Phosphatidylinositol 3,4,5- tris phosphate: an early mediator of insulin-stimula - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:12 |只看该作者 |倒序浏览 |打印

作者：Nicolas Markadieu, Daniel Blero, Alain Boom, Christophe Erneux, and Renaud Beauwens作者单位：1 Department of Cell Physiology, 2 Interdisciplinary Research Institute, 3 Laboratory of Histology, Neuroanatomy and Neuropathology, Université Libre de Bruxelles, Campus Erasme, 1070 Bruxelles, Belgium



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   Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP 3 and PI( 3, 4 )P 2 but not PI( 4, 5 )P 2 stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP 3 levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP 3 was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP 3 also increased PKB phosphorylation. Taken together, the present results establish that the D -3-phosphorylated phosphoinositides PIP 3 and PI( 3, 4 )P 2 mediate the effect of insulin on sodium transport across A6 cell monolayers.
      【关键词】 epithelial Na channel protein kinase B phosphatase and TENsin homolog deleted on chromosome kidney, [1 |1 J* S1 ~- [2 S
   BY REGULATING TOTAL body sodium, the kidney controls extracellular fluid volume and blood pressure. The fine tuning of this homeostasis is performed by the distal nephron and, more particularly, the cortical collecting duct where the principal cells express the amiloride-sensitive epithelial Na channels (ENaC) at the apical side, allowing entry of Na and Na -K -ATPase, extruding Na at the basolateral side ( 34, 36 ). The concerted action of these two membrane proteins results in transepithelial sodium absorption from the urinary to the extracellular space. The activity of ENaC is usually the rate-limiting step of the sodium transport and is under the control of various hormones, mainly aldosterone ( 2, 4, 7, 10, 13, 16, 41 ), insulin ( 3, 13, 23, 35, 40 ), and antidiuretic hormone ( 22, 46 ). The signal transduction pathways linking each of the hormone receptors to ENaC are still under investigation. Insulin stimulates the sodium transport by upregulating the number of apical ENaCs, or by increasing the open probability, or by a combination of both mechanisms ( 1, 3, 19, 30 ). The A6 cell line derived from the kidney of Xenopus laevis oocytes is currently used as a model to investigate sodium transport. When grown on a permeable support, A6 cells form a polarized tight epithelium that displays a high epithelial electrical resistance. After binding to its receptor at the basolateral membrane ( 5 ), insulin appears to stimulate phosphatidylinositol 3-kinase (PI 3-kinase) activity in A6 cells ( 35 ). The PI 3-kinase inhibitors LY-294002 and wortmannin block the insulin-stimulated sodium transport in a dose-dependent manner ( 35 ). The present study aims to further establish the importance of phosphorylated lipid products of PI 3-kinase by adding their permeant forms directly to cells. Our results support the importance of PIP 3 and PI( 3, 4 )P 2 in mediating the increase in sodium transport.
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EXPERIMENTAL PROCEDURES
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Materials. Phosphatidylinositol phosphate derivatives [diC16-PIP 3 sodium salt, diC16-PI( 3, 4 )P 2 ammonium salt and sodium salt, diC16-PI( 3, 5 )P 2 sodium salt, diC16-PI( 4, 5 )P 2 ammonium salt, and diC16-PI3P sodium salt], fluorescent NBD-conjugated phosphatidylinositol phosphate derivatives [NBD-diC16-PIP 3, NBD-diC16-PI( 3, 4 )P 2, and NBD-diC16-PI( 4, 5 )P 2 ], and histone polyamine carrier were purchased from Echelon Biosciences (Salt Lake City, UT). Porcine insulin and DABCO reagent were purchased from Sigma (St. Louis, MO). Gelvatol was purchased from Monsanto. Protease inhibitor tablets were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Millicell inserts were purchased from Millipore (Bedford, MA). Transwell inserts were purchased from Corning Costar (Cambridge, MA). Tissue culture flasks were purchased from Sarstedt (Nümbrecht, Germany). Media was purchased from Invitrogen Life Technologies (Carlsbad, CA). Electrophysiological measurements were realized by use of an epithelial voltohmmeter (EVom) from World Precision Instruments (Sarasota, PA). The current evaluated by this technique is called an epithelial open circuit Na current. A polyclonal antibody to phosphatase and TENsin homolog deleted on chromosome 10 (PTEN; used at a 1,000-fold dilution) was purchased from Alexis Biochemicals (San Diego, CA). PhosphoPKB Thr 308 and Ser 473 (at a 250-fold dilution) and total PKB (at a 500-fold dilution) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal antibodies to phosphotyrosine (at a 1,000-fold dilution) and to PI 3-kinase p85 subunit (at a 250-fold dilution) were purchased from Upstate (Lake Placid, NY). Protein G Sepharose was purchased from Amersham (Arlington Heights, IL). Peroxidase-labeled secondary antibody was purchased from DAKO (Glostrup, Denmark). Geneticin, Lipofectamine 2000 used as a transfection reagent, and the cDNA encoding human PTEN in pcDNA3 were purchased from Invitrogen Life Technologies. EGFP-C1 vector was purchased from Clontech (Palo Alto, CA). X. laevis oocytes were a generous gift from Prof. W. Van Driessche (Department of Physiology, Katholiek Universiteit te Leuven, Belgium)./ V4 i3 v  B; `

Cell culture. A6 cells were received from Prof. W. Van Driessche (Department of Physiology, Katholiek Universiteit te Leuven, Belgium) and originated from Prof. J. P. Johnson (University of Pittsburgh, PA). A6 cells were grown in 28°C in a humidified incubator gassed with 1% CO 2 in O 2. Cells were cultured in 34% Ham's F-12, 34% Leibovitz's L-15, 20% water, 10% FBS serum, 3.8 mM L -glutamine, 87 IU penicillin, 87 µg/ml streptomycin, and 8 mM NaHCO3 medium. The osmolality of the growth medium was 260 mosmol/kgH 2 O. Cells were plated twice a week. For biochemical experiments, cells were cultured in 75-cm 2 plastic culture flasks, in 60-cm 2 plastic culture dishes, or subcultured onto 100-mm Costar Transwell inserts. Cells were used 7-14 days after seeding. For electrophysiological experiments, A6 cells were subcultured onto 24-mm Millicell inserts and incubated overnight in serum-free medium followed by a 4-h incubation in amphibian Ringer solution before electrical measurements. Amphibian Ringer solution had the following composition (in mM): 115 NaCl, 10 glucose, 5 Tris·HEPES, 2.5 KHCO 3, 1 CaCl 2, 1 MgCl 2, pH 7.7, osmolality 260 mosmol/kgH 2 O.
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Cell delivery of phosphorylated phosphoinositides. Delivery of anionic phosphatidylinositol phosphate derivatives across the cell membrane by complexing with positive lysine-rich histone followed the procedure described by the manufacturer (Echelon Biosciences). Cell monolayers grown to confluence on Millicell insert were incubated overnight in serum-free medium that was replaced on the following day by amphibian Ringer solution, and 4 h later phosphoinositides were added together with histone as a carrier. Di-C16 phosphoinositides were chosen as long carbon chain derivatives appear more abundant membrane constituents than similar derivatives with a shorter carbon chain ( 42 ). The phosphoinositide derivatives were complexed with a lysine-rich histone (10-min incubation with vortexing). The concentration ratio of phosphoinositides (20 µM) to histone (5 µM) used in most experiments ( 32, 39 ) was in the range suggested by the manufacturer (Echelon Biosciences), i.e., 1:0.1 to 1:1 (phosphoinositides:histone). The concentrations of histone used never exceeded 5 µM as preliminary experiments showed that a higher concentration slightly increased sodium transport.
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Electrophysiology. Confluent monolayers of A6 cells grown on Millicell and Transwell 4,200 ·cm 2 and a mean transepithelial potential difference 20 mV. Resistance and potential difference were measured using an EVom with chopstick electrodes made of Ag-AgCl pellets. Sodium transport (I Na  ) was calculated from the transepithelial potential difference and resistance. Amiloride added to the apical bathing medium completely inhibited this current validating such computation of I Na . Insulin (100 nM) was always added to the basolateral bathing medium.
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Confocal microscopy. For the determination of the intracellular uptake of permeant phospholipids, we used fluorescent derivatives complexed with histone. A6 cells were seeded onto 12-mm clear Transwell inserts and treated as described in Cell culture. Monolayers were incubated for different times with 20 µM of permeant NBD-diC16PIP 3, NBD-diC16PI( 3, 4 )P 2, and NBD-diC16PI( 4, 5 )P 2. After incubation, inserts were transferred on ice and rinsed 10 times with ice-cold Ringer solution. The A6 cells monolayers were then fixed for 10 min at room temperature in 4% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.4. Inserts were rinsed three times in Ringer solution and mounted with a drop of gelvatol solution containing 100 mg/ml DABCO reagent. Cells were observed under a Zeiss Axiovert fluorescence microscope (MRC 1000, Bio-Rad, Hercules, CA) equipped with an argon-krypton laser (excitation wavelength of 488 nm). Confocal images were analyzed using the Laser-Sharp software (Bio-Rad), National Institutes of Health Image 1.62, and Image software program (Adobe Photoshop).

Western blot analysis. Immunodetection of phosphoPKB on Thr 308 and Ser 473 residues was performed on extracts of cells grown for 10 days on 100-mm Transwell inserts incubated in the presence or absence of insulin (100 nM) added to the basolateral medium for various periods of time as indicated in each experiment. Cells were scraped in ice-cold lysis buffer (150 mM KCl, 10 mM Tris·HCl, 2 mM EDTA, 1 mM sodium orthovanadate, 0.1 mM NaF, 10 nM okadaic acid, 0.5% NP-40, 0.1% -mercaptoethanol, 300 µg/ml pefabloc, and 10 µg/ml leupeptin, pH 7.4) and harvested quickly at 4°C. Immunodetection of PTEN was performed on cells grown at confluence in 75-cm 2 plastic culture flasks and lysed.

Cell extracts were maintained at 4°C for 1 h, and the pellet was discarded by centrifugation (13,000 rpm; 20 min). Protein concentration was determined using the Bradford method ( 8 ). SDS-PAGE was performed as previously described ( 15 ). Briefly, the extracts were solubilized in sample buffer (10 mM Tris·HCl, 1.5% SDS, 0.6% DTT, 6% glycerol, 0.1% bromophenol blue, pH 6.8) and denatured by heating at 95°C for 3 min. Proteins were separated on 7.5% acrylamide gels and transferred to nitrocellulose membrane. These membranes were blocked and incubated overnight with a primary antibody (2% BSA or 0.1% milk, 150 mM NaCl, 50 mM phosphate buffer, 0.05% Tween 20, 0.1% NaN 3, pH 7.4) at 4°C and then washed and incubated for 1 h at room temperature with the appropriate peroxidase-labeled secondary antibody (DAKO). Detection was performed by exposure to enhanced chemiluminescence (Amersham). Membrane incubated overnight with anti-phosphoPKB antibodies was washed and reprobed with total PKB.5 h( l2 ^: f. F" ^
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X. laevis tissues. Heart, kidney, liver, and brain of X. laevis were homogenized in the following ice-cold buffer ( 31 ): 20 mM Tris·HCl (pH 7.5), 0.25 M sucrose, 0.01% NaN 3, 1 mM Na 3 VO 4, 1 nM okadaic acid, 1 mM EDTA, and a cocktail of proteases inhibitors (Roche tablets). The homogenates were centrifuged and the supernatant was used for Western blot analysis using anti-PTEN antibodies.
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Transfection. About 10 6 cells seeded on 60-cm 2 plastic culture dishes reached 70% confluence after 24 h and were then transfected. Briefly, cells were washed twice in a penicillin-streptomycin serum-free medium and the mixture of human PTEN cDNA (10 µg) or empty vector (10 µg) with 30 µl lipofectamine 2000 reagent was added to this incubation medium and left overnight. Cells were then washed, and the transfection medium was replaced by amphibian serum medium. When cells reached confluence, they were plated at a 1/10 dilution into 2 mg/ml Geneticin amphibian serum medium. Complete selection occurred after 10 days. In preliminary experiments, GFP in pEGFP-c1 was transfected into A6 cells and treated with a similar Geneticin-selective pressure. It yielded a transfection efficiency of 40% as checked by fluorescence microscopy. PTEN overexpression was stable only for a few passages. Each physiological experiment was therefore carried out with a new batch of transfected A6 cells. PTEN overexpression was checked by Western blot analysis for each physiological experiment.8 g$ b: {" f) A+ n3 I
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Immunoprecipitation. To test the transfection of PTEN cDNA and the incubation with permeant PIP 3 alter PI 3-kinase activity, the proteins containing phosphorylated tyrosine residue in A6 cell extracts were immunoprecipitated by anti-phosphotyrosine antibodies and the presence of the p85 regulatory subunit in the precipitate was taken as an index of PI 3-kinase stimulation.* O8 y" W8 T% S: E% a

One-hundred microliters of protein G Sepharose, first washed with cold PBS, were incubated with 4 µl of anti-phosphotyrosine antibodies for 2 h at 4°C. The complex was rinsed with ice-cold lysis buffer and incubated overnight at 4°C with 1.2 mg of A6 cell extract. The supernatant was discarded by centrifugation. The pellet was washed and resuspended in 20 µl of sample buffer and boiled for 5 min. The supernatant was collected and loaded onto a polyacrylamide gel. After immunoblotting with anti-phosphotyrosine antibodies, the membrane was stripped and probed with an anti-p85 subunit followed by incubation with anti-mouse antibodies. The level of p85 recruitment by tyrosine-phosphorylated insulin receptor substrates was compared within different samples.

Statistics. Electophysiological data are presented as means ± SD. Paired t -tests were used to compare experimental vs. control groups.

RESULTS
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Permeant PIP 3 increases sodium transport. The effect of different phosphorylated phosphoinositides on sodium transport across confluent monolayers of A6 cells was investigated by adding permeant forms of phosphatidylinositol phosphate derivatives (20 µM) complexed with histone carrier (5 µM) directly to one of the two compartments bathing the monolayer. Apical addition of permeant (i.e., complexed) PI( 4, 5 )P 2, PI( 3, 5 )P 2, and PIP 3 did not modify sodium transport ( Fig. 1 A ), whereas apical addition of PIP 3 and to a lesser extent PI( 3, 4 )P 2 increased sodium transport with a lag time of 60 min ( Fig. 1 B ). No further increase in sodium transport was observed upon simultaneous addition of PI( 3, 4 )P 2 and PIP 3 compared with the addition of PIP 3 alone (data not shown). Apical addition of PIP 3 in the absence of the histone carrier completely failed to increase the current. Compared with the concentration of 20 µM tested, higher concentration of permeant PIP 3 (50 µM) did not induce a significantly greater increase in sodium transport ( Fig. 2 ). Whatever the histone-phosphoinositide complex tested, their addition to the basolateral medium never increased sodium transport.# ^  q8 j% t# O! _3 `
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Fig. 1. Sodium transport (INa ) was evaluated (µA/cm 2 ) as described in EXPERIMENTAL PROCEDURES. Phosphoinositides (20 µM) were made permeant by complexation with a histone carrier (5 µM) and added to the apical bathing medium. A : permeant PI( 3, 5 )P 2, permeant PIP 3, permeant PI( 4, 5 )P 2, and carrier alone did not increase the sodium transport. PI, phosphatidylinositol; P, phosphate. Insulin (100 nM) was added to the basolateral bathing medium. Results are means ± SD; n = 3. B : permeant PIP 3 and permeant PI( 3, 4 )P 2 increased sodium transport after a lag time of 60 min and reached a maximum at 90 to 120 min. Carrier alone did not stimulate the transport. Insulin (100 nM) was added to the basolateral bathing medium. Results are means ± SD; n = 3.

Fig. 2. INa  was evaluated (µA/cm 2 ) as described in EXPERIMENTAL PROCEDURES. Permeant PIP 3 (dotted lines) was added at different concentrations (from top to bottom : 50, 20, 10, 5, 1, and 0.1 µM) to the apical bathing medium; 50 µM did not produce significantly greater effect than 20 µM. The concentrations of 1 and 0.1 µM were not significantly different from the carrier alone. Results are means ± SD; n = 3.

Permeant phospholipids penetrate into A6 epithelial cells. To demonstrate that these permeant phospholipid complexes indeed penetrate into A6 epithelial cells, we used three fluorescent permeant derivatives: NBD-diC16-PIP 3, NBD-diC16-PI( 3, 4 )P 2, and NBD-diC16-PI( 4, 5 )P 2 (20 µM) complexed with histone (5 µM). Permeant PIP 3 and PI( 3, 4 )P 2 added in the apical bathing medium labeled the A6 cells after only 10-min incubation ( Fig. 3, A and G ), but the labeling was further increased after 67 min ( Fig. 3, B and H ). The localization of PIP 3 in A6 cells appears in cytoplasmic vesicles, mostly concentrated around the nucleus ( Fig. 3 B ). Similar observations were done with permeant PI( 4, 5 )P 2 and PI( 3, 4 )P 2, although the labeling was more diffuse in the latter case ( Fig. 3, E and H ). On the contrary, fluorescent permeant PIP 3, PI( 3, 4 )P 2, and PI( 4, 5 )P 2 added to the basolateral side and incubated for 67 min did not enter into the A6 cells ( Fig. 3, C, I, F ). Furthermore, no uptake of fluorescent PIP 3 was observed when it was added to the apical bathing medium without complexing with the histone carrier ( Fig. 3 D ).& o- s8 H+ U, F/ X

Fig. 3. Determination of the uptake of some phosphatidylinositol phosphate derivatives. A6 cell monolayers were ground to confluence for 7 days on clear Transwell inserts and were used only if transepithelial resistance was 1 k ·cm 2. Fluorescent permeant NBD-PIP 3, NBD-PI( 4, 5 )P 2, and NBD-PI( 3, 4 )P 2 (20 µM) complexed or not with histone carrier (5µM) were added to the A6 cell monolayer for different times. A : NBD-PIP 3 (apical side 10 min). B : NBD-PI( 3, 4 )P 2 (apical side 10 min). C : NBD-PIP 3 (apical side 67 min). D : NBD-PI( 3, 4 )P 2 (apical side 67 min). E : NBD-PI( 4, 5 )P 2 (apical side 67 min). F : NBD-PIP 3 (basolateral side 67 min). G : NBD-PI( 3, 4 )P 2 (basolateral side 67 min). H : NBD-PI( 4, 5 )P 2 (basolateral side 67 min). I : NBD-PIP 3 without histone carrier (apical side 67 min)." j+ a5 _8 i( V5 S7 G

Insulin-stimulated sodium transport is decreased in PTEN-overexpressing cells. The transient nature of the increase in sodium transport brought about by permeant PIP 3 could be explained by the action of PIP 3 5- or 3-phosphatase. Western blot analysis shows the presence of the inositol 3-phosphatase PTEN in A6 cells and as well in several X. laevis tissues (heart, kidney, liver, brain) ( Fig. 4 A ). Overexpression of PTEN in A6 cells was achieved by transfecting human PTEN cDNA ( Fig. 4 B ). PTEN overexpression was lost after passages 4 and 5 of the cells, and therefore each experiment was carried out with a new batch of transfected A6 cells. The rate of PTEN overexpression was assessed independently for each experiment ( Fig. 4 B ). In vivo PTEN converts mainly PIP 3 into PI( 4, 5 )P 2, whose addition (as its permeant form) did not raise sodium transport ( Fig. 1 A ). Basal (unstimulated) sodium transport was not significantly different in PTEN-overexpressing monolayers vs. control monolayers. The stimulation of sodium transport induced by insulin was also reduced in PTEN-overexpressing monolayers compared with those transfected with empty vector ( Fig. 5 A ). The difference between stimulated and basal current was estimated at the peak of the response (Peak INa  = peak INa  - basal INa ), i.e., 60 min after addition of insulin and was found 30% higher in empty vector cells compared with PTEN-transfected cells ( Fig. 5 B ). Thus overexpression of PTEN was sufficient to decrease insulin-induced stimulation of sodium transport, most probably by decreasing the levels of PIP 3 in response to insulin.

Fig. 4. Immunodetection of PTEN. A : A6 cell extracts and several Xenopus laevis tissues. From left to right (in µg protein loaded/lane): CHO-IR cells (50 µg) were used as positive control, A6 cells (50 µg), PTEN-overexpressed A6 cells (50 µg), 100 µg of different X. laevis tissues (heart, kidney, liver, and brain), and 20 µg of mouse brain-soluble fractions used also as positive control. B : extracts from A6 cells overexpressing PTEN. From left to right (in µg protein loaded/lane): CHO-IR cell extract used as positive control (50 µg), extracts of wild-type A6 cells (50 µg), of A6 cells transfected with a plasmid containing PTEN cDNA (50 µg), and of A6 cells transfected with a plasmid containing an empty vector (50 µg) were analyzed by SDS-PAGE and transferred to a nitrocellulose sheet probed with anti-PTEN antibodies.
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Fig. 5. INa  was evaluated (µA/cm 2 ) as described in EXPERIMENTAL PROCEDURES. A6 monolayers either overexpressing PTEN or transfected with an empty vector. A : sodium transport was stimulated by insulin (100 nM) or by addition of permeant PIP 3 (20 µM). The increase in sodium transport following insulin stimulation was lower in PTEN-overexpressing cells vs. control A6 cells. PTEN-overexpressing cells also show a lower stimulation of sodium transport than the control A6 cells when stimulated with permeant PIP 3. Results are representative of 3 PTEN transfections. Results are means ± SD; n = 3. B : maximal stimulation of INa  by insulin and by PIP 3. The difference in sodium transport between unstimulated and insulin-stimulated monolayers ( INa ) was evaluated 60 min (Peak INa ) after insulin addition and shows a 30% decrease in the PTEN-transfected cells compared with the empty vector cells. Similarly, the difference in sodium transport between unstimulated (carrier alone) and permeant PIP 3 -stimulated monolayers ( INa ) was evaluated 120 min (Peak INa ) after its addition and shows a 30% decrease in the PIP 3 -stimulated PTEN-transfected cells compared with the PIP 3 -stimulated control cells. Results are means ± SD; n = 3, P

The effect of permeant PIP 3 added to the apical medium was also tested in PTEN-overexpressing cells. The increase in sodium transport induced by permeant PIP 3 was also reduced in A6 monolayers overexpressing PTEN compared with control monolayers ( Fig. 5 A ). The difference between basal current and maximal current stimulated by exogenous PIP 3 (Peak INa ), evaluated at the peak of the response, was also reduced by 30% in PIP 3 -stimulated PTEN-transfected cells compared with the PIP 3 -stimulated control cells ( Fig. 5 B ). Thus overexpression of PTEN in A6 cells reduces similarly the stimulation of sodium transport induced either by insulin or by exogenous PIP 3. Altogether, these results strongly support that PIP 3 plays a major role in insulin-stimulated sodium transport.2 C) a. j1 l3 g  f

PI 3-kinase activity. Stimulation of PI 3-kinase by insulin results from the recruitment of the p85 regulatory subunit by tyrosine-phosphorylated insulin receptor substrate. We therefore immunoprecipitated tyrosine-phosphorylated proteins in extracts from A6 epithelial cells stimulated with insulin for various times and detected by Western blotting whether the p85 subunit was coprecipitated as an indirect assessement of PI 3-kinase activity as performed by others ( 11, 21, 45 ). A6 cell monolayers were stimulated with insulin (100 nM) for 0, 2, and 5 min and extracts were immunoprecipitated by anti-phosphotyrosine antibodies ( Fig. 6 A ). The level of recruitment of the p85 subunit was weak in unstimulated A6 cells but increased after 2- and 5-min stimulation with insulin ( Fig. 6 B ). Crude extracts not submitted to immunoprecipitation showed no difference in the level of PI 3-kinase regulatory subunit ( Fig. 6 B ). Also PTEN transfection did not affect the activity of PI 3-kinase. The recruitment of the p85 subunit was comparable in PTEN-overexpressing cells vs. empty vector-transfected cells ( Fig. 6 C ). Furthermore, incubation with permeant PIP 3 did not affect the recruitment of p85 in anti-phosphotyrosine immunoprecipitates ( Fig. 6 C ).

Fig. 6. Immunoprecipitation (IP) and immunoblotting (IB) of A6 cell extracts. A : (from left to right ) lanes 1 - 3 : phosphotyrosine-containing proteins (PY) of A6 cell extract (1,200 µg protein) from unstimulated and insulin stimulated (2 and 5 min) A6 cells were immunoprecipitated (IP PY) and then immunoblotted with anti-phosphotyrosine. Lanes 4 - 6 : A6 cell crude extracts (100 µg protein loaded/lane) were immunoblotted with anti-phosphotyrosine. Overnight incubation with primary antibody and 20-s film exposure; n = 3. B : recruitment of the phosphatidylinositol 3-kinase (PI 3-kinase) p85 subunit in unstimulated and stimulated A6 cells. The same blot was stripped and reprobed with anti-p85 subunit of PI 3-kinase. CHO-IR cell extract (30 µg protein loaded/lane) was used as positive control. The level of recruitment of PI 3-kinase is weak for unstimulated A6 cells but increases after 2- and 5-min insulin stimulation. There is no difference in the level of recruitment in the crude extracts. Overnight incubation with primary antibody and 5-min film exposure; n = 3. C : recruitment of the p85 subunit in PTEN-overexpressed cells vs. empty vector control cells and in unstimulated vs. PIP 3 -stimulated A6 cells. The phosphotyrosine-containing proteins of A6 cell lysate (1,200 µg protein) from unstimulated and insulin-stimulated transfected A6 cells were immunoprecipitated, immunoblotted with anti-phosphotyrosine, stripped, and reprobed with anti-p85. Lane 1 : CHO-IR cells crude extract (30 µg protein loaded/lane) used as positive control. Lanes 2 - 4 : unstimulated and insulin-stimulated (2 and 5 min) empty vector control A6 cells extract. Lanes 5 - 7 : unstimulated and insulin-stimulated (2 and 5 min) PTEN-overexpressing A6 cells extract. Lane 8 : unstimulated A6 cells extract. Lane 9 : PIP 3 -stimulated (67 min) A6 cells extract. The level of recruitment was the same between PTEN vs. empty vector-transfected cells and between unstimulated vs. PIP 3 -stimulated A6 cells; n = 3.4 k, N" A$ K9 D, [# u1 ]
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PKB phosphorylation by insulin in A6 cells. Three major pathways have been shown to be activated by insulin: PI 3-kinase, MAP kinase, and CAP/Cbl/TC10 cascades as least in adipocytes ( 38, 50 ). The PI 3-kinase pathway is constantly associated with an increase in PKB activity ( 9 ) that can be detected by its phosphorylation on Thr 308 and Ser 473./ s; [, u6 Z, m
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Confluent A6 cell monolayers grown on 60-cm 2 Transwell inserts were incubated for 18 h in serum-free medium and then stimulated with insulin (100 nM) for 0, 5, 7, 10, 15, and 30 min. Insulin-stimulated and -unstimulated Chinese hamster ovary cells overexpressing the insulin receptor (CHO-IR) cell extracts were used, respectively, as positive and negative control for phosphorylated-PKB ( 6 ). In the absence of insulin, no phosphorylated band was detectable. Five minutes after addition of insulin, PKB was already phosphorylated on Thr 308 and Ser 473 residues. After 7 min, the intensity of the phosphorylated bands was maximal and decreased slightly thereafter ( Fig. 7, A and B ), whereas the amount of total PKB reprobed on the same membranes was identical at the various times examined.% c$ x# H/ W- L; Y5 [8 e5 q
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Fig. 7. Immunodetection of phospho-protein kinase B (PKB; top ) and PKB ( bottom ). A : anti-phosphoPKB-Thr 308 ( top ) and anti-PKB ( bottom ) antibodies. Top : from left to right (in µg protein loaded/lane): lane 1 : CHO-IR (45 µg) stimulated with 100 nM insulin for 5 min used as a positive control. Lane 2 : unstimulated CHO-IR (45 µg) used as a negative control. Lane 3 : unstimulated A6 cells (60 µg). Lanes 4 - 8 : A6 cells (60 µg) stimulated with 100 nM insulin, respectively, for 5, 7, 10, 15, and 30 min (overnight incubation with primary antibody and 10-min film exposure; n = 3). Bottom : same membrane reprobed for total PKB antibody (overnight incubation with primary antibody and 1-min film exposure; n = 3). B : anti-phosphoPKB-Ser 473 ( top ) and anti-PKB antibodies. Top : from left to right (in µg protein loaded/lane): lane 1 : CHO-IR (45 µg) stimulated with 100 nM insulin for 5 min used as a positive control. Lane 2 : unstimulated CHO-IR (45 µg) used as a negative control. Lane 3 : unstimulated A6 cells (60 µg). Lanes 4 - 8 : A6 cells (60 µg) stimulated with 100 nM insulin, respectively, for 5, 7, 10, 15, and 30 min (overnight incubation with primary antibody and 10-min film exposure; n = 3). Bottom : same membrane reprobed for total PKB antibody (overnight incubation with primary antibody and 1-min film exposure; n = 3).* U' [! N4 I% P# J- w' x- C6 T/ m
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The level of PKB phosphorylation after 7-min insulin stimulation was compared in PTEN-overexpressing cells vs. empty vector-transfected cells. PKB phosphorylation was reduced in PTEN-overexpressing cells, the difference being stronger in phosphorylation of the Thr 308 residue than in the phosphorylation of the Ser 473 residue ( Fig. 8 A ).3 U  W7 g, u# t4 e! Y
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Fig. 8. Immunodetection of phosphoPKB ( top ) and PKB ( bottom ) in PTEN-overexpressed cells vs. empty vector control cells and in unstimulated vs. PIP 3 -stimulated A6 cells. A : anti-phosphoPKB-Thr 308 ( top ) and anti-PKB ( bottom ) antibodies. Top : from left to right (in µg protein loaded/lane): lane 1 : CHO-IR (45 µg) stimulated with 100 nM insulin for 5 min used as a positive control. Lane 2 : unstimulated CHO-IR (45 µg) used as a negative control. Lane 3 : unstimulated empty vector cells (60 µg). Lane 4 : empty vector cells (60 µg) stimulated with 100 nM insulin for 7 min. Lane 5 : unstimulated PTEN-overexpressed cells (60 µg). Lane 6 : PTEN-overexpressed cells (60 µg) stimulated with 100 nM insulin for 7 min. Lane 7 : unstimulated A6 cells (60 µg). Lane 8 : A6 cells stimulated with permeant PIP 3 for 67 min (60 µg) (overnight incubation with primary antibody and 10-min film exposure; n = 3). Bottom : same membrane reprobed for total PKB antibody (overnight incubation with primary antibody and 1-min film exposure; n = 3). B : anti-phosphoPKB-Ser473 ( top ) and anti-PKB ( bottom ) antibodies. Top : from left to right (in µg protein loaded/lane): lane 1 : CHO-IR (45 µg) stimulated with 100 nM insulin for 5 min used as a positive control. Lane 2 : unstimulated CHO-IR (45 µg) used as a negative control. Lane 3 : unstimulated empty vector cells (60 µg). Lane 4 : empty vector cells (60 µg) stimulated with 100 nM insulin for 7 min. Lane 5 : unstimulated PTEN-overexpressed cells (60 µg). Lane 6 : PTEN-overexpressed cells (60 µg) stimulated with 100 nM insulin for 7 min. Lane 7 : unstimulated A6 cells (60 µg). Lane 8 : A6 cells stimulated with permeant PIP 3 for 67 min (60 µg) (overnight incubation with primary antibody and 10-min film exposure; n = 3). Bottom : same membrane reprobed for total PKB antibody (overnight incubation with primary antibody and 1-min film exposure; n = 3).6 }  K7 e. G' m7 Q& _4 u

A6 cells grown on 60-cm 2 Transwell inserts were incubated with permeant PIP 3 added to the apical solution for 67 min, a time sufficient to raise sodium transport. Permeant PIP 3 induced the phosphorylation of PKB on the Thr 308 residue but no phosphorylation could be detected on Ser 473 residue ( Fig. 8 B ).( \5 y( t4 O1 S4 G" n

DISCUSSION
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Despite ample evidence that sodium transport is regulated by insulin, the intracellular signaling cascade triggered on insulin binding to its receptor is largely unknown in the kidney. Dysregulation may lead to enhanced sodium reabsorption and hypertension. A6 cells cultured on a permeable support have been used as a model epithelium to demonstrate this natriferic effect of insulin ( 19 ), and subsequently this effect has been attributed to an increased number of active ENaC within the apical membrane and/or their open probability. More recently, Blazer-Yost's group ( 35 ) implicated the stimulation of PI 3-kinase as one of the early events triggered by insulin receptor occupancy. This was based on the detection of PIP 3 1 min after insulin stimulation by HPLC analysis of lipid extracts. This increase in PIP 3 as well as the sodium current were both prevented by LY-294002, an inhibitor of PI 3-kinase ( 47 ). However, the basal current was also sensitive to this drug, questioning its specificity in insulin stimulation ( 33 ). Indeed, such an inhibition was also observed in the early rise in the current elicited by the mineralocorticoid hormone aldosterone ( 4 ) and by antidiuretic hormone ( 17 ). Thus the suggestion has been made that LY-294002 might inhibit the maintenance of sodium transport by affecting the insertion of ENaC within the apical membrane, i.e., the fusion or trafficking of ENaC-containing vesicles regardless of hormonal stimulation ( 14 ). More recently, however, Blazer-Yost et al. ( 1 ) showed that insulin induces a redistribution of ENaC to both apical and lateral membranes. At the latter site, there was a colocalization of ENaC and PI 3-kinase regulatory subunits, suggesting that this could represent a storage pool of ENaC that can be recruited and targeted to the apical membrane.
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The major observation of the present study can be summarized as follows: both PIP 3 and to a lesser extent PI( 3, 4 )P 2 reproduced the insulin-induced increase in sodium transport when added with a carrier directly to the apical side of the A6 epithelium. However, when these phosphoinositides were added together, there was no greater effect than with PIP 3 alone, suggesting that the molecules are interacting at the same site. On the other hand, PI( 4, 5 )P 2 does not seem to play a major role in this mechanism. This is at variance with some results obtained by patch-clamp analysis, which suggested a role for PI( 4, 5 )P 2, although only in the presence of GTP ( 52 ). In this particular setting of excised patches, PI( 4, 5 )P 2 is important to alleviate a spontaneous rundown effect, i.e., spontaneous disappearance of ENaC activity ( 29 ). Yue et al. ( 52 ) found no effect of PIP 3 but in this inside-out patch-clamp setting, critical intermediate signaling molecules might have been lost. However, consistent with the present results, Tong et al. ( 44 ) very recently demonstrated that PIP 3 and PI( 3, 4 )P 2 both activate ENaC in excised patches from CHO cells cotransfected with the three human ENaC subunits. The lag time observed in the present study between PIP 3 addition and the final effect on sodium transport is most probably explained by the slow diffusion of PIP 3 into the cell. Although a 10-min incubation time was already sufficient to detect NBD-PIP 3 inside the cells ( Fig. 3 A ), higher intracellular labeling was observed after 67 min ( Fig. 3 B ) and is probably required to build up sufficient PIP 3 to trigger the increase in sodium transport. Our confocal studies on A6 cells show an apparent inhomogeneity of the fluorescent probe from cell to cell within the same monolayer. The reason for this is not understood, but such a variability from cell to cell was also observed in the confocal studies of Blazer-Yost et al. ( 1 ). The critical role of PIP 3 was further underlined by transfection of the inositol 3-phosphatase PTEN, which led to decreased stimulation of sodium transport induced by insulin as well as by permeant PIP 3. The results provide an independent set of data suggesting a role of PIP 3 in mediating insulins effect on sodium transport. However, unlike LY-294002, PTEN transfection did not change the rate of basal (unstimulated) sodium transport.

Thus, as shown by Blazer-Yost et al. ( 1 ), insulin receptor occupancy induces the recruitment of PI 3-kinase to the plasma membrane, which activates its catalytic activity and produces 3-phosphorylated inositide lipids such as PIP 3 and PI( 3, 4 )P 2 that are the principal mediators of PI 3-kinase stimulation. In adipocytes as well as in many other tissues, PIP 3 activates PDK1 and possibly PDK2, leading to phosphorylation of PKB on both Thr 308 and Ser 473 residues. Those phosphorylation steps that activate PKB are necessary but not sufficient to target GLUT4 to the adipocyte plasma membrane ( 49 ). The GLUT4 translocation in adipocytes and muscle cells required not only activation of PKB ( 49 ) but also the stimulation of CAP/Cbl/TC10 cascade, a PI 3-kinase-independent pathway ( 18, 24 ). Yet, the presence and role of PKB in A6 epithelial cells or in the collecting duct have not been questioned so far, whereas emphasis has been placed on another protein kinase, namely, serum and glucocorticoid-inducible kinase (sgk). This kinase plays a major role in the aldosterone stimulation of sodium reabsorption, and its expression is indeed induced by aldosterone and by hyposmotic shock in A6 cells ( 37 ). Although transfection of a dominant kinase dead sgk inhibited basal and insulin-stimulated sodium transport, overexpression of sgk did not increase insulin-stimulated sodium transport nor did transfection of a mutant form of sgk that cannot be phosphorylated by PDK-2 decrease insulin-stimulated sodium transport ( 13, 20 ). Using Western blot analysis, we were able to demonstrate phosphorylation of PKB under the control of insulin addition and that the observed time course is compatible with a physiological mediator role in the enhancement of sodium transport. Furthermore, the inositol 3-phosphatase PTEN ( 25, 48 ) was also detected in the A6 cells. In vivo, this 3-phosphatase has for major substrate PIP 3 which is degraded into PI( 4, 5 )P 2 ( 27, 28 ). PTEN knockout mice have greatly elevated basal levels of PIP 3 and phosphorylated basal PKB activity ( 43 ). To investigate the role of PI 3-kinase without perturbing the system with an inhibitor, we used permeant derivatives of different phosphatidylinositol phosphates, using the procedure developed by Echelon Biosciences. The intracellular delivery of the membrane-impermeable anionic inositol phosphate lipids is greatly improved by complexation with cationic polyamines. Preincubation of phosphoinositides with carrier polyamines produces complexes that enter into living eukaryotic cells such as mammalian, plant, yeast, bacterial, and protozoal cells ( 32 ). This phosphoinositide delivery method has been proven efficient to investigate the signaling role of PI 3-kinase ( 26 ) and PIP kinases ( 39, 51 ). Delivery of PIP 3 to its intracellular target into A6 cells is attested by the intracellular uptake of fluorescent NBD-PIP 3, by the phosphorylation of PKB, and by the increase in Na transport, which, at variance with GLUT4 translocation ( 24 ), was induced by addition of permeant PIP 3 alone. When A6 cells were incubated with permeant PIP 3, we detected phosphorylation on the Thr 308 residue but not on the Ser 473 residue. Furthermore, the reduction of PKB phosphorylation observed in PTEN-overexpressing cells stimulated with insulin was more pronounced on the Thr 308 residue than on the Ser 473 residue. This agrees with the reported greater sensitivity to PIP 3 of the Thr 308 residue ( 39 ) than the Ser 473 residue.; I; e6 I: s* ?/ L- r. C: [$ C

In conclusion, the present study demonstrates that PIP 3 and to a lesser extent PI( 3, 4 )P 2 mediate the increase in sodium transport induced by insulin in A6 epithelium. Furthermore, insulin also phosphorylates PKB. This downstream effector of PIP 3 could represent an important intermediate step leading to the increase in sodium transport. Increased sodium reabsorption by the distal nephron under the influence of hyperinsulinemia could then constitute the pathological factor responsible for arterial hypertension in the metabolic X syndrome.( J+ U% o# j/ h8 d

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This work was presented in an abstract form at the Special FEBS 2003 Meeting on Signal Transduction (Brussels, June 2003). D. Blero is an aspirant of the Fonds National de la Recherche Scientifique. This work was supported by grants from the Fonds Alphonse et Jean Forton, Action de Recherche Concertée of the Communauté Française de Belgique, and the Fondation pour la Recherche Scientifique Médicale./ q# f$ o+ m9 `3 ?

ACKNOWLEDGMENTS

The authors acknowledge the numerous scientific suggestions of C. Moreau and A. Poinas.
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