Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Center for Diabetes Research, Indianapolis, Indiana 46202
Submitted 25 February 2003 ; accepted in final form 23 September 2003
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ABSTRACT |
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vesicular trafficking; signal transduction; sphingolipids
Biochemical and morphological techniques have revealed that several proteins involved in regulating GLUT4 translocation appear corralled in certain regions or "domains" known as lipid rafts and caveolae (6). These include the receptors for insulin and ET-1 as well as intermediary proteins such as Gq/11 and TC10 (12, 18, 53). Lipid rafts and caveolae are particularly rich in cholesterol and sphingomyelin (9). Intriguingly, hydrolysis of sphingomyelin by sphingomyelinase activates GLUT4 translocation and glucose transport (14, 48). Although the molecular nature of this action remains poorly defined, sphingomyelin turnover appears to augment the level of GLUT4 in the plasma membrane by a PI3K-independent signal similar to that reported for osmotic shock and ET-1 (10, 14, 48, 56).
We and others have explored whether the degradation products of sphingomyelin might mimic the stimulatory action of sphingomyelinase (8, 25, 47, 48, 51). Ceramide is the main second messenger produced by sphingomyelinase-mediated hydrolysis of sphingomyelin. Although a ceramide-mediated signaling route activating GLUT4 translocation in 3T3-L1 adipocytes has been proposed (51), conflicting data from 3T3-L1 adipocytes and skeletal muscle suggest otherwise (25, 48). Alternatively, sphingosine and sphingosine-1-phosphate are derived from ceramide. Our studies with skeletal muscle suggested that sphingosine, but not ceramide, was an intermediate player. However, the pattern of action differs from that of sphingomyelinase, and thus it was concluded that sphingosine does not directly mediate the sphingomyelinase-induced augmentation of glucose transport (47, 48).
A collection of evidence from experimental models and human metabolic disorders indicates that cholesterol and sphingomyelin levels are coordinately regulated (38). Generally, it has been observed that altering the cellular content of sphingomyelin or cholesterol results in corresponding changes in mass and/or synthesis of the other lipid. In the case of cholesterol synthesis and trafficking, sphingomyelin regulates the capacity of membranes to absorb cholesterol and thereby controls sterol flux between the plasma membrane and regulatory pathways in the endoplasmic reticulum. Interestingly, cholesterol depletion has recently been reported to inhibit GLUT4 endocytosis (40, 42). However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor (TnFR) internalization (39, 45). Given the documented interactions between metabolism and intracellular distribution of cholesterol and sphingomyelin (38), the hydrolysis of sphingomyelin by exogenously added sphingomyelinase may increase the abundance of GLUT4 in the plasma membrane by slowing the endocytic process via influencing plasma membrane cholesterol balance. However, this interpretation is complicated by the fact that although sphingomyelinase increased plasma membrane content of GLUT4, no effect on the abundance of GLUT1 glucose transporter isoform, which resides in vesicle compartments distinct from GLUT4, was observed in the plasma membrane of sphingomyelinase-stimulated adipocytes (14).
The present work set out to test whether the insulin mimetic action of sphingomyelinase resulted from changes in plasma membrane cholesterol content. Our data demonstrate that membrane cholesterol content was diminished in cells exposed to sphingomyelinase and that prevention of this by cholesterol-loaded methyl--cyclodextrin (
CD) reversed the stimulatory action of sphingomyelinase on GLUT4 translocation. Furthermore, moderate depletion of membrane cholesterol with sphingomyelinase or low concentrations of
CD occurred without any visible changes in membrane morphology, plasma membrane trafficking of other proteins (GLUT1 and TnFR), or the endocytic retrieval of GLUT4. We questioned whether the apparent increase in the rate of GLUT4 exocytosis involved known GLUT4-signaling intermediates. The subsequent report provides a detailed account of these studies.
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MATERIALS AND METHODS |
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Antibodies and cDNAs. Polyclonal rabbit caveolin-1 antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit and antimouse antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit phospho-Akt (Ser473) and phospho-Cbl (Tyr774) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal rabbit GLUT1 antibody was obtained from Biogenesis (Kingston, NH). Monoclonal mouse transferrin receptor antibody was obtained from Zymed Laboratories (San Francisco, CA). Monoclonal phosphotyrosine antibody (PY20:HRP) was purchased from BD Transduction Laboratories (Lexington, KY). Rhodamine red-X-conjugated donkey anti-rabbit or anti-mouse antibodies were from Jackson ImmunoResearch (West Grove, PA). Polyclonal rabbit GLUT4 antibody, enhanced green fluorescent protein (EGFP)-tagged GLUT4, and PH/Grp-1 plasmid DNAs were kindly provided by Dr. Jeffrey E. Pessin (State University of New York at Stony Brook, Stony Brook, NY). Anti-Akt 2-specific antibody was a generous gift from Dr. Morris J. Birnbaum (University of Pennsylvania, Philadelphia, PA).
Cell culture and treatments. Murine 3T3-L1 preadipocytes were cultured in DMEM containing 25 mM glucose and 10% calf serum at 37°C in an 8% CO2 atmosphere. Confluent cultures were induced to differentiate into adipocytes as previously described (25). All studies were performed on adipocytes, which were between 8 and 12 days after differentiation. Before all experimental treatments, the differentiated adipocytes were serum starved in DMEM containing 25 mM glucose for 3 h at 37°C unless otherwise indicated. The concentrations of additions to the medium were 200 mU of sphingomyelinase/ml, 100 µM C2-ceramide, 100 µM C6-ceramide, 50 µM D-sphingosine, 10 µM sphingosine-1-phosphate, 110 mM CD, and 1050 µM nystatin. C2-ceramide, C6-ceramide, and sphingosine were dissolved in DMSO and added in such a way that the medium contained 0.1% DMSO. The corresponding control medium always contained an equivalent concentration of DMSO. Sphingosine-1-phosphate was dissolved in MeOH. Addition of sphingosine-1-phosphate resulted in 0.19% MeOH in the medium, and, therefore, the corresponding control medium always contained an equivalent concentration of MeOH.
CD and nystatin were dissolved directly in DMEM. The incubation periods lasted 5120 min as indicated. During the last 5 or 30 min of incubation, the cells were either left unstimulated or stimulated with 1 or 100 nM insulin as indicated. The preparation of
CD-cholesterol complex was performed essentially by the method of Christian et al. (11), with minor modifications. Briefly, 96.7 µl of cholesterol from 5 mg/ml stock in chloroform-methanol (1:1, vol:vol) were added to a glass tube. The solvent was evaporated under a gentle stream of nitrogen gas, and a dried cholesterol film was formed on the bottom of the tube. Next, 10 ml of 1 mM
CD was added, vortexed, and sonicated (bath sonicator). This 100% saturated
CD-cholesterol solution was incubated in a 37°C water bath for 48 h with vigorous shaking. This mixed solution was then filtered through a 0.45-µm syringe filter (Millipore) before use. In the cholesterol replenishment experiments, cells were preincubated with this solution for 30 min before treatment with sphingomyelinase or
CD and were continually exposed to the
CD-cholesterol solution during the treatment period.
Plasma membrane sheet assay. Preparation of plasma membrane sheets from the adipocytes was as previously described (25). After the isolation of plasma membrane sheets, these purified membranes were used for sphingomyelin content determination or indirect immunofluorescence. The sheets were fixed for 20 min at 25°C in a solution containing 2% paraformaldehyde, 70 mM KCl, 30 mM HEPES, pH 7.5, 5 mM MgCl2, and 3 mM EGTA. The sheets were then blocked in 5% donkey serum for 60 min at 25°C and incubated for 60 min at 25°C with a 1:1,000 dilution of polyclonal rabbit GLUT4 antibody, 1:100 dilution of polyclonal rabbit GLUT1 antibody, 1:50 dilution of polyclonal rabbit caveolin-1 antibody, or 1:50 dilution of monoclonal mouse transferrin receptor antibody, followed by incubation with a 1:50 dilution of rhodamine red-X-conjugated donkey anti-rabbit or anti-mouse immunoglobulin G for 60 min at 25°C.
Subcellular fractionation. Plasma membrane fractions were obtained by using a differential centrifugation method previously described (46). Briefly, control and insulin-stimulated 3T3-L1 adipocytes were washed and resuspended in HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, and 255 mM sucrose containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 5 µg/ml leupeptin). Cell lysates were prepared by shearing the cells through a 22-gauge needle 10 times. Lysates were then centrifuged at 19,000 g for 20 min at 4°C. The crude plasma membrane pellet was resuspended in HES buffer and layered onto a 1.12 M sucrose cushion for centrifugation at 100,000 g for 60 min. The plasma membrane layer was removed from the sucrose cushion and centrifuged at 40,000 g for 20 min. Pelleted plasma membrane was resuspended in a detergent-containing lysis buffer and assayed for soluble protein content.
Sphingomyelin and cholesterol analyses. Determination of [14C]choline-labeled sphingomyelin content was performed as described by Vrtovsnik et al. (50). Cells grown in six-well plates were incubated in 10% FBS-DMEM for 32 h with [14C]choline (1 µCi/ml). At the end of this period, cells were washed three times with PBS and incubated for 16 h in this serum-free medium enriched with [14C]choline (1 µCi/ml). After serum starvation, the cells were washed three times with serum-free DMEM and either left untreated or treated with 0.2 mU/ml sphingomyelinase for 5, 15, 30, 60, and 120 min. As described in detail in Plasma membrane sheet assay, sheets were prepared and lipids from these highly purified plasma membrane fractions were extracted in chloroform-methanol mixture (2:1, vol/vol). Extracts were then evaporated to dryness under a nitrogen stream and solubilized in a chloroform-methanol mixture (2:1, vol/vol). Lipids were separated on heat-activated, high-performance, thin-layer chromatography plates (Analtech, Newark, DE) using chloroform-methanol-acetic acid-H2O mixture (50:30:8:4, vol/vol) as developing solvent. Individual components were detected by iodine vapors and identified by comparison with authentic standards. The spots were scraped off, transferred to scintillation vials, and counted by liquid scintillation counting.
For determination of plasma membrane cholesterol content, we used differential centrifugation method described in Subcellular fractionation to obtain plasma membrane fractions. Plasma membrane pellets were resuspended in 0.25 ml of HES buffer, and cholesterol content was determined by using an enzymatic, colorimetric kit for the quantitative determination of total cholesterol (catalog no. 276-64909; Wako Chemicals, Richmond, VA). The assay was performed exactly as described in the kit's supplied procedure. Briefly, 0.2 ml of the resuspended plasma membrane pellet was vigorously mixed with 5 ml of chloroform-methanol (2:1, vol:vol) extraction solution for 10 min. The mixture was then centrifuged (3,000 rpm, 10 min), and 0.3 ml of the supernatant was added to a glass tube and evaporated via a 100°C water bath. The residue was reconstituted with 0.2 ml of an isopropanol-Triton X-100 solution (9:1, vol/vol), and 3.0 ml of a color reagent solution were added. After a 15-min incubation at 37°C, absorbance was measured at 505 nm.
Transient transfection. Differentiated adipocytes were electroporated (0.16 kV and 960 µF) as previously described (46). Transfection experiments were performed with 50 µg of EGFP-tagged plasmid DNA for analysis of EGFP fluorescence. After electroporation, the adipocytes were replated on glass coverslips and allowed to recover for 1618 h before use.
Preparation of total cell extracts. Total cell extracts were prepared from 100-mm plates of 3T3-L1 adipocytes after treatment. Cells from each plate were washed two times with ice-cold PBS and scraped into 1 ml of ice-cold lysis buffer (25 mM Tris, pH 7.4, 50 mM NaF, 10 mM Na3P2O7, 137 mM NaCl, 10% glycerol, and 1% NP40) containing 2.0 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 5 µg/ml aprotinin, 10 µM leupeptin, and 1 µM pepstatin A by rotation for 15 min at 4°C. Insoluble material was separated from the soluble extract by microcentrifugation for 15 min at 4°C. Protein concentration was determined, and samples were subjected directly to SDS-polyacrylamide electrophoresis.
Electrophoresis and immunoblotting. Whole cell lysates were separated by 7.5% SDS-polyacrylamide gel, and plasma membrane fractions (GLUT4 analyses) were separated by 10% SDS-polyacrylamide gel. The resolved proteins were transferred to Immobilon P membrane (Millipore, MA) or nitrocellulose membrane (GLUT4 analyses) and immunoblotted with a monoclonal phosphotyrosine antibody, a phosphotyrosine-specific Cbl antibody, a phosphoserine-specific Akt antibody, an anti-Akt 2-specific antibody, or a GLUT4 antibody. All immunoblots were subjected to enhanced chemiluminescence detection (Amersham, NJ).
Statistical analysis. All values are presented as means ± SE. Analysis of variance was used to determine differences among groups. Where a significant difference was indicated, the Tukey test was used to determine significant differences between groups. P < 0.05 was considered to be statistically significant.
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RESULTS |
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To confirm the effects of sphingomyelinase on the accumulation of GLUT4 in isolated plasma membrane sheets, we used differential centrifugation to isolate plasma membrane from 3T3-L1 adipocytes. Plasma membrane fractions prepared from cells treated with a physiological dose of insulin (1 nM) displayed a characteristic increase in GLUT4 compared with plasma membrane fractions prepared from untreated cells (Fig. 1C, compare lanes 1 and 2). Consistent with the microscopic analysis, immunoblot analysis showed that sphingomyelinase treatment for 60 min markedly increased the basal state level of GLUT4 in the plasma membrane (Fig. 1C, compare lanes 1 and 3). As we previously reported (48), the insulin effect was enhanced in the presence of sphingomyelinase (Fig. 1C, lane 4). The ability of sphingomyelinase to mobilize GLUT4 to the plasma membrane in the absence of insulin was particularly striking, and thus all subsequent experiments were focused on elucidation of its mechanism.
Degradation products of sphingomyelin do not mimic the insulin-like action of sphingomyelinase. Sphingomyelin turnover involves sphingomyelinase- and ceramidase-stimulated release of ceramide and sphingosine, respectively (2). Ceramidase only degrades ceramides with a long acyl side chain, whereas the short-chain C2-ceramide and C6-ceramide (only 2 and 6 carbons in the side chain) are not degraded (30, 43, 44). We took advantage of this distinction to determine whether measured effects are due to ceramide or its degradation product sphingosine. Exposure of cells to C2-ceramide or sphingosine for 60 min (data not shown) or 120 min did not duplicate the insulin-mimetic action of sphingomyelinase as assessed by plasma membrane sheet GLUT4 immunofluorescence (Fig. 2A, compare panels 13). Under identical ceramide and sphingosine exposure conditions, we observed the antagonizing action of these sphingolipids on insulin action (Fig. 2A, compare panels 46), as we reported previously (25, 48). This observation substantiates the intracellular action of these cell-permeable sphingolipids and their inability to activate the glucose transport process. Using the plasma membrane sheet assay, we also found that C6-ceramide and sphingosine-1-phosphate lacked insulin-mimetic activity (data not shown).
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Because we previously demonstrated that expressed GLUT4-EGFP displays identical subcellular distribution and insulin-stimulated translocation as the endogenous GLUT4 protein (16), we examined the effects of these sphingolipids on GLUT4-EGFP trafficking. As typically observed for the endogenous GLUT4 protein, in the basal state GLUT4-EGFP was localized to the perinuclear region and small vesicles scattered throughout the cytoplasm (Fig. 2B, panel 1). As expected, both insulin and sphingomyelinase treatments for 60 min resulted in the recruitment of GLUT4-EGFP to the plasma membrane detected as a continuous rim of cell surface fluorescence (Fig. 2B, panels 2 and 3). The percentage of cells displaying GLUT4-EGFP plasma membrane rim fluorescence after a 2-h exposure to insulin, sphingomyelinase, and several sphingolipids is presented in Fig. 2C. Sphingomyelinase action was not reproduced by C2-ceramide, C6-ceramide, sphingosine, or sphingosine-1-phosphate treatments (Fig. 2C). Although these results do not exclude downstream metabolites of sphingomyelin conclusively (e.g., long chain ceramides), these findings implicate that the potential mechanism of sphingomyelinase may entail events independent of sphingolipid second messengers. Given previous reports showing altered cellular content of sphingomyelin or cholesterol results in corresponding changes in mass and/or synthesis of the other lipid, we next measured the effect of sphingomyelinase on the plasma membrane content of cholesterol.
Insulin-mimetic action of sphingomyelinase is coupled to plasma membrane cholesterol loss. Reminiscent to the effects of sphingomyelinase on plasma membrane sphingomyelin content over time, the enzyme also induced a time-dependent decrease in plasma membrane cholesterol content (Fig. 3). The kinetic profile of sphingomyelinase-induced cholesterol depletion paralleled the observed hydrolysis of sphingomyelin shown in Fig. 1A, which rapidly reached a saturable loss by 15 min. Although sphingomyelin levels were reduced by 68.5% after 15 min, the extent of cholesterol depletion after this period of time was only 16.6%. Interestingly, continued exposure of the cells to sphingomyelinase for 30, 60, and 120 min, which was associated with an approximate 80% reduction in membrane sphingomyelin (see Fig. 1A) at each of these time points, resulted in a loss of cholesterol between 26.4 and 32.3%. This associated loss of cholesterol from the plasma membrane prompted us to ask whether sphingomyelinase-induced GLUT4 translocation could be prevented by adding back this level of cholesterol.
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Here, we used CD to replenish the depleted plasma membrane cholesterol induced by sphingomyelinase treatment for 60 min. The reduction in plasma membrane cholesterol induced by sphingomyelinase treatment was prevented in cells incubated in medium enriched with
CD preloaded with cholesterol (
CD-Chol) (Fig. 4A). The effect of
CD-Chol did not significantly alter the basal-state level of cholesterol content in the plasma membrane (Fig. 4A). Surprisingly, the ability of sphingomyelinase to mobilize GLUT4 to the plasma membrane was prevented in cells incubated in
CD-Chol (Fig. 4B, compare panels 2 and 4) and the effect of
CD-Chol did not alter the basal-state level of GLUT4 content in the plasma membrane (Fig. 4B, compare panels 1 and 3). In contrast to the inability of sphingomyelinase to activate GLUT4 translocation in the presence of
CD-Chol, the ability of sphingomyelinase to deplete sphingomyelin from the plasma membrane was unaffected by the cholesterol replenishment procedure (Fig. 4C). This provides further support that the insulin-mimetic action of sphingomyelinase is not attributed to its direct action on sphingomyelin per se but, rather, its indirect effect on membrane cholesterol balance. To further examine this novel molecular basis of sphingomyelinase action, we next asked whether reduction of plasma membrane cholesterol could activate GLUT4 translocation.
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Exposure of cells to increasing concentrations of CD (110 mM) resulted in a dose-dependent stimulation of GLUT4 translocation (Fig. 5A, panels 15) and a dose-dependent decrease in plasma membrane cholesterol (Fig. 5B, open bars). As we observed for sphingomyelinase, less than a 50% loss of cholesterol from the plasma membrane induced by 1 mM (20%, P = 0.056) and 2.5 mM
CD (44%, P < 0.05) was also associated with accumulation of plasma membrane GLUT4. In addition, the effects of 2.5 mM
CD on GLUT4 translocation and cholesterol depletion were blocked in cells incubated in the presence of
CD:Chol (Fig. 5A, compare panels 3 and 6, and Fig. 5B, compare 2.5 mM open and solid bars). The effects of two other cholesterol binding agents, nystatin and filipin, were also evaluated, and exposure of cells to increasing concentrations of nystatin (Fig. 5C) and filipin (data not shown) resulted in a dose-dependent stimulation of GLUT4 translocation. Combined, these data strengthen the concept that reduction in plasma membrane cholesterol content positively influences GLUT4 translocation.
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Considering the extensive 60 and 67% removal of cholesterol from the plasma membrane by 5 and 10 mM CD, respectively, we next examined the morphological effects of cholesterol extraction on membrane integrity. Cells exposed to 5 mM
CD, but not 1 or 2.5 mM
CD, displayed nuclear propidium iodide staining (Fig. 6A, panels 14). Consistent with the number of nuclei displayed (Fig. 6A, panel 4), phase contrast images showed
6070 cells per field (data not shown). Although this assessment provided a global view of membrane permeability, we next measured the regional effects of
CD on cholesterol- and sphingomyelin-enriched lipid raft microdomains. A large subset of lipid raft microdomains exists as plasma membrane caveolae that are clustered into higher-order structures in adipocytes, thus making them visible by light microscopy (53). High magnification of plasma membrane sheets from control cells and those incubated with 1 and 2.5 mM
CD displayed an abundance of caveolin-enriched circular rosette structures (Fig. 6B, panels 13). In contrast, caveolin rosettes were not apparent in sheets prepared from cells exposed to 5 mM
CD (Fig. 6B, panel 4). In addition to the absence of an effect of low
CD concentrations, sphingomyelinase or
CD:Chol treatment conditions did not alter plasma membrane integrity (data not shown). Taken together, these results suggest that depletion of cholesterol with 1 and 2.5 mM
CD promotes GLUT4 translocation without adverse effects on cell or caveolar integrity.
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Activation of GLUT4 translocation by moderate cholesterol depletion does not result from impaired endocytosis. Recent studies have documented that cholesterol depletion greater than 50% reduces the rate of internalization of transferrin receptor by more than 85% without affecting intracellular receptor trafficking back to the cell surface (45). In view of the inhibitory effect of extensive cholesterol depletion on endocytosis, we examined the effect of sphingomyelinase and 2.5 mM CD on endocytosis in more detail. By immunofluorescent microscopy of plasma membrane sheets, both the GLUT1 glucose transporter isoform (Fig. 7A) and transferrin receptor (Fig. 7B), which reside in vesicle compartments distinct from GLUT4, displayed a basal state plasma membrane level and an insulin-stimulated translocation as previously reviewed (34). Whereas insulin treatment activated the translocation of these proteins, sphingomyelinase or 2.5 mM
CD treatments had no significant effect (Fig. 7, A and B, panels 3 and 4). Consistent with greater cholesterol depletion having a negative impact on endocytosis, we observed an increase in GLUT1 and TnFR in our plasma membrane sheets from cells treated with 5 mM
CD (Figs. 7, A and B, compare panels 1 and 5). To further verify that endocytosis was not being affected by moderate cholesterol depletion, we next examined the effects of 2.5 mM
CD on the endocytic retrieval of insulin-recruited plasma membrane GLUT4. Insulin stimulation resulted in a marked translocation of GLUT4 to the plasma membrane in the absence of 2.5 mM
CD (Fig. 8, panels 1 and 2). After removal of insulin and a 30-min recovery period in the absence of insulin, there was a decrease in plasma membrane GLUT4 indicative of endocytic retrieval of the transporter (Fig. 8, panel 3). As previously observed, the presence of 2.5 mM
CD resulted in increased levels of plasma membrane-associated GLUT4 (Fig. 8, panel 4). As observed in the presence of sphingomyelinase (Fig. 1C, lane 4), insulin stimulation was further enhanced by the presence of 2.5 mM
CD (Fig. 8, panel 5). The insulin-stimulated increase in plasma membrane GLUT4 levels in adipocytes exposed to 2.5 mM
CD decreased subsequent to insulin removal and recovery (Fig. 8, panel 6). Consistent with a previous report (22), we did not detect loss of sheets after insulin removal and recovery. These observations and the finding that sphingomyelinase and 2.5 mM
CD specifically augments the plasma membrane content of GLUT4, but not GLUT1 or TnFR, suggest that the effect on this transporter is attributed to a specific exocytosis event for the GLUT4 protein rather than a reduction in the overall rate of endocytic trafficking.
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Key signaling molecules of insulin action are not engaged by sphingomyelinase or CD. It was recently demonstrated that sphingomyelinase treatment increased PI3K activity in a rat 2 fibroblast cell line (8). Therefore, we determined whether the insulin mimetic action of sphingomyelinase or
CD in 3T3-L1 adipocytes could be attributed to activation of insulin-signaling events. Insulin stimulation resulted in increased tyrosine phosphorylation of the insulin receptor
-subunit and IRS1 (Fig. 9A, compare lanes 1 and 2). In contrast to the effect of insulin, sphingomyelinase or
CD stimulation had no effect on the phosphorylation of these proteins at 30 min (Fig. 9A, compare lanes 1, 3, and 4) or at 5-, 15-, 60-, and 120-min treatment intervals (data not shown). In addition to IRS1, insulin increases the tyrosine phosphorylation of Cbl, an event required for efficient GLUT4 recruitment to the plasma membrane (5, 37). As previously reported, in unstimulated cells there was no detectable Cbl tyrosine phosphorylation, whereas insulin induced a marked tyrosine phosphorylation of Cbl (Fig. 9B, compare lanes 1 and 2). In contrast, there was no detectable tyrosine phosphorylation of Cbl in response to sphingomyelinase or
CD at 30 min (Fig. 9B, compare lanes 1, 3, and 4) or at 5-, 15-, 60-, and 120-min treatment intervals (data not shown). To test whether the greater effect of insulin in the presence of sphingomyelinase or
CD (see Figs. 1C and 8) resulted from enhanced signaling events, cells were treated with sphingomyelinase or
CD for 30 min before further incubation for 5 min in the presence or absence of 100 nM insulin. The insulin stimulated tyrosine phosphorylation states of the insulin receptor
-subunit, IRS-1, or Cbl were not significantly affected by sphingomyelinase or
CD (Fig. 9, A and B, compare lanes 2, 5, and 6). Insulin receptor
-subunit, IRS-1, and Cbl immunoblotting of those same membranes demonstrated the presence of equal amounts of insulin receptor
-subunit, IRS-1, and Cbl protein (Fig. 9, C and D, compare lanes 16).
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Although the signaling events distal to the Cbl cascade remain unclear, a likely downstream candidate molecule of the IRS cascade involved in GLUT4 translocation is the Akt serine/threonine kinase. Akt 1 activity was assessed by using an anti-phospho-Ser473 antibody that is specific for the serine phosphorylated activated Akt 1 isoform, whereas the activity of Akt 2 was determined by monitoring the mobility shift indicative of the phosphorylated and active enzyme (25). Insulin stimulated both Akt 1 (Fig. 10A, top immunoblot, compare lanes 1 and 2) and Akt 2 (Fig. 10A, bottom immunoblot compare lanes 1 and 2). In contrast, exposure of cells to sphingomyelinase and CD for 30 min did not result in phosphorylation of either Akt isoform (Fig. 10A, compare lanes 1, 3, and 4). Analyses performed with cells treated for 5, 15, 60, and 120 min also did not show an effect of sphingomyelinase and
CD (data not shown). Consistent with these findings, we found that sphingomyelinase and
CD did not activate PI3K. This activity was assessed by monitoring the in vivo production of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by taking advantage of the specific, high-affinity interaction of the Grp1 pleckstrin homology (PH) domain with PIP3 (23, 49). This was accomplished by utilizing a fusion protein consisting of the EGFP tag fused to this PH domain (EGFP-PH/Grp1) as previously reported (25). In the absence of insulin, expression of EGFP-PH/Grp1 resulted in a predominant nuclear localization with a smaller amount distributed throughout the cell cytoplasm (Fig. 10B, panel 1). The presence of the EGFP-PH/Grp1 fusion protein in the nucleus is a property of EGFP in 3T3-L1 adipocytes since expression of just EGFP itself also results in a predominant nuclear localization. Insulin stimulation resulted in the accumulation of the EGFP-PH/Grp1 fusion protein at the cell surface membrane, indicative of PIP3 formation at the plasma membrane (Fig. 10B, panel 2). Sphingomyelinase treatment did not result in the accumulation of the EGFP-PH/Grp1 fusion protein at the cell surface membrane (Fig. 10B, panel 3). Quantification of these data demonstrated that insulin, but not sphingomyelinase, treatment resulted in a greater percentage of cells displaying cell surface EGFP-PH/Grp1 fluorescence (Fig. 10C). Similar to the effects of sphingomyelinase,
CD treatment did not result in the accumulation of the EGFP-PH/Grp1 fusion protein at the cell surface membrane (data not shown). As observed for the insulin-stimulated states of the insulin receptor
-subunit, IRS-1, or Cbl, activation of the Akt isoforms by insulin was not altered by sphingomyelinase or
CD (Fig. 10A, compare lanes 2, 5, and 6). Collectively, these data indicate that sphingomyelinase and
CD activate GLUT4 translocation via signaling pathways that are independent of known signaling intermediates of insulin action.
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DISCUSSION |
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The use of cyclodextrins for manipulating cellular cholesterol content has been described (11). The exposure of cells to high concentrations of CD (10100 mM) results in cholesterol efflux far in excess of those achieved with physiological cholesterol acceptors such as HDL. At lower concentrations (
1 mM),
CD has been shown to function as a cholesterol shuttle that can catalyze the exchange of cholesterol between cells and serum lipoproteins (4). The mechanism that allows
CD to remove cholesterol from cell membranes so efficiently is related to its ability to reduce the activation energy for cholesterol efflux from
20 kcal/mol required for movement of cell cholesterol to phospholipid acceptors to a value of 79 kcal/mol (57). This difference has been attributed to the need for cholesterol molecules in the plasma membrane to desorb completely into the aqueous phase before being absorbed by HDL particles or phospholipid liposomes, whereas the membrane cholesterol molecules can incorporate directly into the hydrophobic cavity of the
CD molecule without the necessity of traveling through any intermediate aqueous phase (35, 57). It is this ability to directly transfer cholesterol between cell membranes and
CD that enabled us to ask whether the insulin-mimetic effect of sphingomyelinase resulted from the coordinately regulated sphingomyelin and cholesterol levels. As expected, the plasma membrane content of cholesterol exposed to sphingomyelinase was reduced. Remarkably, restoration of this cholesterol loss by
CD-Chol abolished the insulin-mimetic action of sphingomyelinase. Importantly, this replenishment procedure did not alter the hydrolysis of sphingomyelin by sphingomyelinase. This further strengthens our findings that sphingomyelin-derived lipids do not mediate sphingomyelinase action.
Labeling with antibodies against the caveolar structural protein caveolin has revealed that control 3T3-L1 adipocyte membranes typically display an abundance of caveolar structures of 50- to 100-nm diameters and that incubation for 60 min with CD (220 mM) resulted in a concentration-dependent decrease in caveolar structure (32). Consistent with the importance of this plasma membrane domain in insulin action, propagation of the insulin signal was inhibited in cells exposed to 10 mM
CD (32). More recently, it was demonstrated that treatment of 3T3-L1 adipocytes with 10 mM
CD resulted in a clear time-dependent dissolution of caveolin structures, which was most pronounced after 30 min of
CD exposure (53). Taken together, these findings show that low concentrations of
CD and short exposure periods markedly minimize the negative effects of
CD on caveolar morphology and insulin signaling. In line with these findings, our results clearly show that moderate depletion of cholesterol promotes GLUT4 translocation without adverse effects on cell or caveolar integrity. In addition, we found that 2.5 mM
CD did not affect insulin signal transduction or that the same exposure level did not impair the endocytic retrieval of plasma membrane proteins. In direct agreement with these disturbances with higher concentrations of
CD, we found that exposure of cells to 5 and 10 mM
CD resulted in the reported alterations in insulin signaling (32) and endocytosis (40, 42, 45).
Our findings are supported by studies published intermittently over the past 35 years. For example, cholesterol-complexing drug experiments performed in the late 1960s and early 1970s demonstrated that removal of cholesterol from the plasma membrane augmented basal glucose uptake and metabolism (1, 26). Because cholesterol causes a highly ordered, gel-like state (9), it is not surprising that a cell's responsiveness to insulin may depend on the cell surface membrane fluidity. In agreement with this postulate, studies have documented that moderate increases in plasma membrane fluidity increase glucose transport (13, 36, 54). Furthermore, it has been shown that basal glucose transport is not fully active in fat cells and can be increased further by augmenting fluidity (13). In direct support of that finding, insulin-stimulated glucose transport is decreased when fluidity diminishes (36). Interestingly, recent data suggests that the antidiabetic drug metformin, by increasing membrane fluidity, may correct a protein configuration(s) disturbed by the diabetic state (28, 55). New insight into the membrane basis of metformin action suggests that metformin, via stimulation of 5'-AMP-activated kinase (AMPK; Refs. 20, 29, 58), may attenuate cholesterol synthesis by suppressing the expression of a sterol regulatory element binding protein, SREBP-1 (58). SREBP-1 belongs to a family of key lipogenic transcription factors directly involved in the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (41).
Studies using lipid analogs and lipid-anchored proteins with varying fluidity preferences have recently provided data consistent with the idea that the plasma membrane consists mostly of lipid raft domains with small regions of fluid lipids intercalated among mostly ordered lipid domains (19). We postulate that, within the plasma membrane, a subset of rafts may consist of novel signaling components that slow the constitutive exocytic movement of GLUT4 to the plasma membrane in the absence of insulin. Dispersion of these domains would thus remove this restraint and result in GLUT4 trafficking to the plasma membrane. One could envision that part of the complex insulin-signaling network that activates GLUT4 translocation also disengages this suppression signal. On the other hand, the fact that sphingomyelinase augments the effects of insulin may imply that insulin action does not turn off this negative signal, and any manipulation that does disengage the suppression machinery enhances insulin action. In contrast to the cholesterol-dependent event being associated with a signal transduction mechanism per se, a nonsignaling basis may exist. For example, recent studies have shown that phosphatidylinositol 4,5-bisphosphate (PIP2) accumulates at cholesterol-dependent rafts, where it regulates actin dynamics at the cell surface (17, 27). Therefore, it is possible that changes in raft properties may be coupled to actin cytoskeleton. Indeed, a role for actin in insulin-stimulated GLUT4 translocation has been implicated by several studies. Treatment with the actin-depolymerizing agent cytochalasin D or the actin monomer-binding Red Sea sponge toxins latrunculin A or B inhibited insulin-stimulated GLUT4 translocation (31, 52). Importantly, it has been shown that insulin elicits actin filament (F-actin) formation (3, 7, 21, 31, 33). Thus insulin signaling to polymerize cortical F-actin apparently represents a required pathway for optimal movement or fusion of GLUT4-containing vesicle membranes to the cell surface membrane.
In summary, the present study demonstrates that exogenous sphingomyelinase stimulates GLUT4 translocation in 3T3-L1 adipocytes. This action does not appear to be mediated by the degradation products of sphingomyelin or steps in the insulin signal transduction pathway. These data suggest that sphingomyelinase exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT4 translocation. Future work is needed to understand the influence of the plasma membrane architecture on insulin action and the glucose transport process.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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