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Osmotic Regulation of Cellular Glucose Uptake
C H A P T E R
T W E N T Y
Osmotic Regulation of Cellular Glucose Uptake Philippe Gual, Teresa Gonzalez, Thierry Gremeaux, Yannick Le Marchand-Brustel, and Jean-Franc¸ois Tanti Contents 1. Introduction 2. Hyperosmolarity and Glucose Transport 2.1. Differentiation of 3T3-L1 adipocytes 2.2. Glucose uptake induced in response to hyperosmolarity 2.3. Study of pathways involved in the hyperosmotic effect on glucose uptake 2.4. Inhibition of insulin-stimulated glucose uptake by hyperosmotic stress 3. Hyperosmolarity and Membrane Ruffling 3.1. Membrane ruffling assay 4. Hyperosmolarity and Signaling Pathways 4.1. Preparation of total cell lysates 4.2. Immunoprecipitation of docking proteins (Gab1 or IRS1) 4.3. Western blotting assays 5. Hyperosmolarity and Phosphatidylinositol 3-Kinase Activity 5.1. Preparation of phosphatidylinositol 5.2. Immunoprecipitation of phosphatidylinositol 3-kinase 5.3. Measurement of PI 3-kinase activity in immunoprecipitation 6. Conclusion Acknowledgments References
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Abstract This chapter describes various approaches allowing the study of hyperosmolarity in the functions of 3T3-L1 adipocytes. Hyperosmolarity mimics insulin responses, such as glucose uptake and membrane ruffling, but also antagonizes these insulin effects, which can be evaluated in 3T3-L1 adipocytes. The molecular mechanisms of these effects can be also investigated by measuring the
INSERM U 568; University of Nice Sophia-Antipolis, Nice, France Methods in Enzymology, Volume 428 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)28020-6
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activation of different signaling pathways: (i) the phosphorylation of docking proteins on tyrosine and serine residues (serines 307 and 632), (ii) the phosphorylation of serine/threonine kinases, and (iii) the activation of phosphatidylinositol 3-kinase.
1. Introduction Exposure of many mammalian cell types to metabolic or osmotic stress results in an acute increase in the rate of glucose uptake. This adaptive response allows the cells to maintain or regain their ATP levels by increasing flux through the glycolytic pathway. However, like several other insulinomimetic agents, hyperosmolarity not only partly activates several insulinspecific biological responses, but also induces a state of insulin resistance. Conditions dehydrating insulin target tissues such as hyperosmolarity or amino acid deprivation are frequently associated with insulin resistance. Sepsis and burn injury are associated with dehydration-induced insulin resistance. Furthermore, in rat epididymal fat cells, hyperosmotic stress markedly reduces in vitro insulin-induced glucose transport (Komjati et al., 1988). In perfused rat liver, hyperosmolarity impairs insulin-mediated cell swelling and reverses the proteolysis inhibition induced by insulin (Vom Dahl et al., 1991). In 3T3-L1 adipocytes, pretreatment with sorbitol strongly decreases the ability of insulin to stimulate glucose uptake, lipogenesis, glycogen synthesis, and membrane ruffling (Chen et al., 1999; Gual et al., 2003a). Glucose uptake induced by insulin in muscle and adipose tissues is due to the translocation of the glucose transporter Glut 4 from an intracellular pool to the plasma membrane (Bryant et al., 2002; Saltiel and Kahn, 2001). These biological responses require tyrosine phosphorylation of insulin receptor substrate-1 (IRS1), which leads to the binding and activation of phosphatidylinositol 3-kinase (PI 3-kinase). Downstream effectors of PI 3-kinase such as protein kinase B (PKB) or atypical PKC are involved in Glut 4 translocation. Furthermore, it has been shown that insulininduced Glut 4 translocation also requires activation of a second pathway, which is completely independent of PI 3-kinase activity. In adipocytes, the Cbl protooncogene, associated with Cbl-associated protein and adapter protein containing PH and SH2 domain, is phosphorylated in response to insulin and regulates glucose uptake. Once phosphorylated, Cbl recruits the adapter protein Crk-II in a complex with C3G, a GDP to GTP exchange factor for TC10, a Rho family GTPase, allowing for its activation, which regulates the traffic of Glut 4-containing vesicles. GTP-bound TC10 could participate in Glut 4 translocation through a modification of cortical actin or a stimulation of actin polymerization at the level of Glut 4 compartments
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(Scheme 20.1) (Dugani and Klip, 2005; Gual et al., 2003b; Saltiel and Pessin, 2002; Watson et al., 2004). In both adipocyte (Scheme 20.1) and muscle cells, hyperosmolarity promotes glucose uptake by multiple mechanisms that do not require the PI 3-kinase/PKB pathway (Gual et al., 2003b) but are dependent on the cell type. In muscle, osmotic stress induces glucose uptake by stimulation of AMPkinase and/or inhibition of Glut 4 endocytosis. In adipocytes, activation of the Grb2-associated binder-1 (Gab1)-dependent signaling pathway plays an important role in osmotic stress-mediated glucose uptake (Gual et al., 2003a; Janez et al., 2000). Upon sorbitol stimulation, the phosphorylated Gab1 recruits Crk-II via its SH2 domain. The Crk-II SH3 domains are constitutively associated with C3G, a GDP-to-GTP exchange factor for several small GTP-binding proteins, including TC10 (Gual et al., 2002). The activation of TC10 and remodeling of cortical actin are required for osmotic shock-mediated Glut 4 translocation and glucose uptake (Gual et al., 2002). Apart from its insulin-like effects, hyperosmolarity leads to cellular insulin resistance (Fig. 20.1) mediated by both prevention of PKB activation Adipocyte Glucose Hyperosmotic Caveolin stress Lipid raft TC10 C3G Src
Actin
Hyperosmotic stress
PLD
GTP
ERKs
GDP
aPKC
P CrkII P Y Y Gab1Y P PI3-K Y
Pyk2
P
PLCg Cbl Y P CrkII
Glut 4 vesicles (VAMP2-positive)
Scheme 20.1 Signaling pathways activated by hyperosmotic stress to induce Glut 4 translocation in 3T3-L1 adipocytes and adipose cells. Osmotic stress induces glucose uptake by activation of Gab1-dependent signaling pathways. Sorbitol promotes the activation of cytosolic Src kinase, which phosphorylates Gab1 on the tyrosine residue. Phosphorylated Gab1 recruits the Crk-II/C3G complex. C3G, a guanine nucleotide exchange factor, exchanges GDP for GTP on TC10. Activated GTP-bound TC10 could then modify the cortical actin structure or stimulate the actin polymerization on Glut 4 compartments. Osmotic stress recruits vesicles containing both Glut 4 and VAMP2 in adipocytes. Glut 4 translocation stimulated by hyperosmotic stress could also depend on PYK2 activity, which leads to the activation of ERK, PLD, and finally atypical PKC. Adapted from Gual et al. (2003b).
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14 *
2-DOG uptake (fold stimulation)
12 10
*
8
*
6 4 2 0 Basal
Insulin
Sorbitol
Sorbitol + Insulin
Figure 20.1 Hyperosmolarity and glucose transport. After serum starvation, 3T3-L1 adipocytes were treated without or with insulin (100 nM) or sorbitol (600 mM) for 20 min (empty bars) or pretreated with sorbitol (600 mM) for 40 min before a 20-min insulin stimulation (100 nM) (gray bar). Uptake of [2-3H]deoxyglucose was measured during a 3-min period. Means SEM of three independent experiments are shown.
IB:
Sorbitol:
−
−
+
+
a-pS307
a-pS632
IRS1
a-IRS1
a-IRS1
IRS1
IP a-IRS1
IP a-IRS1
Figure 20.2 Hyperosmotic stress triggers the phosphorylation of IRS1 on serines 307 and 632. 3T3-L1 adipocytes were stimulated without or with 600 mM sorbitol for 20 min at 37. Cell lysates were then immunoprecipitated (IP) using anti-IRS1 antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and blotted (IB) using antipSer307 IRS1 or anti-pSer632 IRS1 antibodies, as indicated. The membrane was then stripped and probed with anti-IRS1 antibodies. Representative autoradiographs are shown.
(Chen et al., 1999) and inhibition of the IRS1 function (Gual et al., 2003a). Chen et al. (1999) have reported that hyperosmolarity prevents insulininduced PKB activation. They suggest that a calyculin A- or okadaic acidsensitive protein phosphatase leads to the deactivation of PKB (Chen et al., 1999). Furthermore, a short-term osmotic stress induces the phosphorylation of IRS1 on serine 307 by an mTOR-dependent pathway (Gual et al., 2003a) but also on serine 632 (Fig. 20.2). This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations (Gual et al., 2003a). However, prolonged osmotic stress alters IRS1 and IRS2 functions by inducing their degradation (Gual et al.,
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2003a,b; Rui et al., 2001), which could contribute to the downregulation of insulin action.
2. Hyperosmolarity and Glucose Transport In muscle and adipose cells, hyperosmolarity triggers the cell surface accumulation of Glut 4, leading to an increase in glucose transport (Dugani and Klip, 2005; Gual et al., 2003b; Saltiel and Pessin, 2002; Watson et al., 2004). While the hyperosmotic stress mimics insulin responses, it also antagonizes insulin effects. Both effects can be evaluated in 3T3-L1 adipocytes (see Scheme 20.1).
2.1. Differentiation of 3T3-L1 adipocytes 3T3-L1 fibroblasts are a continuous substrain of 3T3 (Swiss albino) developed through clonal isolation (CL-173, ATCC). The cells undergo a preadipose to adipose-like conversion as they progress from a rapidly dividing to a confluent and contact inhibited state. After differentiation, Glut 4 translocation and glucose uptake can be induced in response to insulin. The differentiation protocol is as follows. 3T3-L1 fibroblasts are grown in six-well plates or 100-mm dishes in Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM glucose and 10% calf serum and induced to differentiate in adipocytes. Two days after confluence, medium is changed for a differentiation buffer, which includes DMEM, 25 mM glucose, 10% fetal calf serum supplemented with isobutylmethylxanthine (0.5 mM), dexamethasone (0.25 mM), thiazolidinediones (10 mM), and insulin (5 mg/ml). The medium is removed after 2 days and replaced with DMEM, 25 mM glucose, 10% fetal calf serum supplemented with insulin, and thiazolidinediones (10 mM) for 2 more days. Then, the cells are fed every 2 days with DMEM, 25 mM glucose, and 10% fetal calf serum. 3T3-L1 adipocytes are used 8 to 15 days after the beginning of the differentiation protocol. Sixteen hours before each experiment, the medium is changed to serum-free DMEM supplemented with 0.5% bovine serum albumin (BSA).
2.2. Glucose uptake induced in response to hyperosmolarity The technique allows for the measurement of glucose uptake using a glucose analog (2-deoxyglucose, DOG). The labeled 2-DOG is transported into the cells with high affinity, phosphorylated but not further metabolized. The labeled 2-DOG phosphate is trapped in the cell, and the rate of uptake can be taken as a measure of unidirectional transport.
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1. After serum starvation, cells (six-well plates) are washed three times with 1 ml of Krebs–Ringer phosphate (KRP) buffer (10 mM phosphate buffer, pH 7.4, 1.25 mM MgSO4, 1.25 mM CaCl2, 136 mM NaCl, 4.7 mM KCl) at 37 . 2. Cells are incubated in 900 ml of KRP buffer supplemented with 0.2% BSA without or with sorbitol (600 mM) for 20 min. 3. Measurement of glucose transport is initiated by the addition of 100 ml of KRP buffer supplemented with 0.2% BSA and containing 1 mM deoxyD-glucose and 0.5 mCi of 2-[3H]deoxy-D-glucose. 4. The reaction is stopped after 3 min by rapid aspiration, and cells are washed four times with ice-cold phosphate-buffered saline (PBS). 5. Cells are lysed with 700 ml of KRP and 1% Triton X-100. After brief sonication, 300 ml of lysates is mixed with 3 ml of liquid scintillation, and the radioactivity associated with the cells is counted in a b-counter using the tritium program. 6. Radioactivity is normalized by measuring the protein concentration in each sample by the bicinchoninic acid (BCA) assay (Pierce). Do not forget to count an aliquot of the radioactive deoxyglucose solution (using a commercial scintillation fluid), which will allow expressing the results in absolute amounts of glucose taken up by the cells.
2.3. Study of pathways involved in the hyperosmotic effect on glucose uptake To determine the pathways required for hyperosmotic stress-induced glucose transport or the study agents capable of preventing this response, 3T3-L1 adipocytes are pretreated with a specific inhibitor or agents (at step 2) before stimulation with sorbitol (600 mM ). Deoxyglucose uptake is then measured as described earlier. The TC10 activity per se can be inhibited by Clostridium difficile toxin B (1 mg/ml), an inhibitor of the Rho family proteins. Latrunculin B (10 mM ) (Calbiochem), an agent that sequesters actin monomers, or jasplakinolide (10 mM ) (Molecular Probes, Eugene, OR), a stabilizer of filamentous actin, also inhibits sorbitol-induced glucose uptake. It is important to note that latrunculin B does not alter the early proximal osmotic shock signaling events such as Gab1 phosphorylation and its association with Crk-II. After serum starvation, 3T3-L1 adipocytes are treated without or with C. difficile toxin B (1 mg/ml), latrunculin B (10 mM ), or jasplakinolide (10 mM ) for 120 min at 37 . Cells are then either left untreated or stimulated with 600 mM sorbitol for 20 min at 37 .
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2.4. Inhibition of insulin-stimulated glucose uptake by hyperosmotic stress Like several other insulinomimetic agents, hyperosmolarity not only partially activates several insulin-specific biological responses, but also induces a state of insulin resistance. To evaluate this, the following protocol is used. 1. After serum starvation, cells (six-well plates) are then washed three times with 1 ml of KRB buffer at 37 . 2. Cells are incubated in 800 ml of KRP buffer supplemented with 0.2% BSA without or with sorbitol (600 mM) for 40 min. 3. One hundred microliters of insulin (5 nM in KRP buffer supplemented with 0.2% BSA) is added per well of six-well plates for 15 min. 4. Deoxyglucose uptake is then determined as described previously.
3. Hyperosmolarity and Membrane Ruffling Hyperosmotic stress induced an increase of nearly twofold in the number of cells with membrane ruffles (Gual et al., 2003a; Janez et al., 2000). Insulin stimulation promoted a fourfold increase in the number of cells harboring membrane ruffles, an effect that was totally abolished by sorbitol treatment. This indicates that sorbitol pretreatment inhibits insulininduced membrane ruffling markedly, suggesting that inhibition of IRS1associated PI 3-kinase activity by hyperosmotic stress appears sufficient to alter this insulin effect (Gual et al., 2003a,b).
3.1. Membrane ruffling assay As described previously (Barres et al., 2006; Gual et al., 2003a), 3T3-L1 adipocytes are grown and differentiated on glass coverslips. After overnight serum starvation, the medium is replaced by serum-free medium supplemented without or with 600 mM sorbitol before 20 min of insulin stimulation (0.5 nM ). Cells are washed twice with ice-cold PBS and fixed with 4% paraformaldehyde for 20 min on ice. After two washes with ice-cold PBS, cells are permeabilized with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin for 30 min at room temperature. After three washes with ice-cold PBS, cells are incubated with Texas red-phalloidin (Molecular Probes, Inc.) in PBS/0.1% Triton X-100/1% BSA for 30 min at room temperature. Cells are then washed twice with ice-cold PBS, and coverslips are mounted in 20 ml of Mowiol onto glass slides. Cells are examined using a Leica confocal microscope equipped with a Leica confocal laser-scanning imaging. Cells are studied at a magnification of 40 using a 1.0 to 0.50 oil immersion objective. Series of images are collected along the z axis and
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examined. In each condition, 200 cells in random fields should be examined by two different persons blind to the origin of the images. Cells that show clustering of actin staining at the periphery are scored as positive for membrane ruffles.
4. Hyperosmolarity and Signaling Pathways In 3T3-L1 adipocytes, osmotic stress promotes the activation of cytosolic Src kinase, which phosphorylates Gab1 on the tyrosine residue. Phosphorylated Gab1 recruits the Crk-II/C3G, leading to the activation of TC10, which could then modify the cortical actin structure or stimulate the actin polymerization on Glut 4 compartments. Glut 4 translocation stimulated by hyperosmotic stress could also depend on PYK2 activity, which leads to the activation of ERK, PLD, and finally atypical PKC (Gual et al., 2003b). Acute osmotic stress (from 10 to 30 min of sorbitol cell treatment) induces the phosphorylation of IRS1 on Ser307 by a mTOR-dependent pathway (Gual et al., 2003a), which in turn leads to an impairment of IRS1 functions induced by physiological insulin concentrations (Gual et al., 2003a, 2005). Hyperosmotic stress also promotes activation of a PKB phosphatase that maintains PKB in an inactive state in response to insulin. However, prolonged osmotic stress (4 h of sorbitol cell treatment) alters IRS function by inducing their degradation, thus contributing to the downregulation of insulin action (Gual et al., 2003a, 2005).
4.1. Preparation of total cell lysates The activation of SRC, ERK, JNK, p38 mitogen-activated protein kinase, PKC, and PKB can be evaluated directly by determining their phosphorylation levels by Western blotting from total cell lysates using phosphospecific antibodies. In parallel, the total amount of IRS proteins can be evaluated directly by Western blotting from total cell lysates. 3T3-L1 adipocytes are serum starved overnight in DMEM/0.5% BSA. 3T3-L1 adipocytes are incubated in serum-free medium supplemented or not with 600 mM sorbitol and subsequently treated with or without a low concentration of insulin (0.2 nM). To study the effect of pharmacological inhibitors, cells are pretreated for 30 min with various inhibitors in serumfree medium followed by incubation in serum-free medium without or with 600 mM sorbitol and pharmacological inhibitors. Cells are subsequently washed with ice-cold buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 10 mM Na4P2O7, and 2 mM sodium orthovanadate) before solubilization for 30 min at 4 in lysis buffer containing phosphatase and protease inhibitors to keep on the level of phosphorylation of proteins
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(20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 150 mM NaF, 2 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], protease inhibitors cocktail, 100 nM okadaic acid, and 1% Triton X-100). Clarified lysates are obtained after centrifugation (15 min at 15,000g at 4 ), and protein concentration is measured by the BCA assay. The addition of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases PP1 and PP2A, in the lysis buffer preserves the phosphorylation of proteins on serine/threonine residues.
4.2. Immunoprecipitation of docking proteins (Gab1 or IRS1) The phosphorylation levels of Gab1 and IRS1 and their association with signaling molecules can be evaluated after specific immunoprecipitation followed by immunoblotting with appropriate antibodies (Fig. 20.2). Clarified lysates (0.5–1 mg of proteins) prepared as described earlier are incubated for 3 h at 4 with appropriate antibodies preadsorbed on protein G–Sepharose (4 mg of antibodies/sample). After washes with lysis buffer, immune pellets are resuspended in Laemmli buffer and proteins are separated by SDS-PAGE using a 7.5 or 10% resolving gel and transferred to a polyvinylidene difluoride membrane.
4.3. Western blotting assays The membrane is blocked with saline buffer (10 mM Tris, pH 7.4, and 140 mM NaCl) containing 5% (w/v) bovine serum albumin for 2 h at room temperature and blotted overnight at 4 with commercial antibodies at the dilution indicated in the manufacturer’s instructions and at 1 mg/ml for the anti-pS307 or anti-pS632-IRS1 antibodies (home made). After incubation with horseradish peroxidase-conjugated secondary antibodies, proteins are detected by enhanced chemiluminescence. In some cases, the membrane is stripped for 30 min at 50 in 62 mM Tris, pH 6.8, 100 mM 2-mercaptoethanol, and 2% SDS and is reprobed with the appropriate antibodies.
5. Hyperosmolarity and Phosphatidylinositol 3-Kinase Activity The insulin effect on glucose uptake requires the tyrosine phosphorylation of IRS1 and the recruitment and activation of the PI 3-kinase. Hyperosmotic stress antagonizes insulin-mediated IRS1 phosphorylation, IRS1-associated PI 3-kinase activity, and subsequently Glut 4 translocation and glucose uptake (Gual et al., 2003a,b). The following assay evaluates the PI 3-kinase activity associated with IRS1 or p85 in vitro.
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5.1. Preparation of phosphatidylinositol L-a-Phosphatidylinositol
(bovine liver in ammonium salt in chloroform) can be purchased from Sigma. The required amount of PI (10-mg/sample, prepare amount for n þ 4 samples) is added to a 1.5-ml microcentrifuge tube, and the organic solvent is removed by evaporation using a Speed-Vac apparatus. Five microliters per sample of sonication buffer (10 mM HEPES, pH 7.4, 1 mM EGTA) is added, and the PI is suspended by sonication on ice for 10 min (20 30-s pulses, the samples are placed on ice to avoid overheating); alternatively, a sonication water bath can be used.
5.2. Immunoprecipitation of phosphatidylinositol 3-kinase 3T3-L1 adipocytes are starved overnight in 2 ml of serum-free medium containing 0.5% BSA. Cells are then stimulated with 600 mM sorbitol for 40 min and then stimulated without or with 0.2 nM insulin for 5 min at 37 . Each condition is realized in triplicate. Plates are put on ice and the medium is removed quickly. Cells are washed twice with 2 ml of ice-cold freshly prepared buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, and 2 mM sodium orthovanadate). Buffer A is removed, and cells are scraped in 1 ml of ice-cold lysis buffer (buffer A containing 1% Nonidet P-40, 10 mg/ml aprotinin, 1 mM PMSF) and are transferred to 1.5-ml microcentrifuge tubes. The tubes are rocked for 1h at 4 and are then centrifuged at 15,000g for 15 min at 4 to sediment insoluble material. The supernatant fraction is transferred to new 1.5-ml microcentrifuge tubes, and the protein concentration is measured in each sample. Three hundred to 500 mg of proteins is then immunoprecipitated with 5 mg of rabbit antibodies against the p85 subunit of the PI 3-kinase or against IRS1 (Upstate Biotechnology, Lake Placid, NY) preadsorbed on protein A–Sepharose beads (wet volume of protein A–Sepharose beads: 20 ml). Tubes are shaken for 3 h at 4 and then the immunoprecipitates are collected by centrifugation for 2 min at 15,000g. Tubes are placed on ice, and the immunoprecipitates are washed twice with each of the following ice-cold buffers, which have to be freshly prepared: i. Phosphate-buffered saline containing 200 mM sodium orthovanadate and 1% Nonidet P-40 ii. 100 mM Tris-HCl, pH 7.4, 500 mM LiCl, 200 mM sodium orthovanadate iii. 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 200 mM sodium orthovanadate Then, the last wash is removed completely using a Hamilton syringe and 5 ml (10 mg) of sonicated L-a-phosphatidylinositol in 10 mM HEPES, pH 7.5, 1 mM EGTA is added directly on dried protein A–Sepharose beads.
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5.3. Measurement of PI 3-kinase activity in immunoprecipitation All the following steps should be performed in agreement with the guidelines for handling radioactive samples. PI 3-kinase activity is measured at room temperature by the addition of 25 ml of a reaction mixture consisting of 40 mM HEPES, pH 7.4, 20 mM MgCl2, 80 mM ATP, 5 mCi [g-32P]ATP (Amersham BioSciences, PB10168, 10 mCi/ml, 13,000 Ci/mmol). The reaction is stopped after 20 min by the addition of 40 ml 4 N HCl, and phospholipids are extracted into chloroform by the addition of 160 ml of chloroform:methanol (1:1, vol/vol) with shaking for 10 min. The organic and aqueous phases are separated by centrifugation at 15,000g for 2 min, and organic phase-containing phospholipids (70–80 ml) are collected in microcentrifuge tubes using a Hamilton syringe or an automatic pipette. The organic phase could be stored at 20 if necessary on a practical point of view. All the preceding steps have to be performed on fresh cells. For each sample, the chloroformic phase is evaporated using a Speed-Vac apparatus, and the phospholipids are resuspended in 10 ml of chloroform and are spotted onto silica gel 60 TLC plates at 3 cm of the bottom of the plate (a maximum of 15 samples are spotted on a plate). TLC plates are developed by chromatography in chloroform/methanol/ammoniac/water (60 ml/ 47 ml/4.4 ml /8.8 ml) until the migration front reaches 1 cm of the top of the plate (this step should be performed under a chemical hood). The plate is dried and radiolabeled lipids are visualized by autoradiography. Quantification is performed by densitometry scanning of the autoradiogram.
6. Conclusion This chapter summarizes the current approaches permitting the study of hyperosmolarity on membrane ruffling, Glut 4 translocation, glucose transport, and signaling pathways in 3T3-L1 adipocytes. It should be noted that hyperosmotic stress (usually induced by high extracellular concentrations of sorbitol) is not a physiological stimulus, but is used as a tool that could lead to the discovery of novel molecular mechanisms of glucose transport and also for a better understanding of the molecular mechanisms of cellular insulin resistance.
ACKNOWLEDGMENTS This work was supported by grants from the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM, France), the University of Nice, the Fondation Bettencourt- Schueller, the Re´gion Provence Alpes Coˆte d’Azur, the Conseil Ge´ne´ral des Alpes-Maritimes, and the Comite´ Doyen Jean Le´pine (Nice, France). Part of the work was supported by a grant from ALFEDIAM-Takeda Laboratories (Puteaux, France) to J.-F. Tanti. P. Gual was successively supported by a Fellowship from La Ligue Contre le Cancer and from ALFEDIAM.
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REFERENCES Barres, R., Gre´meaux, T., Gual, P., Gonzalez, T., Gugenheim, J., Tran, A., Le MarchandBrustel, Y., and Tanti, J. F. (2006). Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation. Mol. Endocrinol. 20, 2864–2875. Bryant, N. J., Govers, R., and James, D. E. (2002). Regulated transport of the glucose transporter GLUT4. Nat. Rev. Mol. Cell. Biol. 3, 267–277. Chen, D., Fucini, R. V., Olson, A. L., Hemmings, B. A., and Pessin, J. E. (1999). Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state. Mol. Cell. Biol. 19, 4684–4694. Dugani, C. B., and Klip, A. (2005). Glucose transporter 4: Cycling, compartments and controversies. EMBO Rep. 6, 1137–1142. Gual, P., Gonzalez, T., Gre´meaux, T., Barres, R., Le Marchand-Brustel, Y., and Tanti, J. F. (2003a). Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes. J. Biol. Chem. 278, 26550–26557. Gual, P., Le Marchand-Brustel, Y., and Tanti, J. F. (2003b). Positive and negative regulation of glucose uptake by hyperosmotic stress. Diabetes Metab. 29, 566–575. Gual, P., Le Marchand-Brustel, Y., and Tanti, J. F. (2005). Positive and negative regulation of insulin signaling through IRS-1 phosphorylation. Biochimie 87, 99–109. Gual, P., Shigematsu, S., Kanzaki, M., Gre´meaux, T., Gonzalez, T., Pessin, J. E., Le Marchand-Brustel, Y., and Tanti, J. F. (2002). A Crk-II/TC10 signaling pathway is required for osmotic shock-stimulated glucose transport. J. Biol. Chem. 277, 43980–43986. Heydrick, S. J., Gautier, N., Olichon-Berthe, C., Van Obberghen, E., and Le MarchandBrustel, Y. (1995). Early alteration of insulin stimulation of PI 3-kinase in muscle and adipocyte from gold thioglucose obese mice. Am. J. Physiol. 268, E604–E612. Janez, A., Worrall, D. S., Imamura, T., Sharma, P. M., and Olefsky, J. M. (2000). The osmotic shock-induced glucose transport pathway in 3T3-L1 adipocytes is mediated by Gab-1 and requires Gab-1-associated phosphatidylinositol 3-kinase activity for full activation. J. Biol. Chem. 275, 26870–26876. Komjati, M., Kastner, G., Waldhausl, W., and Bratusch-Marrain, P. (1988). Detrimental effect of hyperosmolality on insulin-stimulated glucose metabolism in adipose and muscle tissue in vitro. Biochem. Med. Metab. Biol. 39, 312–318. Rui, L., Fisher, T. L., Thomas, J., and White, M. F. (2001). Regulation of insulin/insulinlike growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2. J. Biol. Chem. 276, 40362–40367. Saltiel, A. R., and Kahn, C. R. (2001). Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414, 799–806. Saltiel, A. R., and Pessin, J. E. (2002). Insulin signaling pathways in time and space. Trends Cell Biol. 12, 65–71. Vom Dahl, S., Hallbrucker, C., Lang, F., Gerok, W., and Ha¨ussinger, D. (1991). Regulation of liver cell volume and proteolysis by glucagon and insulin. Biochem. J. 278, 771–777. Watson, R. T., Kanzaki, M., and Pessin, J. E. (2004). Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes. Endocr. Rev. 25, 177–204.
T W E N T Y
Osmotic Regulation of Cellular Glucose Uptake Philippe Gual, Teresa Gonzalez, Thierry Gremeaux, Yannick Le Marchand-Brustel, and Jean-Franc¸ois Tanti Contents 1. Introduction 2. Hyperosmolarity and Glucose Transport 2.1. Differentiation of 3T3-L1 adipocytes 2.2. Glucose uptake induced in response to hyperosmolarity 2.3. Study of pathways involved in the hyperosmotic effect on glucose uptake 2.4. Inhibition of insulin-stimulated glucose uptake by hyperosmotic stress 3. Hyperosmolarity and Membrane Ruffling 3.1. Membrane ruffling assay 4. Hyperosmolarity and Signaling Pathways 4.1. Preparation of total cell lysates 4.2. Immunoprecipitation of docking proteins (Gab1 or IRS1) 4.3. Western blotting assays 5. Hyperosmolarity and Phosphatidylinositol 3-Kinase Activity 5.1. Preparation of phosphatidylinositol 5.2. Immunoprecipitation of phosphatidylinositol 3-kinase 5.3. Measurement of PI 3-kinase activity in immunoprecipitation 6. Conclusion Acknowledgments References
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Abstract This chapter describes various approaches allowing the study of hyperosmolarity in the functions of 3T3-L1 adipocytes. Hyperosmolarity mimics insulin responses, such as glucose uptake and membrane ruffling, but also antagonizes these insulin effects, which can be evaluated in 3T3-L1 adipocytes. The molecular mechanisms of these effects can be also investigated by measuring the
INSERM U 568; University of Nice Sophia-Antipolis, Nice, France Methods in Enzymology, Volume 428 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)28020-6
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2007 Elsevier Inc. All rights reserved.
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activation of different signaling pathways: (i) the phosphorylation of docking proteins on tyrosine and serine residues (serines 307 and 632), (ii) the phosphorylation of serine/threonine kinases, and (iii) the activation of phosphatidylinositol 3-kinase.
1. Introduction Exposure of many mammalian cell types to metabolic or osmotic stress results in an acute increase in the rate of glucose uptake. This adaptive response allows the cells to maintain or regain their ATP levels by increasing flux through the glycolytic pathway. However, like several other insulinomimetic agents, hyperosmolarity not only partly activates several insulinspecific biological responses, but also induces a state of insulin resistance. Conditions dehydrating insulin target tissues such as hyperosmolarity or amino acid deprivation are frequently associated with insulin resistance. Sepsis and burn injury are associated with dehydration-induced insulin resistance. Furthermore, in rat epididymal fat cells, hyperosmotic stress markedly reduces in vitro insulin-induced glucose transport (Komjati et al., 1988). In perfused rat liver, hyperosmolarity impairs insulin-mediated cell swelling and reverses the proteolysis inhibition induced by insulin (Vom Dahl et al., 1991). In 3T3-L1 adipocytes, pretreatment with sorbitol strongly decreases the ability of insulin to stimulate glucose uptake, lipogenesis, glycogen synthesis, and membrane ruffling (Chen et al., 1999; Gual et al., 2003a). Glucose uptake induced by insulin in muscle and adipose tissues is due to the translocation of the glucose transporter Glut 4 from an intracellular pool to the plasma membrane (Bryant et al., 2002; Saltiel and Kahn, 2001). These biological responses require tyrosine phosphorylation of insulin receptor substrate-1 (IRS1), which leads to the binding and activation of phosphatidylinositol 3-kinase (PI 3-kinase). Downstream effectors of PI 3-kinase such as protein kinase B (PKB) or atypical PKC are involved in Glut 4 translocation. Furthermore, it has been shown that insulininduced Glut 4 translocation also requires activation of a second pathway, which is completely independent of PI 3-kinase activity. In adipocytes, the Cbl protooncogene, associated with Cbl-associated protein and adapter protein containing PH and SH2 domain, is phosphorylated in response to insulin and regulates glucose uptake. Once phosphorylated, Cbl recruits the adapter protein Crk-II in a complex with C3G, a GDP to GTP exchange factor for TC10, a Rho family GTPase, allowing for its activation, which regulates the traffic of Glut 4-containing vesicles. GTP-bound TC10 could participate in Glut 4 translocation through a modification of cortical actin or a stimulation of actin polymerization at the level of Glut 4 compartments
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(Scheme 20.1) (Dugani and Klip, 2005; Gual et al., 2003b; Saltiel and Pessin, 2002; Watson et al., 2004). In both adipocyte (Scheme 20.1) and muscle cells, hyperosmolarity promotes glucose uptake by multiple mechanisms that do not require the PI 3-kinase/PKB pathway (Gual et al., 2003b) but are dependent on the cell type. In muscle, osmotic stress induces glucose uptake by stimulation of AMPkinase and/or inhibition of Glut 4 endocytosis. In adipocytes, activation of the Grb2-associated binder-1 (Gab1)-dependent signaling pathway plays an important role in osmotic stress-mediated glucose uptake (Gual et al., 2003a; Janez et al., 2000). Upon sorbitol stimulation, the phosphorylated Gab1 recruits Crk-II via its SH2 domain. The Crk-II SH3 domains are constitutively associated with C3G, a GDP-to-GTP exchange factor for several small GTP-binding proteins, including TC10 (Gual et al., 2002). The activation of TC10 and remodeling of cortical actin are required for osmotic shock-mediated Glut 4 translocation and glucose uptake (Gual et al., 2002). Apart from its insulin-like effects, hyperosmolarity leads to cellular insulin resistance (Fig. 20.1) mediated by both prevention of PKB activation Adipocyte Glucose Hyperosmotic Caveolin stress Lipid raft TC10 C3G Src
Actin
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P CrkII P Y Y Gab1Y P PI3-K Y
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Scheme 20.1 Signaling pathways activated by hyperosmotic stress to induce Glut 4 translocation in 3T3-L1 adipocytes and adipose cells. Osmotic stress induces glucose uptake by activation of Gab1-dependent signaling pathways. Sorbitol promotes the activation of cytosolic Src kinase, which phosphorylates Gab1 on the tyrosine residue. Phosphorylated Gab1 recruits the Crk-II/C3G complex. C3G, a guanine nucleotide exchange factor, exchanges GDP for GTP on TC10. Activated GTP-bound TC10 could then modify the cortical actin structure or stimulate the actin polymerization on Glut 4 compartments. Osmotic stress recruits vesicles containing both Glut 4 and VAMP2 in adipocytes. Glut 4 translocation stimulated by hyperosmotic stress could also depend on PYK2 activity, which leads to the activation of ERK, PLD, and finally atypical PKC. Adapted from Gual et al. (2003b).
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14 *
2-DOG uptake (fold stimulation)
12 10
*
8
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6 4 2 0 Basal
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Figure 20.1 Hyperosmolarity and glucose transport. After serum starvation, 3T3-L1 adipocytes were treated without or with insulin (100 nM) or sorbitol (600 mM) for 20 min (empty bars) or pretreated with sorbitol (600 mM) for 40 min before a 20-min insulin stimulation (100 nM) (gray bar). Uptake of [2-3H]deoxyglucose was measured during a 3-min period. Means SEM of three independent experiments are shown.
IB:
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+
a-pS307
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a-IRS1
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Figure 20.2 Hyperosmotic stress triggers the phosphorylation of IRS1 on serines 307 and 632. 3T3-L1 adipocytes were stimulated without or with 600 mM sorbitol for 20 min at 37. Cell lysates were then immunoprecipitated (IP) using anti-IRS1 antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and blotted (IB) using antipSer307 IRS1 or anti-pSer632 IRS1 antibodies, as indicated. The membrane was then stripped and probed with anti-IRS1 antibodies. Representative autoradiographs are shown.
(Chen et al., 1999) and inhibition of the IRS1 function (Gual et al., 2003a). Chen et al. (1999) have reported that hyperosmolarity prevents insulininduced PKB activation. They suggest that a calyculin A- or okadaic acidsensitive protein phosphatase leads to the deactivation of PKB (Chen et al., 1999). Furthermore, a short-term osmotic stress induces the phosphorylation of IRS1 on serine 307 by an mTOR-dependent pathway (Gual et al., 2003a) but also on serine 632 (Fig. 20.2). This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations (Gual et al., 2003a). However, prolonged osmotic stress alters IRS1 and IRS2 functions by inducing their degradation (Gual et al.,
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2003a,b; Rui et al., 2001), which could contribute to the downregulation of insulin action.
2. Hyperosmolarity and Glucose Transport In muscle and adipose cells, hyperosmolarity triggers the cell surface accumulation of Glut 4, leading to an increase in glucose transport (Dugani and Klip, 2005; Gual et al., 2003b; Saltiel and Pessin, 2002; Watson et al., 2004). While the hyperosmotic stress mimics insulin responses, it also antagonizes insulin effects. Both effects can be evaluated in 3T3-L1 adipocytes (see Scheme 20.1).
2.1. Differentiation of 3T3-L1 adipocytes 3T3-L1 fibroblasts are a continuous substrain of 3T3 (Swiss albino) developed through clonal isolation (CL-173, ATCC). The cells undergo a preadipose to adipose-like conversion as they progress from a rapidly dividing to a confluent and contact inhibited state. After differentiation, Glut 4 translocation and glucose uptake can be induced in response to insulin. The differentiation protocol is as follows. 3T3-L1 fibroblasts are grown in six-well plates or 100-mm dishes in Dulbecco’s modified Eagle’s medium (DMEM) containing 25 mM glucose and 10% calf serum and induced to differentiate in adipocytes. Two days after confluence, medium is changed for a differentiation buffer, which includes DMEM, 25 mM glucose, 10% fetal calf serum supplemented with isobutylmethylxanthine (0.5 mM), dexamethasone (0.25 mM), thiazolidinediones (10 mM), and insulin (5 mg/ml). The medium is removed after 2 days and replaced with DMEM, 25 mM glucose, 10% fetal calf serum supplemented with insulin, and thiazolidinediones (10 mM) for 2 more days. Then, the cells are fed every 2 days with DMEM, 25 mM glucose, and 10% fetal calf serum. 3T3-L1 adipocytes are used 8 to 15 days after the beginning of the differentiation protocol. Sixteen hours before each experiment, the medium is changed to serum-free DMEM supplemented with 0.5% bovine serum albumin (BSA).
2.2. Glucose uptake induced in response to hyperosmolarity The technique allows for the measurement of glucose uptake using a glucose analog (2-deoxyglucose, DOG). The labeled 2-DOG is transported into the cells with high affinity, phosphorylated but not further metabolized. The labeled 2-DOG phosphate is trapped in the cell, and the rate of uptake can be taken as a measure of unidirectional transport.
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1. After serum starvation, cells (six-well plates) are washed three times with 1 ml of Krebs–Ringer phosphate (KRP) buffer (10 mM phosphate buffer, pH 7.4, 1.25 mM MgSO4, 1.25 mM CaCl2, 136 mM NaCl, 4.7 mM KCl) at 37 . 2. Cells are incubated in 900 ml of KRP buffer supplemented with 0.2% BSA without or with sorbitol (600 mM) for 20 min. 3. Measurement of glucose transport is initiated by the addition of 100 ml of KRP buffer supplemented with 0.2% BSA and containing 1 mM deoxyD-glucose and 0.5 mCi of 2-[3H]deoxy-D-glucose. 4. The reaction is stopped after 3 min by rapid aspiration, and cells are washed four times with ice-cold phosphate-buffered saline (PBS). 5. Cells are lysed with 700 ml of KRP and 1% Triton X-100. After brief sonication, 300 ml of lysates is mixed with 3 ml of liquid scintillation, and the radioactivity associated with the cells is counted in a b-counter using the tritium program. 6. Radioactivity is normalized by measuring the protein concentration in each sample by the bicinchoninic acid (BCA) assay (Pierce). Do not forget to count an aliquot of the radioactive deoxyglucose solution (using a commercial scintillation fluid), which will allow expressing the results in absolute amounts of glucose taken up by the cells.
2.3. Study of pathways involved in the hyperosmotic effect on glucose uptake To determine the pathways required for hyperosmotic stress-induced glucose transport or the study agents capable of preventing this response, 3T3-L1 adipocytes are pretreated with a specific inhibitor or agents (at step 2) before stimulation with sorbitol (600 mM ). Deoxyglucose uptake is then measured as described earlier. The TC10 activity per se can be inhibited by Clostridium difficile toxin B (1 mg/ml), an inhibitor of the Rho family proteins. Latrunculin B (10 mM ) (Calbiochem), an agent that sequesters actin monomers, or jasplakinolide (10 mM ) (Molecular Probes, Eugene, OR), a stabilizer of filamentous actin, also inhibits sorbitol-induced glucose uptake. It is important to note that latrunculin B does not alter the early proximal osmotic shock signaling events such as Gab1 phosphorylation and its association with Crk-II. After serum starvation, 3T3-L1 adipocytes are treated without or with C. difficile toxin B (1 mg/ml), latrunculin B (10 mM ), or jasplakinolide (10 mM ) for 120 min at 37 . Cells are then either left untreated or stimulated with 600 mM sorbitol for 20 min at 37 .
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2.4. Inhibition of insulin-stimulated glucose uptake by hyperosmotic stress Like several other insulinomimetic agents, hyperosmolarity not only partially activates several insulin-specific biological responses, but also induces a state of insulin resistance. To evaluate this, the following protocol is used. 1. After serum starvation, cells (six-well plates) are then washed three times with 1 ml of KRB buffer at 37 . 2. Cells are incubated in 800 ml of KRP buffer supplemented with 0.2% BSA without or with sorbitol (600 mM) for 40 min. 3. One hundred microliters of insulin (5 nM in KRP buffer supplemented with 0.2% BSA) is added per well of six-well plates for 15 min. 4. Deoxyglucose uptake is then determined as described previously.
3. Hyperosmolarity and Membrane Ruffling Hyperosmotic stress induced an increase of nearly twofold in the number of cells with membrane ruffles (Gual et al., 2003a; Janez et al., 2000). Insulin stimulation promoted a fourfold increase in the number of cells harboring membrane ruffles, an effect that was totally abolished by sorbitol treatment. This indicates that sorbitol pretreatment inhibits insulininduced membrane ruffling markedly, suggesting that inhibition of IRS1associated PI 3-kinase activity by hyperosmotic stress appears sufficient to alter this insulin effect (Gual et al., 2003a,b).
3.1. Membrane ruffling assay As described previously (Barres et al., 2006; Gual et al., 2003a), 3T3-L1 adipocytes are grown and differentiated on glass coverslips. After overnight serum starvation, the medium is replaced by serum-free medium supplemented without or with 600 mM sorbitol before 20 min of insulin stimulation (0.5 nM ). Cells are washed twice with ice-cold PBS and fixed with 4% paraformaldehyde for 20 min on ice. After two washes with ice-cold PBS, cells are permeabilized with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin for 30 min at room temperature. After three washes with ice-cold PBS, cells are incubated with Texas red-phalloidin (Molecular Probes, Inc.) in PBS/0.1% Triton X-100/1% BSA for 30 min at room temperature. Cells are then washed twice with ice-cold PBS, and coverslips are mounted in 20 ml of Mowiol onto glass slides. Cells are examined using a Leica confocal microscope equipped with a Leica confocal laser-scanning imaging. Cells are studied at a magnification of 40 using a 1.0 to 0.50 oil immersion objective. Series of images are collected along the z axis and
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examined. In each condition, 200 cells in random fields should be examined by two different persons blind to the origin of the images. Cells that show clustering of actin staining at the periphery are scored as positive for membrane ruffles.
4. Hyperosmolarity and Signaling Pathways In 3T3-L1 adipocytes, osmotic stress promotes the activation of cytosolic Src kinase, which phosphorylates Gab1 on the tyrosine residue. Phosphorylated Gab1 recruits the Crk-II/C3G, leading to the activation of TC10, which could then modify the cortical actin structure or stimulate the actin polymerization on Glut 4 compartments. Glut 4 translocation stimulated by hyperosmotic stress could also depend on PYK2 activity, which leads to the activation of ERK, PLD, and finally atypical PKC (Gual et al., 2003b). Acute osmotic stress (from 10 to 30 min of sorbitol cell treatment) induces the phosphorylation of IRS1 on Ser307 by a mTOR-dependent pathway (Gual et al., 2003a), which in turn leads to an impairment of IRS1 functions induced by physiological insulin concentrations (Gual et al., 2003a, 2005). Hyperosmotic stress also promotes activation of a PKB phosphatase that maintains PKB in an inactive state in response to insulin. However, prolonged osmotic stress (4 h of sorbitol cell treatment) alters IRS function by inducing their degradation, thus contributing to the downregulation of insulin action (Gual et al., 2003a, 2005).
4.1. Preparation of total cell lysates The activation of SRC, ERK, JNK, p38 mitogen-activated protein kinase, PKC, and PKB can be evaluated directly by determining their phosphorylation levels by Western blotting from total cell lysates using phosphospecific antibodies. In parallel, the total amount of IRS proteins can be evaluated directly by Western blotting from total cell lysates. 3T3-L1 adipocytes are serum starved overnight in DMEM/0.5% BSA. 3T3-L1 adipocytes are incubated in serum-free medium supplemented or not with 600 mM sorbitol and subsequently treated with or without a low concentration of insulin (0.2 nM). To study the effect of pharmacological inhibitors, cells are pretreated for 30 min with various inhibitors in serumfree medium followed by incubation in serum-free medium without or with 600 mM sorbitol and pharmacological inhibitors. Cells are subsequently washed with ice-cold buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 10 mM Na4P2O7, and 2 mM sodium orthovanadate) before solubilization for 30 min at 4 in lysis buffer containing phosphatase and protease inhibitors to keep on the level of phosphorylation of proteins
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(20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 150 mM NaF, 2 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], protease inhibitors cocktail, 100 nM okadaic acid, and 1% Triton X-100). Clarified lysates are obtained after centrifugation (15 min at 15,000g at 4 ), and protein concentration is measured by the BCA assay. The addition of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases PP1 and PP2A, in the lysis buffer preserves the phosphorylation of proteins on serine/threonine residues.
4.2. Immunoprecipitation of docking proteins (Gab1 or IRS1) The phosphorylation levels of Gab1 and IRS1 and their association with signaling molecules can be evaluated after specific immunoprecipitation followed by immunoblotting with appropriate antibodies (Fig. 20.2). Clarified lysates (0.5–1 mg of proteins) prepared as described earlier are incubated for 3 h at 4 with appropriate antibodies preadsorbed on protein G–Sepharose (4 mg of antibodies/sample). After washes with lysis buffer, immune pellets are resuspended in Laemmli buffer and proteins are separated by SDS-PAGE using a 7.5 or 10% resolving gel and transferred to a polyvinylidene difluoride membrane.
4.3. Western blotting assays The membrane is blocked with saline buffer (10 mM Tris, pH 7.4, and 140 mM NaCl) containing 5% (w/v) bovine serum albumin for 2 h at room temperature and blotted overnight at 4 with commercial antibodies at the dilution indicated in the manufacturer’s instructions and at 1 mg/ml for the anti-pS307 or anti-pS632-IRS1 antibodies (home made). After incubation with horseradish peroxidase-conjugated secondary antibodies, proteins are detected by enhanced chemiluminescence. In some cases, the membrane is stripped for 30 min at 50 in 62 mM Tris, pH 6.8, 100 mM 2-mercaptoethanol, and 2% SDS and is reprobed with the appropriate antibodies.
5. Hyperosmolarity and Phosphatidylinositol 3-Kinase Activity The insulin effect on glucose uptake requires the tyrosine phosphorylation of IRS1 and the recruitment and activation of the PI 3-kinase. Hyperosmotic stress antagonizes insulin-mediated IRS1 phosphorylation, IRS1-associated PI 3-kinase activity, and subsequently Glut 4 translocation and glucose uptake (Gual et al., 2003a,b). The following assay evaluates the PI 3-kinase activity associated with IRS1 or p85 in vitro.
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5.1. Preparation of phosphatidylinositol L-a-Phosphatidylinositol
(bovine liver in ammonium salt in chloroform) can be purchased from Sigma. The required amount of PI (10-mg/sample, prepare amount for n þ 4 samples) is added to a 1.5-ml microcentrifuge tube, and the organic solvent is removed by evaporation using a Speed-Vac apparatus. Five microliters per sample of sonication buffer (10 mM HEPES, pH 7.4, 1 mM EGTA) is added, and the PI is suspended by sonication on ice for 10 min (20 30-s pulses, the samples are placed on ice to avoid overheating); alternatively, a sonication water bath can be used.
5.2. Immunoprecipitation of phosphatidylinositol 3-kinase 3T3-L1 adipocytes are starved overnight in 2 ml of serum-free medium containing 0.5% BSA. Cells are then stimulated with 600 mM sorbitol for 40 min and then stimulated without or with 0.2 nM insulin for 5 min at 37 . Each condition is realized in triplicate. Plates are put on ice and the medium is removed quickly. Cells are washed twice with 2 ml of ice-cold freshly prepared buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, and 2 mM sodium orthovanadate). Buffer A is removed, and cells are scraped in 1 ml of ice-cold lysis buffer (buffer A containing 1% Nonidet P-40, 10 mg/ml aprotinin, 1 mM PMSF) and are transferred to 1.5-ml microcentrifuge tubes. The tubes are rocked for 1h at 4 and are then centrifuged at 15,000g for 15 min at 4 to sediment insoluble material. The supernatant fraction is transferred to new 1.5-ml microcentrifuge tubes, and the protein concentration is measured in each sample. Three hundred to 500 mg of proteins is then immunoprecipitated with 5 mg of rabbit antibodies against the p85 subunit of the PI 3-kinase or against IRS1 (Upstate Biotechnology, Lake Placid, NY) preadsorbed on protein A–Sepharose beads (wet volume of protein A–Sepharose beads: 20 ml). Tubes are shaken for 3 h at 4 and then the immunoprecipitates are collected by centrifugation for 2 min at 15,000g. Tubes are placed on ice, and the immunoprecipitates are washed twice with each of the following ice-cold buffers, which have to be freshly prepared: i. Phosphate-buffered saline containing 200 mM sodium orthovanadate and 1% Nonidet P-40 ii. 100 mM Tris-HCl, pH 7.4, 500 mM LiCl, 200 mM sodium orthovanadate iii. 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 200 mM sodium orthovanadate Then, the last wash is removed completely using a Hamilton syringe and 5 ml (10 mg) of sonicated L-a-phosphatidylinositol in 10 mM HEPES, pH 7.5, 1 mM EGTA is added directly on dried protein A–Sepharose beads.
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5.3. Measurement of PI 3-kinase activity in immunoprecipitation All the following steps should be performed in agreement with the guidelines for handling radioactive samples. PI 3-kinase activity is measured at room temperature by the addition of 25 ml of a reaction mixture consisting of 40 mM HEPES, pH 7.4, 20 mM MgCl2, 80 mM ATP, 5 mCi [g-32P]ATP (Amersham BioSciences, PB10168, 10 mCi/ml, 13,000 Ci/mmol). The reaction is stopped after 20 min by the addition of 40 ml 4 N HCl, and phospholipids are extracted into chloroform by the addition of 160 ml of chloroform:methanol (1:1, vol/vol) with shaking for 10 min. The organic and aqueous phases are separated by centrifugation at 15,000g for 2 min, and organic phase-containing phospholipids (70–80 ml) are collected in microcentrifuge tubes using a Hamilton syringe or an automatic pipette. The organic phase could be stored at 20 if necessary on a practical point of view. All the preceding steps have to be performed on fresh cells. For each sample, the chloroformic phase is evaporated using a Speed-Vac apparatus, and the phospholipids are resuspended in 10 ml of chloroform and are spotted onto silica gel 60 TLC plates at 3 cm of the bottom of the plate (a maximum of 15 samples are spotted on a plate). TLC plates are developed by chromatography in chloroform/methanol/ammoniac/water (60 ml/ 47 ml/4.4 ml /8.8 ml) until the migration front reaches 1 cm of the top of the plate (this step should be performed under a chemical hood). The plate is dried and radiolabeled lipids are visualized by autoradiography. Quantification is performed by densitometry scanning of the autoradiogram.
6. Conclusion This chapter summarizes the current approaches permitting the study of hyperosmolarity on membrane ruffling, Glut 4 translocation, glucose transport, and signaling pathways in 3T3-L1 adipocytes. It should be noted that hyperosmotic stress (usually induced by high extracellular concentrations of sorbitol) is not a physiological stimulus, but is used as a tool that could lead to the discovery of novel molecular mechanisms of glucose transport and also for a better understanding of the molecular mechanisms of cellular insulin resistance.
ACKNOWLEDGMENTS This work was supported by grants from the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM, France), the University of Nice, the Fondation Bettencourt- Schueller, the Re´gion Provence Alpes Coˆte d’Azur, the Conseil Ge´ne´ral des Alpes-Maritimes, and the Comite´ Doyen Jean Le´pine (Nice, France). Part of the work was supported by a grant from ALFEDIAM-Takeda Laboratories (Puteaux, France) to J.-F. Tanti. P. Gual was successively supported by a Fellowship from La Ligue Contre le Cancer and from ALFEDIAM.
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