimulated PKB/Akt activation. Collectively, this study indicates the involvement of Rab5 in insulin signaling.

Both environmental and genetic factors contribute to the development of type 2 diabetes mellitus. Although detailed mechanisms underlying the development of this disorder remain unknown, impairment of insulin action has been clearly established as an early defect in the pathogenesis of type 2 diabetes (1,2). Binding of insulin to the insulin receptor (IR) 2 causes receptor autophosphorylation and activation of its intrinsic kinase activity. Activated IR phosphorylates a variety of intracellular substrates, most notably the insulin receptor substrate (IRS) proteins (3,4). Tyrosine phosphorylation of these proteins provides binding sites for downstream effectors with Src homolog 2 domains or phosphotyrosine binding domains that activate multiple signaling pathways required for insulin-stimulated glucose uptake and maintenance of energy homeostasis (5,6). Type 1A phosphatidylinositol 3-kinase (PI3K) represents a primary target of insulin action (7). Phosphorylation of IRS1 and/or IRS2 on tyrosine residues by activated IR creates recognition sites for the p85 regulatory subunit of PI3K, thereby allowing the activation of the p110 catalytic subunit (8). PI3K-generated phosphoinositides PI(3,4,5)P 3 and PI(3,4)P 2 recruit protein kinase B (PKB)/Akt and 3-phosphoinositide-dependent kinase to the membrane, resulting in PKB/Akt phosphorylation and activation (9 -11). Recent studies also demonstrated a second requisite pathway that involves recruitment of tyrosine-phosphorylated Cbl to lipid rafts and activation of the lipid raft-associated small G protein TC10 in differentiated 3T3-L1 adipocytes (1,12,13). Synergistic cooperation of these two signaling pathways leads to the full activation of GLUT4 translocation to the plasma membrane (PM) and glucose uptake in adipocytes (1,12,13).
After binding to insulin, the activated receptor complex is internalized into endosomes from which receptors are either recycled back to the PM or transported to lysosomes for degradation (14,15). Thus, receptor internalization represents a mechanism for insulin degradation and receptor deactivation. However, receptor internalization may be required for full activation of receptor signaling because internalization may be important for activated receptors to access their downstream effectors, which may not reside at the PM/cytosol interface (16). Previous studies using various models have suggested that insulin stimulation results in the endosomal accumulation and concentration of IR kinase that is both tyrosine-phosphorylated and active toward exogenous substrates (17,18). It has also been shown that endosomes are important sites for IRS-1 phosphorylation and PI3K activation in rat adipocytes and 3T3-L1 adipocytes (19,20). Subcellular fractionation studies have demonstrated that insulin causes a small increase in the level of p85␣ in the PM-containing fraction but a much larger increase in the microsomal fraction (21). Collectively, these studies suggest the functional importance of spatial compartmentalization exhibited by signaling molecules in insulin signaling and indicate that trafficking through the endosomal apparatus might be important for proper insulin action. However, expression of a GTPase-deficient dynamin (K44A) decreases the internalization of IR in adipocytes but has no effect on both insulin-stimulated Akt and MAPK activation (22). On the contrary, K44A/ dynamin partially inhibits insulin-stimulated MAPK activation without affecting Akt activation in hepatoma cells (23). These results suggest that full activation of distinct insulin signaling pathways may require different thresholds for the amounts of internalized IR in different biological environments.
Rab5 is a protein localized to the PM, clathrin-coated vesicles, and early endosomes and is ubiquitously expressed (24). Rab5 has been implicated in membrane receptor internalization and in cytoskeletal-dependent organelle movement (25,26). GTP-Rab5 also coordinates endosome fusion by interacting with several Rab5 effectors such as PI3Ks, Rabaptin-5, EEA-1, and Rabenosyn-5 (27,28). Recently, Rab5 has been shown to be a key regulator connecting the lipid raft-dependent endocytosis and the clathrin-dependent endocytosis pathways (29). The presence of a subgroup of three Rab5 proteins (Rab5a, Rab5b, and Rab5c) in mammalian cells has been demonstrated from yeast to mammals. Original studies demonstrated that the three Rab5 isoforms share similar subcellular localization and regulatory functions in the early endocytic pathway (30). Although they are similar functionally, the three Rab5 proteins are differentially phosphorylated in vitro (31). Down-regulation of Rab5 isoforms utilizing RNA interference (RNAi) suggested that down-regulation of a single Rab5 isoform has no effect on either EGF or transferrin (Tfn) internalization (32). However, down-regulation of all three Rab5 isoforms significantly decreased the internalization of both EGF and Tfn (32). Experiments utilizing dominant negative Rab5A suggest a requirement for Rab5 activity in both IR and EGF internalization (14,33).
Rab5A interacts directly with two distinct PI3Ks, type I PI3K and hVps34 (34). More recent research showed that p85␣ binds to both GDP and GTP forms of Rab5A and that it functions as a GTPase-activating Protein for Rab5A (35)(36)(37). Identification of PI4-, and PI5-phosphatases as novel Rab5 effectors reinforced the involvement of Rab5A in phosphoinositide turnover in the endocytic pathway (29). Because temporal and spatial distribution of phosphoinositides is critical for insulin-stimulated PKB/ Akt activation, Rab5 and its effectors have emerged as important regulators of insulin action. Here we employed RNAi to examine the effects of Rab5 on insulin signaling in NIH3T3/hIR cells.
Cell Culture of NIH3T3/hIR Cells and Stimulation with Insulin-NIH3T3/hIR cells were cultured in DMEM containing 10% fetal calf serum, 100 units/ml penicillin G sodium, and 100 g/ml streptomycin sulfate at 37°C with 5% CO 2 . Cells were starved by incubation in DMEM without serum overnight. Starved cells were stimulated with pre-warmed DMEM with the indicated concentrations of insulin for 10 min at 37°C.
Protein Extraction, Immunoprecipitation, and Western Blot-Cell monolayers were washed twice with ice-cold phosphatebuffered saline and lysed at 4°C for 30 min with a buffer containing 50 mM Tris⅐HCl, pH 7.6, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1% protease inhibitor mixture solution (Sigma). The cell lysates were clarified by centrifugation at 10,000 ϫ g for 10 min at 4°C prior to separation by SDS-PAGE. The resolved proteins were transferred to nitrocellulose membranes, and the membranes were blocked with powdered milk solution (5% (w/v)). Individual proteins were detected with specific antibodies and visualized by blotting with horseradish peroxidase-conjugated secondary antibodies as described. For immunoprecipitation, 100 g of clarified lysate protein was incubated with antibodies (0.5 g) overnight at 4°C. The immune complexes were precipitated with protein G-agarose (Sigma) for 1 h at 4°C, and the beads were washed extensively with lysis buffer before solubilization in SDS sample loading buffer.
Horseradish Peroxidase Uptake in NIH3T3/hIR Cells-NIH3T3/hIR cells cultured in 6-well plates were transfected with siRNA and starved as described above. The cells were incubated for 5 min at 37°C in 1 ml of DMEM in the presence or absence of 10 nM insulin. Horseradish peroxidase (HRP) uptake was initiated by adding 1 ml of DMEM containing 4 mg/ml HRP and 1% bovine serum albumin. The uptake was carried out at 37°C for 30 min. The cells were washed and lysed, and the resulting cell lysate was assayed for HRP activity as described previously (38).
Biotinylation and Internalization of Cell Surface Insulin Receptor-Cell surface insulin receptor was labeled with biotin as described (39). Briefly, confluent NIH3T3/hIR cells pretreated with siRNAs were washed three times with ice-cold PBS-Ca-Mg (PBS, pH 7.4, 0.1 mM CaCl 2 , 1 mM MgCl 2 ) followed by incubation with a solution containing 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce) in PBS-Ca-Mg at 4°C for 30 min. The reaction was quenched by washing the cells three times with ice-cold PBS-Ca-Mg containing 15 mM glycine. The cells were then incubated in binding buffer (120 mM NaCl, 1.2 mM MgSO 4 , 2.5 mM KCl, 15 mM NaAc, 10 mM glucose, 1 mM EDTA, 50 mM HEPES, pH 7.8, 10 mg/ml BSA) in the absence or presence of 10 nM insulin for 20 min at 37°C. The cells were then washed with ice-cold PBS and treated with cold DMEM containing HEPES (20 mM, pH 7.4) and Pronase (Roche Applied Science) for 1 h at 4°C. Cells were then washed three times with cold PBS containing BSA (10 mg/ml) to quench the Pronase activity. The collected cell pellets were lysed as described above, and the biotinylated proteins were purified using Mag-naBind TM streptavidin beads (Pierce) as described in the product instructions.
Protein Extraction from White Adipose Tissue of Zucker Rats-Female obese Zucker (fa/fa) rats and lean congenic controls (5-6 weeks old) were housed and maintained with a 12-h light/ 12-h dark photoperiod. Animals were sacrificed (asphyxiated by CO 2 ), and inguinal fat pads (WAT) were collected as described (40). Proteins from WAT were extracted as described previously (40) and stored at Ϫ70°C until used for Western blot analysis.

RESULTS
siRNAs against Rab5 Attenuate Akt Activation by Insulin without Affecting MAPK Activity-Previous studies suggested that Rab5 might interact with PI3Ks and thus mediate insulin signaling (34,35). To determine whether specific isoforms of Rab5 affect insulin signaling, siRNAs directed against Rab5a, -5b, and -5c were synthesized. siRNA duplexes were transfected into NIH3T3/hIR cells. Forty eight hours later, the cells were lysed, and the extent of Rab5 silencing was quantitated by Western blotting. The siRNAs effectively depleted the appropriate Rab5 isoform by over 90% of levels observed in the scrambled negative control siRNA-transfected cells (Fig. 1A). No off-target silencing was observed (Fig. 1A). To verify the specificities of the Rab5 antibodies used in this study, Western blots were carried out on purified His tag Rab5a, -5b, and -5c proteins. The results in Fig. 1B showed that the antibodies specifically detected their target proteins.
Utilizing RNAi-mediated depletion of Rab5 GTPases, we examined whether Rab5 GTPases are required for insulin-stimulated activation of PKB/Akt and MAPK. NIH3T3/hIR cells were treated with siRNA against Rab5a, -5b, or -5c prior to starvation and insulin stimulation. Total cell lysates were prepared in the presence of protease and phosphatase inhibitors and subjected to Western blot analysis. Knockdown of a single Rab5 isoform had no significant effects on insulin-stimulated PKB/Akt activation ( Fig. 2A). Moreover, insulin-stimulated MAPK activation was also not affected by siRNA against a single isoform of Rab5 ( Fig. 2A). A recent study demonstrated that Rab5a is redundant, and inhibition of EGF and Tfn required the depletion of all three Rab5 isoforms (32). Accordingly, NIH3T3/hIR cells were treated with a nucleotide duplex pool of siRNAs against all three Rab5 isoforms. Interestingly, depletion of all three Rab5 isoforms dramatically decreased insulin-stimulated PKB/Akt activation (Fig. 2, B and C). This effect was likely not due to the regulation of the IR expression level because Rab5 depletion did not alter the total amount of IR (Fig. 3). On the contrary, this result predicts a role for Rab5 in the phosphorylation cascade of insulin signaling. To reveal whether Rab5 is required for different insulin signaling pathways, the effect of Rab5 depletion on MAPK activation was studied. Interestingly, depletion of all three Rab5 isoforms did not decrease insulin-stimulated MAPK activation, but slightly increased MAPK activation under basal conditions in the absence of insulin (Fig. 2B). Collectively, these results demonstrate that Rab5 has distinct effects on different insulin-signaling pathways. These results also suggest the cooperative and redundant regulation of insulin signaling by three Rab5 isoforms. Rab5 Is Required for Insulin-stimulated Association of IRS1 and p85␣ but Not the Phosphorylation of IR nor IRS1-Binding of insulin to IR results in the auto-phosphorylation of IR, which is followed by the recruitment and phosphorylation of IRS1. To explore the mechanism underlying Rab5-mediated PKB/Akt activation by insulin, the effects of Rab5 on insulin-stimulated phosphorylation of IR and IRS1 were examined. NIH3T3/hIR cells, pretreated with scrambled negative control siRNA or siR-NAs against Rab5a, -5b, and -5c, were starved and treated with 10 nM insulin for 10 min. Total lysates were prepared, and the IR or IRS1 proteins were immunoprecipitated. The resulting immunoprecipitates were subjected to Western blot analysis using a phosphotyrosine antibody. Elimination of Rab5 proteins did not alter the phosphorylation of IR or IRS1 by insulin (Fig. 3), suggesting that the target of Rab5 lies downstream of IRS1 phosphorylation.
Recruitment of the p85 subunit of type I PI3K to IRS1 represents a crucial step for the activation of PI3K by insulin. It was therefore of interest to test whether IRS1 was immunoprecipitated from cell lysates, and the resulting immunoprecipitates were subjected to Western blot using an antibody against p85␣. siRNAs against Rab5 significantly decreased the amount of p85␣ that was pulled down by IRS1 (Fig. 4). Reciprocally, downregulation of all Rab5 isoforms decreased the amount of IRS1 that was co-immunoprecipitated with p85␣ using anti-p85␣ antibody (Fig. 4). These results demonstrate that the insulinstimulated association between IRS1 and p85␣ requires Rab5. The data suggest that the impaired association between IRS1 and p85␣ results in decreased PKB/Akt activation.
GAPex-5 Contributes to Rab5-mediated PKB/Akt Activation by Insulin-Previous studies suggested that a pool of IRS1 proximal to plasma membrane is important for downstream insulin signaling, whereas the cytosolic pool is totally dispensable (41). Because our results indicate that the recruitment of p85 to IRS1 requires Rab5, a pool of Rab5 localized proximal to the PM might be activated by insulin treatment. Rab5 can be activated The whole cell lysates were prepared and subjected to immunoblot (IB) analysis with antibodies directed against each specific protein as indicated. A, individual Rab5 isoform (5A, 5B, or 5C) was down-regulated in NIH3T3/hIR cells. Cells were stimulated with insulin, and whole cell lysates were subjected to immunoblot analysis utilizing antibodies recognizing pAkt (Ser-473) or pMAPK. B, all three Rab5 isoforms (5A, 5B, and 5C) were down-regulated in NIH3T3/hIR cells. Cells were stimulated with insulin, and whole cell lysates were subjected to immunoblot analysis utilizing antibodies recognizing pAkt (Ser-473), total Akt, pMAPK, and total MAPK. C, quantification of the mean Ϯ S.E. of three independent experiments. Akt phosphorylations were normalized to control with 10 nM insulin. *, phosphorylation levels differed significantly ( p Ͻ 0.01) compared with insulin-stimulated control samples. An unpaired Student's t test was used in all statistical analyses. FIGURE 3. Down-regulation of all three Rab5 isoforms has no effect on insulin-stimulated tyrosine phosphorylation of IR and IRS1. NIH3T3/hIR cells were transfected with negative control (NC ) siRNA or siRNAs against Rab5 (5A, 5B, and 5C ) as indicated. Cells were starved and stimulated with 10 nM insulin at 37°C for 10 min as described under "Experimental Procedures." The total lysates were prepared, and the IR or IRS1 proteins were immunoprecipitated. The resulting immunoprecipitates (IP) or whole cell lysates (WCL) were subjected to immunoblot (IB) analysis utilizing antibodies as indicated. The experiment was repeated three times with similar results. SEPTEMBER 22, 2006 • VOLUME 281 • NUMBER 38 by various exchange factors, including Rin1 (42), Alsin (ALS2) (43,44), and Rabex-5 (45). Rin1 and ALS2 are absent in NIH3T3 fibroblasts, and Rabex-5 is likely localized in the intracellular endosomes (43,(45)(46)(47). Depletion of Rabex-5 has no effect on insulin signaling in NIH3T3/hIR cells (data not shown). Thus, there are likely other Rab5 exchange factors contributing to insulin-stimulated Rab5 activation. GAPex-5, a recently identified vps9 domain containing exchange factor (Fig. 5A), has been shown to be recruited to PM by CIP4 and to activate Rab5a on the PM upon insulin stimulation in 3T3-L1 adipocytes. 3 Moreover, a Caenorhabditis elegans homolog of GAPex-5, rme-6, has been shown recently to localize to PM and be a regulator of Rab5 at the clathrin-coated pit (48). We therefore predicted that GAPex-5 might be involved in Rab5-mediated PKB/Akt activation after exposure to insulin. Accordingly, we explored the effects of GAPex-5 on insulin-stimulated PKB/Akt activation by employing RNAi-mediated gene silencing. siRNA against GAPex-5 decreased its protein expression level with over 90% efficiency (Fig. 5B). Interestingly, down-regulation of GAPex-5 dramatically blocked the activation of PKB/Akt when the cells were exposed to 0.1 nM insulin (Fig. 5, C and D). Surprisingly, 10 nM insulin almost overcame the inhibitory effect of GAPex-5 (Fig. 5, C and D). However, MAPK activation by insulin was not affected by GAPex-5 down-regulation at either 0.1 or 10 nM insulin (Fig. 5, C and D). These results suggested that Rab5 activation on the PM by GAPex-5 contributed to insulin-stimulated Akt activation. Rab5 is localized at multiple sites of the endocytic pathway, including the PM, clathrin-coated vesicles, endocytic vesicles, and early endosomes. Our results indicate that at least one pool of Rab5 is activated upon insulin stimulation, and this activation is required in the insulin signaling pathway leading to PKB/Akt activation.

Rab5 Regulates Insulin Signaling
Insulin-stimulated Fluid Phase Endocytosis Requires Rab5 and GAPex-5-Previous studies demonstrated that fluid phase endocytosis (as measured by HRP uptake) was activated by insulin in multiple insulin-responsive cell lines (49). Moreover, the active form of Rab5a, but not other Rab GTPases, induced . Down-regulation of all three Rab5 isoforms impair insulinstimulated interaction between IRS1 and p85␣ subunit of PI3K. NIH3T3/ hIR cells were transfected with negative control (NC) siRNA or siRNAs against Rab5 (5A, 5B, and 5C) as indicated. Cells were starved and stimulated with 10 nM insulin at 37°C for 10 min as described under "Experimental Procedures." The total lysates were prepared, and the IRS1 or p85␣ proteins were immunoprecipitated. The resulting immunoprecipitates (IP) or whole cell lysates (WCL) were subjected to immunoblot (IB) analysis utilizing indicated antibodies. The experiment was repeated twice with similar results.

FIGURE 5. Down-regulation of GAPex-5 inhibits insulin-stimulated PKB/ Akt activation without affecting MAPK activation.
A, domain structure of mammalian GAPex-5. B, NIH3T3/hIR cells were transfected with negative control (NC) siRNA or siRNAs against GAPex-5 (G5) as indicated. The whole cell lysates were prepared and subjected to immunoblot (IB) analysis utilizing antibodies as indicated. C, after siRNA treatment, NIH3T3/hIR cells were starved and stimulated with insulin at indicated concentrations as described under "Experimental Procedures." The whole cell lysates were prepared, and immunoblot analysis was performed utilizing antibodies recognizing pAkt (Ser-473), total Akt, pMAPK, and total MAPK. D, quantification of the mean Ϯ S.E. of three independent experiments. Akt phosphorylations were normalized to control with 10 nM insulin. *, phosphorylation levels differed significantly ( p Ͻ 0.01) compared with insulin-stimulated control samples. An unpaired Student's t test was used in all statistical analyses. fluid phase endocytosis, whereas its dominant negative isoform had the opposite effect (38). When HRP uptake in NIH3T3/hIR cells stimulated with insulin was examined, we found that it was dose-dependent, peaking at a concentration of 10 nM (Fig. 6A). To confirm the involvement of Rab5 proteins in insulin-stimulated HRP uptake, all three isoforms of Rab5 were depleted followed by measurement of HRP uptake. Depletion of Rab5 decreases the basal HRP uptake and totally blocks the insulinstimulated HRP uptake (Fig. 6B). This result suggests that insulin activates Rab5, which is required for HRP uptake. Next, we examined whether GAPex-5 was required for insulin-stimulated HRP uptake. GAPex-5 was depleted in NIH3T3/hIR cells prior to insulin stimulation, and the HRP uptakes were measured in the presence or absence of insulin. Down-regulation of GAPex-5 had no effect on HRP uptake in the absence of insulin (Fig. 6B). Insulin increased HRP uptake by 90% in NIH3T3/hIR cells, which has been transfected with negative control RNA duplexes (Fig. 6B). Interestingly, depletion of GAPex-5 dramatically decreased insulin-induced HRP uptake (Fig. 6B). It seems likely that a pool of Rab5 is activated by insulin on or near the PM that is important for Akt activation by insulin.
Ligand-stimulated Insulin Receptor Endocytosis Requires Rab5 and GAPex-5-Rab5 plays a central role in internalization of receptor tyrosine kinases and G protein-coupled receptors by regulating the formation and dynamics of early endosomes, the fusion of endocytic vesicles, and their movements along microtubules (50,51). Insulin receptors were found to be internalized upon insulin binding, and the rate of internalization was decreased by overexpression of the dominant negative form of Rab5a (14). Therefore, internalized IR was analyzed as described under "Experimental Procedures." At 37°C, insulin receptors were internalized in the absence of insulin, whereas no internalized insulin receptors were detected at 4°C (Fig. 7A). Insulin dramatically increased the rate of receptor internalization at 37°C. Maximal internalization of IR was achieved at 10 nM insulin (Fig. 7A). To study the involvement of Rab5 and GAPex-5 on insulin receptor internalization, all three Rab5 isoforms or GAPex-5 was depleted prior to the examination of receptor internalization. Consistent with previous results utilizing dominant negative Rab5A (14), depletion of Rab5 iso-FIGURE 6. Down-regulation of Rab5 or GAPex-5 inhibits insulin-stimulated horseradish peroxidase uptake. A, NIH3T3/hIR cells were starved and incubated for 5 min at 37°C in DMEM in the presence or absence of the indicated concentrations of insulin. HRP uptake was measured as described under "Experimental Procedures." The results represent mean Ϯ S.E. of three independent experiments. B, NIH3T3/hIR cells were transfected with negative control (NC) siRNA, siRNAs against Rab5A, -5B, and -5C (R5), or siRNA against GAPex-5 (G5). Cells were starved and incubated for 5 min at 37°C in DMEM in the presence or absence of 10 nM insulin. HRP uptake assays were performed as described above. The results represent mean Ϯ S.E. of three independent experiments. FIGURE 7. Down-regulation of Rab5 or GAPex-5 inhibits insulin-stimulated receptor internalization. Cell surface insulin receptor was labeled with biotin as described under "Experimental Procedures." The cells were then incubated with Pronase (2.5 mg/ml) and washed three times with cold PBS containing BSA (10 mg/ml). The biotinylated proteins were purified as described under "Experimental Procedures." The bound proteins were separated by SDS-PAGE and subjected to immunoblot (IB) analysis utilizing an antibody recognizing the ␤-subunit of insulin receptor. A, NIH3T3/hIR cells were treated with the indicated concentrations of insulin at 37°C for the indicated time. Purified biotinylated proteins were analyzed using antibody recognizing the ␤-subunit of insulin receptor. B, NIH3T3/hIR cells were pretreated with negative control (NC) siRNA or siRNAs against Rab5A, -5B, and -5C (R5) or siRNA against GAPex-5 (G5). The cells were incubated at 37°C for 20 min after biotin labeling. Purified biotinylated proteins were analyzed utilizing antibody recognizing the ␤-subunit of insulin receptor. Whole cell lysates (WCL) were analyzed utilizing antibody recognizing tubulin. The experiment was repeated three times with similar results.
forms dramatically decreased insulin-stimulated internalization (Fig. 7B). Interestingly, down-regulation of GAPex-5 also decreased insulin-stimulated receptor internalization (Fig. 7B). These results reinforce the conclusion that GAPex-5 activates a pool of Rab5, which is required for both fluid phase and receptor-mediated endocytosis.
Depletion of Dynamin 2 Blocks Insulin Receptor Internalization but Not Insulin Signaling-A previous study showed that depletion of dynamin 2 decreased the internalization of EGF and Tfn (32). To illustrate whether Rab5-mediated insulin signaling is dependent on receptor internalization, dynamin 2 was depleted in NIH3T3/hIR cells to block IR internalization. The selected siRNA produced over 90% depletion of dynamin 2 (Fig.  8A) and dramatically inhibited insulin-stimulated receptor internalization (Fig. 8B). Blockage of IR internalization by dynamin 2 depletion has no effect on both insulin-stimulated Akt/PKB and MAPK activation. This result suggests that Rab5-mediated insulin signaling does not depend on receptor internalization.

Down-regulation of Rab5 Isoforms in WAT of Obese Zucker
Rats-Impaired activation of insulin-stimulated Akt/PKB by depletion of Rab5 GTPases and GAPex-5 suggests that they may be involved in the development of obesity. Accordingly, we investigated the modulation of Rab5 expression levels in Zucker (fa/fa) obese rats. 5-Week-old female lean and homozygous obese rats were fed ad libitum. Animals were sacrificed, and inguinal fat pads (WAT) were removed for protein extraction. Protein extracts were analyzed for alterations in the expression of Rab5 by immunoblot analysis. Interestingly, Western blots of Rab5a, -5b, and -5c showed the dramatic decrease of all three isoforms of Rab5 in obese animals compared with their congenic lean controls in white adipose tissue (Fig. 9). Collectively, these results demonstrated that Rab5 is less abundant in a commonly utilized genetic model of diabetes and obesity.

DISCUSSION
This study provides several lines of evidence that Rab5 is required for insulin-stimulated PKB/Akt activation. First, although siRNA down-regulation of a single Rab5 isoform had no effect on either PKB/Akt or MAPK activation by insulin, depletion of all three Rab5 isoforms impaired insulin-stimulated PKB/Akt activation without affecting MAPK activation. These results indicate that individual Rab5 isoforms are redundant, consistent with previous results that depletion of all three Rab5 isoforms is necessary to inhibit Tfn and EGF internalization (32). It is also consistent with the cooperative regulatory role of Rab5 isoforms in the early endocytic pathway (30). Second, down-regulation of all three isoforms of Rab5 decreased the insulin-dependent association between IRS1 and the p85␣ subunit of PI3K, indicating Rab5 may be required to recruit p85␣ to tyrosyl-phosphorylated IRS1. Third, elimination of GAPex-5, a Rab5 exchange factor containing a vps9 domain, also desensitized insulin-stimulated PKB/Akt activation. Fourth, insulin-stimulated HRP uptake, a process mediated by Rab5 activity (38), was dramatically blocked by depletion of GAPex-5. Fifth, insulin-stimulated IR internalization, another process mediated by Rab5 activity (8), FIGURE 8. Down-regulation of dynamin 2 blocks insulin receptor internalization but not insulin signaling. A, NIH3T3/hIR cells were transfected with negative control (NC) siRNA or siRNAs against dynamin 2 (Dy2) as indicated. Whole cell lysates were separated by SDS-PAGE and subjected to immunoblot analysis utilizing antibodies recognizing dynamin 2 and tubulin. B, NIH3T3/hIR cells were pretreated with negative control siRNA or siRNAs against dynamin 2. The cells were incubated at 37°C for 20 min after biotin labeling. Purified biotinylated proteins were analyzed utilizing antibody recognizing the ␤-subunit of the insulin receptor. Whole cell lysates (WCL) were analyzed utilizing antibody recognizing tubulin. C, after siRNA treatment, NIH3T3/hIR cells were starved and stimulated with insulin at the indicated concentrations as described under "Experimental Procedures." The whole cell lysates were prepared, and immunoblot (IB) analysis was performed utilizing antibodies recognizing pAkt (Ser-473), total Akt, pMAPK, and total MAPK. The experiment was repeated twice with similar results. FIGURE 9. Down-regulation of Rab5 isoforms in Zucker (fa/fa) obese rats. 5-Week-old female lean and homozygous obese rats were fed ad libitum. Animals were sacrificed, and inguinal fat pads (WAT) were removed for protein extraction as described under "Experimental Procedures." 10 g of proteins were separated by SDS-PAGE and analyzed by immunoblot (IB) with antibodies recognizing Rab5a, -5b, -5c, or tubulin. was also dramatically decreased by depletion of GAPex-5. Collectively, these results suggest that full activation of PKB/Akt by insulin requires Rab5 and that insulin-stimulated activation of Rab5 is mediated by GAPex-5.
The dynamics of insulin signaling has been extensively studied by a variety of approaches over the last 2 decades. James and co-workers (52) provided evidence to suggest that a cytoskeleton-associated intracellular endosome (low density membrane) fraction was enriched in phosphorylated IR and IRS1. Previous studies suggested that the pleckstrin homology domain of IRS1 plays a critical role in localizing PI3K to a particulate fraction of the cell, possibly associated with cytoskeletal elements that are necessary for PKB/Akt activation (53). The importance of membrane-bound IRS1 is reinforced by an in vitro study showing that the cytosolic fraction of IRS1 is dispensable for insulinstimulated PKB/Akt activation (41). Moreover, release of IRS proteins from an intracellular complex coincides with the development of insulin resistance (54). At 4°C, insulin-stimulated tyrosine phosphorylation of IRS1 was as intense as that observed at 37°C (19), indicating that IR internalization was not required for phosphorylation of IRS1 by insulin. A study utilizing dominant negative dynamin to block insulin receptor internalization also suggested that IRS1 was properly phosphorylated without insulin receptor internalization (23). This is consistent with our result in that down-regulation of Rab5, whose activity mediates IR internalization (14), has no effect on insulin-stimulated IRS1 phosphorylation. One possible explanation of how cytoskeleton-associated IRS1 is phosphorylated by IR on the PM before internalization is that IRS1-containing cytoskeletal complexes are located in close proximity to the PM. This model is reinforced by our results that inhibition of IR internalization by depletion of dynamin 2 has no effect on insulin signaling in NIH3T3/hIR cells (Fig. 8).
Upon tyrosyl phosphorylation of IRS1 by IR, the p85 subunit of PI3K is recruited to the membrane, which leads to the activation of PI3K (8). Insulin-stimulated membrane recruitment of p85 was predominantly observed by enrichment of p85 in microsomal fractions and not in PM fractions (21). This study demonstrates that insulin-stimulated association of IRS1 and p85␣ is impaired by depletion of Rab5. Rab5 has been shown to interact with two types of PI3K (34) and to regulate phosphoinositide turnover in the endocytic pathway (29). More recent research showed that p85␣ binds to both GDP and GTP forms of Rab5A and is a GTPaseactivating Protein for Rab5A (35)(36)(37). Our results suggest that Rab5 might recruit p85␣ to IRS1 that is localized to and phosphorylated at the cytoskeleton-membrane interface and may possibly activate a subpopulation of PI3K molecules, which are required for insulin-stimulated PKB/Akt activation. The p85␣ subunit of PI3K has been shown to localize to all subcellular fractions (PM, low density membrane, and cytosol) (41). It is not clear which pool of p85 is indispensable for insulin-stimulated PKB/Akt activation. It is also unclear how Rab5 mediates the interaction between IRS1 and p85␣. There are two possible mechanisms for Rab5mediated recruitment of p85␣ to tyrosyl-phosphorylated IRS1. Rab5 may mediate the fusion of IRS1-associated membranes with p85␣-containing vesicles and enable the spatial proximity between these two components. Another more likely possibility is that p85␣ may interact with Rab5, resulting in a conformational change in p85␣ that enhances interaction between its Src homolog 2 domain and phosphotyrosine of IRS1. Future in vitro experiments using pre-purified components will help answer this question.
GAPex-5 is a vps9 domain containing protein that is relocated from an intracellular storage compartment to the plasma membrane upon insulin stimulation by CIP4 in 3T3-L1 adipocytes. 3 Moreover, a recent study of the C. elegans homolog of GAPex-5, rme6, also suggested a PM localization (48). The exchange activity of GAPex-5, also named RAP6, for Rab5 and its effects on insulin internalization have been studied recently (55). Thus, GAPex-5 might mediate the activation of a pool of Rab5 proximal to the plasma membrane that is necessary to recruit p85␣ to tyrosyl-phosphorylated IRS1. This study showed that depletion of GAPex-5 desensitized the PKB/Akt activation response to insulin. Thus, GAPex-5 may contribute to the activation of a pool of Rab5 by insulin. This point is reinforced by inhibition of insulin-stimulated HRP uptake and insulin receptor internalization by GAPex-5 depletion. It is interesting that a high concentration of insulin (10 nM) totally rescued the inhibition of insulin-mediated PKB/Akt activation by siRNA against GAPex-5. This result suggests the existence of multiple mechanisms underlying insulin-stimulated activation of Rab5.
The presence of Rab5 in multiple intracellular compartments (e.g. PM, clathrin-coated vesicle, early endosome, and microtubules) suggests spatial compartmentalization of this small GTPase. Previous studies demonstrated a critical role of Rab5A in GLUT4 translocation and suggested that insulin signaling inhibits Rab5 activity and the interaction of dynein with microtubules, thus retarding the inward movement of GLUT4 in fat cells (56). It is likely that there are multiple pools of Rab5, which undergo different regulatory mechanisms and support distinct cellular functions. Expression of Rab5A lacking a prenylation site had no effect on insulin-stimulated PKB/Akt activation (57). This result supports the presence of multiple pools of Rab5 and suggests that cytosolic, prenylation-deficient Rab5A does not interfere with Rab5 activity required for insulin-stimulated PKB/Akt activation. However, insulin-stimulated PKB/Akt activation was reported to be inhibited by overexpressing dominant negative Rab5B in THP-1 cells (58). Dominant negative Rab5B may inhibit global Rab5 activity in different pools.
NIH3T3/hIR cells are a model system for studying early insulin receptor signaling and may not faithfully represent what happens in target tissues. However, further evidence for a role of Rab5 in insulin signaling was provided by experiments utilizing genetically diabetic and obese fa/fa Zucker rats. Western blot analysis demonstrated the dramatic decrease of all three isoforms of Rab5 in homozygous Zucker obese fa/fa Zucker rats relative to their congenic lean controls in WAT. The decreased expression levels of Rab5 isoforms may contribute to the impaired insulin signaling in obese fa/fa Zucker rats. Obesity is characterized by a shift in the metabolic fates of fatty acids from catabolic pathways to anabolic pathways, which occur in distinct compartments. Rab5 may represent a novel