Insulin-feedback via PI3K-C2 activated PKB /Akt1 is required for glucose-stimulated insulin secretion

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1 The FASEB Journal Research Communication Insulin-feedback via PI3K-C2 activated PKB /Akt1 is required for glucose-stimulated insulin secretion Barbara Leibiger,* Tilo Moede,* Sabine Uhles,* Christopher J. Barker,* Marion Creveaux,* Jan Domin, Per-Olof Berggren,*,1 and Ingo B. Leibiger*,1 *Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden; and Renal Section, Faculty of Medicine, Imperial College, London, UK ABSTRACT Phosphatidylinositide 3-kinases (PI3Ks) play central roles in insulin signal transduction. While the contribution of class Ia PI3K members has been extensively studied, the role of class II members remains poorly understood. The diverse actions of class II PI3K-C2 have been attributed to its lipid product PI(3)P. By applying pharmacological inhibitors, transient overexpression and small-interfering RNA-based knockdown of PI3K and PKB/Akt isoforms, together with PI-lipid profiling and live-cell confocal and total internal reflection fluorescence microscopy, we now demonstrate that in response to insulin, PI3K-C2 generates PI(3,4)P 2, which allows the selective activation of PKB /Akt1. Knockdown of PI3K-C2 expression and subsequent reduction of PKB /Akt1 activity in the pancreatic -cell impaired glucose-stimulated insulin release, at least in part, due to reduced glucokinase expression and increased AS160 activity. Hence, our results identify signal transduction via PI3K-C2 as a novel pathway whereby insulin activates PKB/Akt and thus discloses PI3K-C2 as a potential drugable target in type 2 diabetes. The high degree of codistribution of PI3K-C2 and PKB /Akt1 with insulin receptor B type, but not A type, in the same plasma membrane microdomains lends further support to the concept that selectivity in insulin signaling is achieved by the spatial segregation of signaling events. Leibiger, B., Moede, T., Uhles, S., Barker, C. J., Creveaux, M., Domin, J., Berggren, P.-O., Leibiger, I. B. Insulin-feedback via PI3K-C2 activated PKB /Akt1 is required for glucosestimulated insulin secretion. FASEB J. 24, (2010). Key Words: insulin signaling diabetes mellitus fluorescence microscopy biosensors Insulin exerts pleiotropic effects involving mitogenic and/or metabolic events that are tissue as well as development dependent. However, the molecular mechanisms that are responsible for transducing the insulin signal from the insulin receptor (IR) to selective end point responses remain poorly understood. The current view on how selectivity is achieved proposes the activation of specific signal-transduction pathways, thereby engaging selective adaptor/effector proteins. In this process, phosphatidylinositide 3-kinases (PI3Ks) are one of the central and most intensely studied groups of enzymes. A possibility to achieve selectivity in signal transduction at the level of PI3K action is the generation of different lipid products by different members of the PI3K family and the subsequent activation/recruitment of selective downstream enzymes and their protein substrates (1 3). To date, 3 classes of mammalian PI3Ks have been identified based on their domain structures, differences in catalytic activity toward defined substrates, and modes of regulation (4). Only members of class Ia and class II PI3Ks have been reported to be activated by insulin. The respective class Ia PI3Ks are heterodimers consisting of the catalytic subunit p110 or p110 and a regulatory subunit of the p85 or p85 gene families (1, 5). Class II PI3Ks were originally identified by sequence homology with other PI3Ks (6), and, so far, 3 mammalian isoforms, i.e., PI3K-C2 (7, 8), PI3K-C2 (9, 10), and PI3K-C2 (11 13) have been described. PI3K-C2 and PI3K-C2 are ubiquitously expressed, while the expression of PI3K-C2 is observed mainly in the liver and prostate and to a lesser degree in the small intestine, pancreas, and kidney (14). Only PI3K-C2 and -C2 have been shown to respond to insulin stimulation (15 18). Class II PI3Ks prefer PI and PI(4)P as substrates (7, 8, 19) and therefore show a different substrate specificity compared with class I PI3Ks. Class II PI3Ks can be distinguished from each other by their different sensitivity toward PI3K inhibitors. PI3K-C2 is sensitive to low nanomolar levels of wortmannin, similar to class Ia PI3Ks, while it shows a reduced sensitivity toward LY (9, 19). In contrast, PI3K-C2 is more resistant to both PI3K inhibitors and higher concentrations are needed to block its activity (7). Studies by us and others have shown that insulinstimulated insulin gene transcription in pancreatic -cells involves the activity of PI3K class Ia (20, 21) and that this signaling pathway is initiated by signaling through the IR-A isoform (20, 22). On the other hand, insulin-stimulated up-regulation of the -cell glucokinase transcription unit ( GK) via the IR-B isoform is neither sensitive to concentrations of wortmannin and LY that block class Ia activity nor to the expression of a dominant-negative acting mutant of PI3K class 1 Correspondence: Rolf Luft Research Center for Diabetes and Endocrinology, L1:03, Karolinska Institutet, SE Stockholm, Sweden. I.B.L., ingo.leibiger@ki.se; P.- O.B., per-olof.berggren@ki.se doi: /fj /10/ FASEB

2 Ia adapter protein p85, i.e., p85 (22). Most interestingly, signal transduction leading to the activation of GK gene transcription requires protein kinase B (PKB) activity, which is dependent on phosphorylation by phosphoinositol-dependent kinase 1 (PDK1) (22). Consequently, these data imply the involvement of a PI3K activity that is different from class Ia PI3K. The aim of the present study was to test whether and, if so, how the class II member PI3K-C2 is involved in insulin-stimulated pancreatic -cell signal transduction via IR-B and PKB. MATERIALS AND METHODS Cell culture HIT-T15 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mm glutamine, and 10% fetal calf serum at 5% CO 2 and 37 C. Before experiments, cells were incubated overnight in fully supplemented culture medium containing 0.1 mm glucose. MIN6m9 cells were cultured in DMEM containing 11.1 mm glucose and supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mm glutamine, 10% FCS, and 75 M -mercaptoethanol at 5% CO 2 and 37 C. Before experiments, cells were incubated for 6 h in fully supplemented culture medium containing 2 mm glucose. Cells were stimulated, if not indicated otherwise, for 10 min with 5 mu/ml insulin in fully supplemented culture medium containing 0.1 mm (HIT-T15) or 2 mm (MIN6m9) glucose. Islet cell and tissue preparation Islets were prepared from 9-mo-old normoglycemic ob/ob mice or 4-mo-old B6 mice. Isolation of pancreatic islets was described by Leibiger et al. (20, 23). Muscle, liver, brain, and fat were obtained from B6 mice, washed 3 times with PBS, resuspended in lysis buffer (137 mm NaCl; 2.7 mm KCl; 1 mm MgCl 2 ; 20 mm Tris-HCl, ph 8.0; 4 mm Na 3 VO 4; 10 mm NaF; 1 mm PMSF; 1 g/ml aprotinin; 1% Triton X-100; and 10% glycerol), and homogenized using a glass-glass homogenizer followed by passing the homogenate 5 times through a syringe needle ( mm/29 G 1/2). The homogenate was centrifuged for 5 min at 600 g to remove cell debris, and the amount of protein in the supernatant was determined by the Bradford method. Expression constructs The construction of the following vectors has been described before: pd2.rins1.dsred2/r GK.GFP (24); prccmvi.hir(a), prccmvi.hir(b), prccmvi.hir(a) 23 -GFP and prccmvi.hir(b) 23-GFP (22); prccmvi.hir(a)-mgfp, prccmvi.hir(b)-mgfp, prccmvi.hir(a)-mcerulean, prccmvi.hir(a)-mvenus, prccmvi.hir(b)-mcerulean, and prccmvi.hir(b)- mvenus (24); prccmvi.hir(b) NPEF -mgfp and prccmvi. hir(b) NPEA -mgfp (25); pfret2-devd (26), pef.gluglu- PI3K-C2 (7), pgfp-2xfyvehrs (27), prset-tdtomato (28), pegfp-ph Akt (29), and pcdna3.1ha-akt2 (30). Plasmid psport1.hakt3 was purchased from ImaGenes (Berlin, Germany). PI3K-C2 constructs pcmv.myc-pi3k-c2 -WT was generated by subcloning the PI3K-C2 -cdna from pef.gluglu-pi3k-c2 in frame into pcmv.myc (Clontech, Palo Alto, CA, USA). Plasmid pcmv. Myc-PI3K-C2 -R1251P was generated by changing the codon for R1251 from CGA to CCA by site-directed mutagenesis. To generate pcmv.myc-pi3k-c2 -CAAX, we introduced an NruIsite in front of the STOP-codon of PI3K-C2 and subcloned the Ki-ras CAAX motif from psg5.pkb -CAAX in frame into Myc- PI3K-C2 via the NruI site. prccmvi.mgfp-pi3k-c2 was constructed by subcloning the PI3K-C2 -cdna from pef.gluglu- PI3K-C2 into prccmvi.mgfp0. prccmvi.mcerulean-pi3k- C2 was obtained by exchanging the mgfp-cassette vs. a mcerulean-cassette. To generate prccmvi.mgfp-pi3k-c2 - CAAX, we introduced a NruI-site in front of the STOP-codon of PI3K-C2 and subcloned the Ki-ras CAAX motif from psg5.pkb - CAAX in frame into mgfp-pi3k-c2 via the NruI site. dtomato-tagged human IR variants prccmvi.hir(a)-dtomato and prccmvi.hir(b)-dtomato were generated by exchanging the mgfp-cdna in prccmvi.hir(a)- mgfp and prccmvi.hir(b)-mgfp vs. dtomato-cdna from prset-tdtomato. PKB-constructs Plasmid prccmvi.mgfp-pkb was generated by subcloning the HA-PKB -cassette from pcmv5.ha-pkb in frame into prccmvi. mgfp0. Plasmid prccmvi.dtomato-pkb was generated by subcloning the HA-PKB -cassette from pcmv5.ha-pkb in frame into prccmvi.dtomato0. Respective constructs for PKB (human Akt2) and PKB (human Akt3) were obtained by exchanging cdnas for the respective PKB variants. Luciferase constructs pgl4.rins1.luc2neo and pgl4.r GK.Luc2neo were generated by subcloning the rat insulin-1 promoter ( 410/ 1 bp) sequence from prins1.gfp (23) and the rat -cell glucokinase promoter ( 278/ 123 bp) sequence from pr GK.CAT (31), respectively, into pgl4.luc2/neo (Promega, Madison, WI, USA). pcmv.hrluccp was generated by subcloning the CMV promoter from prc/cmv (Invitrogen, Stockholm, Sweden) into pgl4.hrluccp (Promega). All mutations were performed by employing the QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA), and the respective oligonucleotides purchased from Sigma (Paris, France). All constructions were verified by DNA sequence analysis. Small interfering (sirna)-mediated knockdown sirnas against mouse p85 (Pik3r1: sirnaid ), PI3K-C2 (Pik3c2a: sirna1id 68525, sirna2id 68710) and validated nontargeting negative control (AM4613) were purchased from Ambion (Applied Biosystems/Ambion, Austin, TX, USA). sirnas against mouse PKB (ON-TARGETplus SMARTpool L ), PKB (ON-TARGETplus SMARTpool L ), PKB (ON-TARGETplus SMARTpool L ), and nontargeting sirna pool (ON-TARGETplus sicontrol D ) were purchased from Dharmacon (Nordic Biolabs, Taby, Sweden). MIN6 cells were first transfected using Lipofectamine 2000 with the sirna and transfected 48 h later for some experiments with other expression constructs of interest. All experiments were performed 96 h after transfection with the sirna. Transfection with the nontargeting negative control sirna (here- PI3K-C2 MEDIATED INSULIN SECRETION 1825

3 after: control sirna) had no effect on protein expression, promoter activation, and insulin secretion (Supplemental Fig. 1). Online monitoring of GFP and DsRed2 expression Expression of DsRed2 and GFP was detected using digital imaging fluorescence microscopy as extensively described by Leibiger et al. (20, 22 24). For transient transfection studies, HIT-T15 and MIN6m9 cells were grown on 24-mm glass coverslips, transfected by the lipofectamine technique, and cultured further for 24 h in fully supplemented culture medium. HIT-T15 cells were incubated overnight in fully supplemented RPMI 1640 culture medium at 0.1 mm glucose. MIN6m9 cells were incubated for 6 h in fully supplemented DMEM culture medium containing 2 mm glucose. Cells were stimulated for 5 min with 5 mu insulin/ml at low glucose in fully supplemented medium. Analysis of PI3K activity HIT-T15 cells were transfected with either prccmvi.hir(a) 23 - mgfp or prccmvi.hir(b) 23 -mgfp. Cell lysates containing mg of protein were subjected to immunoprecipitation with rabbit anti-gfp (A11122, Molecular Probes, Invitrogen) antibody, rabbit anti-pi3 Kinase p85 (Upstate Biotechnology, Lake Placid, NY, USA) or rabbit anti-pi3k-c2 (17). PI3K activity was analyzed in the immunoprecipitates as described by Krook et al. (32) using L- -phosphatidylinositol from Avanti Polar Lipids (Alabaster, AL, USA). Analysis of basal promoter activity HIT-T15 cells were transfected with the combination pgl4.rins1.luc2neo and pgl4.cmv.hrluccp or pgl4. r GK.Luc2neo and pgl4.cmv.hrluccp and the respective PI3K- C2, TC10 constructs or empty vector. Cells were incubated overnight in fully supplemented RPMI 1640 culture medium at 0.1 mm glucose. Firefly and Renilla luciferase luminescence were measured in cell lysates using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer s instructions. Basal promoter activity was calculated by dividing firefly luciferase luminescence by Renilla luciferase luminescence. Analysis of PKB activity Analysis of PKB activity was performed using the Akt1/PKB immunoprecipitation kinase assay kit (Upstate Biotechnology) according to the manufacturer s instructions. PKB was immunoprecipitated with rabbit anti-akt3/pkb antibodies (Upstate Biotechnology) from the same lysates, and kinase activity was measured using the same PKB assay kit. Activity of overexpressed HA-tagged PKB and PKB was analyzed in HA immunoprecipitates (HA.11 Clone 16B12, Covance) using the same PKB assay kit. RNA analysis GK mrna was analyzed by comparative RT-PCR as described by Leibiger et al. (23) using primers 5 -CGACCGGAT- GGTGGATGAGA-3 and 5 -TCACGTCCTCACTGGCTT-3. -Actin mrna was analyzed by RT-PCR using primers 5 - AACTGGAACGGTGAAGGCGA-3 and 5 -AACGGTCTCA- CGTCAGTGTA-3. We chose PCR conditions that guaranteed the amplification of GK and actin fragments within the linear range, as verified by testing various numbers of amplification cycles (cycles 32 to 42). 32 P-labeled PCR products were separated on a 6% polyacrylamide sequencing gel and analyzed by phosphoimaging. Quantification was performed with TINA-software 2.07d (Raytest, Straubenhardt, Germany). Values of GK mrna were normalized by -actin values. PI-lipid profiling MIN6m9 cells were transfected with nontargeting control or C2 sirna as described above. The efficiency of knockdown was analyzed by Western blotting. For experiments devised to measure 3-phosphorylated lipids (3-PIs), the DMEM medium contained a final concentration of 50 M inositol and [ 3 H] inositol at a concentration of 20 Ci/ml, together with 10% dialyzed FBS. Cells were labeled after transfection for 72 h in 92 mm dishes before experiments were carried out. Ninety-six hours after transfection, cells were starved for 6 h in labeling medium containing 2 mm glucose. Then LY was added or not to the medium to a final concentration of 25 M. Ten minutes later insulin (or vehicle) was added to the medium, and the final concentration was 5 mu/ml. At the end of the 10 min incubation with insulin, the media were removed, and the cells were killed with 1 ml of ice-cold 1 M HCl. Inositol lipids were then extracted, deacylated and separated on HPLC exactly as described by Yu et al. (33). Fractions were collected and Ultima Flo AP scintillant (PerkinElmer Sverige AB, Upplands Väsby, Sweden) was added. Radioactivity was determined by a Packard scintillation counter. The 10 min period for insulin stimulation was chosen because PKB activity was maximal after 10 min. Insulin secretion Cells growing in 35-mm dishes were first washed 5 times with Krebs-Ringer bicarbonate (KRB) buffer (16 mm HEPES, 115 mm NaCl, 20 mm NaHC0 3, 4.7 mm KCl, 1.2 mm MgSO 4, 2.56 mm CaCl 2, and 1.2 mm KH 2 PO 4, ph 7.4) containing 0.5 mm glucose and 0.1% BSA and then incubated in the same buffer for1hat5%co 2 and 37 C. The dishes were then transferred to a 37 C water bath, and the cells were washed with the same buffer several times. Then the cells were perifused at 37 C with the buffer for the times indicated in the figures. The buffer was changed every minute, and the perifusate was frozen for later analysis. Cells were stimulated for 10 min with 11.1 mm glucose or 3 min with 30 mm KCl. After the experiment, the cells were washed twice with PBS and lysed and the lysates were frozen for later analyses for insulin content or by Western blotting. Insulin secretion and content were measured by the ArcDia 2-photon fluorescence excitation microparticle fluorometry (TPX) assay for insulin (Arc- Dia Group, Turku, Finland; described at under technologies and TPX-assays ). Western blotting Cells were washed with PBS, lysed with lysis buffer [50 mm Tris, ph 7.5; 1 mm EDTA; 1 mm EGTA; 0.5 mm Na 3 VO 4 ; 0.1% (v/v) 2-mercaptoethanol; 1% Triton X-100; 50 mm NaF; 5 nm sodium pyrophosphate; 10 mm sodium -glycerol phosphate; 0.1 mm PMSF; 1 g/ml of aprotinin; pepstatine leupeptin; and 1 M Microcystin] and homogenized, and the amount of protein was measured in the supernatants by the Bradford method. Equal amounts of protein ( g) were separated over a 10% SDS-polyacrylamide gel (buffering system according to Laemmli), and proteins were electrotransferred to PVDF membrane. In the case of the phosphospecific antibodies, the membranes were probed with the respective antibodies and then stripped and reprobed with antiodies recognizing the respective total protein levels. Rabbit anti-as160 antibody was purchased from Cell Signaling 1826 Vol. 24 June 2010 The FASEB Journal LEIBIGER ET AL.

4 Technology (Danvers, MA, USA), and rabbit anti-phospho- AS160 (Thr642) antibody was purchased from Invitrogen. Further, the following antibodies were used: rabbit anti-pi3k- C2 (17), rabbit anti-pi3 kinase p85 (Upstate Biotechnology), mouse anti-pkb clone SKB1 (Upstate Biotechnology), rabbit anti-pkb (Cell Signaling Technology), rabbit anti-pkb (Upstate Biotechnology), rabbit anti-glucokinase (Abcam, Cambridge, UK), and mouse anti-gapdh (Ambion). Immunoreactivity was detected with horseradish peroxidase-conjugated secondary antibodies using the ECL system (Amersham, Piscataway, NJ, USA). Coimmunoprecipitation MIN6 cells were transfected with IR-B 23 -GFP and myc-pi3k- C2, and 48 h after start of transfection, cells were starved for 6 h in fully supplemented culture medium. After stimulation with insulin (5 mu/ml) for 3 min, the cells were lysed in lysis buffer (20 mm Tris-HCl, ph 8.0; 137 mm NaCl; 15% glycerol; 0.5% Triton X-100; 1 mm PMSF; 10 mm NaF; 4 mm Na 3 VO 4 ; and 1 g/ml aprotinin, leupeptin, and pepstatin each). The lysate was incubated for 30 min at 4 C on a rotator and centrifuged for 15 min at g, and the supernatant was collected. Two milligrams of protein was incubated with 5 g of rabbit polyclonal antibody against GFP (Molecular Probes, Invitrogen) on a rotator for 16 h at 4 C. Fifty microliters of preeliquibrated protein A/G Plus agarose was added and incubated for additional 4 h. Immunoprecipitates were washed 3 times with lysis buffer on ice and then resuspended in 2 SDS sample buffer, boiled, and separated on an SDS-polyacrylamide gel. Western blot analysis was performed with mouse monoclonal anti-myc-tag antibody (clone 9E10; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Laser-scanning confocal and total internal reflection fluorescence (TIRF) microscopy Laser-scanning confocal microscopy was performed using a Leica TCS SP2 (Leica Lasertechnik, Heidelberg, Germany) with the following settings: Leica HCX PL APO 63/1.20/ 0.17 UV objective lens, excitation wavelength 488 nm (Ar laser) and 543 nm (HeNe laser), a 488/543 double dichroic mirror, and detection of GFP at 505 to 525 nm and of Tomato at 605 to 670 nm. TIRF microscopy was performed using a Zeiss Axiovert 200M microscope (Carl Zeiss, New York, NY, USA) equipped with an Plan-Fluar 100/1.45 oil objective, a TIRF-slider, a LASOS 77 laser for excitation, and an AxioCamHS camera for image capture. For detection of GFP fluorescence, the following filter sets from Zeiss were used: for GFP detection, excitation 488/10 nm, dicroic 492, and emission 520/35 nm; for Tomato detection, excition 488/10 nm, dicroic 492, and emission 615/70 nm. Codistribution analysis was performed by using the Image Correlation Analysis (ICA) plug-in for ImageJ provided by Li et al. (34) at the Wright Cell Imaging Facility, University Health Network Research, Canada ( facilities/wcif/fdownload.html). We utilized ICA as described by Li et al. (34) to calculate an intensity correlation quotient (ICQ) for each pair of images. ICA postulates that codistributed signals covary in their intensity, i.e., a bright pixel in channel 1 should correspond to a bright pixel in channel 2. For analysis, product difference from the mean (PDM) for each pixel is calculated. Pixels that in both channels are either very bright or very dark result in a positive PDM value, while pixels that are exclusive result in negative values. If the brightness of either pixel is around the average of the picture, the resulting PDM is 0. The PDM values can be utilized to generate an image that visualizes colocalization. To calculate the ICQ, the number of positive PDM values is divided by the total number of PDM values for each pair of images and 0.5 is subtracted. ICQ can therefore vary between 0.5 and 0.5, with 0.5 indicating perfect covariance, 0.5 perfect mutual exclusion, and 0 random distribution. FRET analysis by acceptor photobleaching FRET analysis by acceptor photobleaching was performed after adapting the method described by Karpova and McNally (35) to the Leica TCS-SP2 confocal microscope. Briefly, cells were transfected with constructs encoding mcerulean- and mvenus-tagged proteins and imaged by live-cell confocal microscopy. For determination of FRET between IR-B and PI3K-C2 under different culture conditions, cells were fixed with 3% paraformaldehyde in PBS before microscopy. Two consecutive images were obtained 1 min apart using the following settings: excitation detection at 458 nm (laser power 15%), double-dicroic at 458/514 nm, and emission detection at nm (mcerulean) and nm (mvenus), further referred to as image 1 and image 2. The FRET-acceptor mvenus was then photobleached for 1 min by exciting it with the 514-nm laser at full power. After that, an additional image was acquired using the settings described above, further referred to as image 3. The values for C F and E F were calculated using the following formulas: C F (CI 2 CI 1 )/CI 1 ; E F (CI 3 CI 2 )/CI 2 ; where CI 1,CI 2,CI 3 are Cerulean intensity of images 1, 2, and 3, respectively. All intensities were background corrected before these calculations. FRET did occur if the value for E F is significantly higher than the value for C F, caused by the fact that photobleaching of the FRET acceptor mvenus would lead to an increase in fluorescence of the FRET donor mcerulean. RESULTS Expression of PI3K-C2 in insulin-producing cells The observation in our previous study (22) that 150 nm wortmannin or 100 M LY was needed to abolish the IR-B/PDK1/PKB-dependent up-regulation of GK transcription by insulin suggested the involvement of the PI3K class II member PI3K-C2 in this specific signaling pathway. Although it is assumed that PI3K- C2 is widely expressed (7), the expression of this enzyme at the protein level in insulin-producing cells has not been verified. We therefore performed Western blot analysis. The 170-kDa form of PI3K-C2 was present in lysates prepared from mouse and human pancreatic islets and insulin-producing cell lines HIT- T15 and MIN6, as well as from classical insulin-target tissues, i.e., muscle, liver, and fat (Fig. 1A) for control. To verify the identity of the recognized band in the Western blots, we performed sirna-mediated knockdown of PI3K-C2 expression in MIN6 cells (Fig. 1A). PI3K-C2 is involved in IR-B-mediated activation of GK gene expression To test the involvement of PI3K-C2 in insulin-stimulated GK promoter activition via IR-B and PKB, we PI3K-C2 MEDIATED INSULIN SECRETION 1827

5 Figure 1. Effect of expression levels of class II PI3K-C2 or class Ia adapter p85 on basal and stimulated insulin- and GK-promoter activities as well as GK protein levels. A) Identification of PI3K-C2 in islets and insulin-producing cell lines by Western blot analysis. Lysates obtained from human and mouse pancreatic islets, insulin-producing HIT T15 cells, MIN6 cells, MIN6 cells treated with sirna against PI3K-C2, and mouse tissue (brain, kidney, liver, fat, and muscle) were probed with an antibody against PI3K-C2. Arrow indicates PI3K-C2 ( 170 kda). B) Effect of overexpressed wild-type (C2 -WT) and kinase-dead mutant (C2 -R1251P) on basal insulin-promoter (solid bars) and GK-promoter (open bars) activities. HIT cells were cotransfected with either rins1.luc2neo/cmv.hrluccp or GK.Luc2neo/CMV.hRlucCP in combination with either empty vector (mock), C2 -WT, or C2 -R1251P, and the ratio of Luc/hRLuc luminescence was obtained. Data are means se (n 3). C) Effect of sirna against PI3K-C2 or p85 on insulin-stimulated insulin or GK expression in MIN6 cells. MIN6 cells were transfected with either control sirna (control sirna), sirna against PI3K-C2 (C2 sirna1 and sirna2), or p85 (p85 sirna). Levels of insulin (filled bars) and GK (open bars) mrnas were measured by comparative RT-PCR and are percentage of mrna levels of nonstimulated control (given as 1.0). Data are means se (n 3). D) Effect of sirna against PI3K-C2 or p85 on insulin-stimulated insulin-promoter (solid bars) or GK-promoter (open bars) activation in MIN6 cells. MIN6 cells were cotransfected with rins1.dsred2/ GK.GFP in combination with either control sirna (control sirna), sirna against PI3K-C2 (C2 sirna1 and sirna2), or p85 (p85 sirna). Changes in promoter activity were measured as ratios of fluorescence obtained at 240 vs. 60 min after stimulation with insulin and are means se (n 14). E) Effect of sirna treatment on PI3K-C2 and p85 protein levels. MIN6 cells were transfected with either control sirna (control), sirna against PI3K-C2 (C2 1 and C2 2), or p85 (p85 sirna), and protein levels were analyzed by Western blotting 96 h after transfection. Protein levels of PI3K-C2 and p85 are shown in relation to GAPDH protein levels. A representative blot out of 3 independent experiments is shown. F, G) Effect of sirna against PI3K-C2 or glucokinase on GK protein levels in MIN6 cells. MIN6 cells were transfected with either control sirna (control sirna), sirna against PI3K-C2 (C2 sirna2), or glucokinase (GK sirna). Lysates were prepared 4 d after transfection and used for Western blot analysis (F). Changes in GK protein levels were measured in Western blots as ratios of glucokinase signal vs. GAPDH signal; data are means se (n 3) (G). A representative blot out of 3 independent experiments is shown. **P 0.01, ***P vs. respective stimulated control. next analyzed the effect of overexpression as well as of knockdown of the enzyme. As shown in Fig. 1B, overexpression of PI3K-C2 led to an increase in basal GK promoter activity, which could not be stimulated further by insulin. In contrast, expression of a catalytically inactive variant of the enzyme, i.e., PI3K-C2 -R1251P (36), did not result in an increase in basal GK promoter activity. Reduction of PI3K-C2 expression levels to 20% by sirna-mediated knockdown abolished the insulin-stimulated increase in GK mrna levels as well as promoter activity (Fig. 1C, D). On the other hand, neither knockdown nor overexpression of PI3K-C2 influenced insulin-stimulated insulin promoter activity (Fig. 1B D). sirna-mediated knockdown of the class Ia PI3K regulatory subunit p85 (expression levels: 40%) did not affect insulin-stimulated GK promoter activity but, as expected (22), reduced insulin-stimulated insulin promoter activation in the same cell (Fig. 1D, E). Finally, we tested the effect of PI3K-C2 knockdown on GK protein levels. As shown in Fig. 1F, G, reduction of PI3K-C2 levels to 20% resulted in a decrease in glucokinase protein levels to 75%. PKB isoform is involved in insulin-mediated activation of GK gene expression and acts downstream of PI3K-C2 Based on the observation that overexpression of either PKB or PDK1 led to a more pronounced activation of the GK promoter and expression of a PDK1-antisense construct having a negative effect, we suggested the 1828 Vol. 24 June 2010 The FASEB Journal LEIBIGER ET AL.

6 involvement of PKB in the IR-B-mediated activation of GK transcription in the pancreatic -cell (22). To test whether PI3K-C2 acts upstream of PKB, we first wanted to identify the PKB isoform involved in insulinstimulated GK expression. Western blot analysis revealed that all 3 PKB isoforms, i.e., PKB /Akt1, PKB / Akt2, and PKB /Akt3, are present in pancreatic islets as well as in insulin-producing cell lines (data not shown). PKB isoform-selective knockdown showed that reduced expression of PKB but not of PKB or PKB affected insulin-stimulated GK promoter activity as well as GK protein levels (66% of control) in MIN6 cells (Fig. 2A, B). Next, we analyzed the effect of PI3K-C2 on insulin-stimulated PKB activities. As shown in Fig. 2C, D, reduction of PI3K-C2 expression levels to 22% almost completely abolished insulinmediated activation of PKB but not that of PKB or PKB in the same preparation. Vice versa, overexpression of PI3K-C2 led to an increase in the basal activity of PKB but not of PKB or PKB (Fig. 2E). These data suggest that PI3K-C2 acts upstream of PKB and contributes to its activation. Figure 2. Role of PKB isoforms in insulinstimulated GK promoter activation and effect of PI3K-C2 levels on PKB activity. A) Effect of sirna treatment on PKB isoform protein levels. MIN6 cells were transfected with either control sirna (control), sirna against PKB (PKB ), PKB (PKB ), or PKB (PKB ), and protein levels were analyzed by Western blotting 96 h after transfection. Protein levels of PKB isoforms and glucokinase are shown in relation to GAPDH protein levels. A representative blot out of 3 independent experiments is shown. B) Effect of sirna against PKB isoforms on insulin-stimulated insulin-promoter (solid bars) or GK-promoter (open bars) activation. MIN6 cells were cotransfected with rins1.dsred2/ GK.GFP in combination with either control sirna (control sirna), sirna against PKB (PKB sirna), PKB (PKB sirna), or PKB (PKB sirna). Changes in promoter activity were measured as ratios of fluorescence obtained at 240 vs. 60 min after stimulation with insulin; data are means se (n 12). C) Effect of sirna against PI3K-C2 on insulin-stimulated activity of PKB, PKB, and PKB. MIN6 cells were transfected with either control sirna (ctrl) or sirna against PI3K-C2 (C2 2) in combination with vectors encoding HA-PKB or HA-PKB and treated or not with insulin (5 mu/ml, 10 min). PKB activity was measured in PKB immunoprecipitates (IP) or in HA immunoprecipitates, as indicated. PKB activity is percentage of PKB activity of the nonstimulated control (given as 100%). Data are means se (n 3). D) Effect of sirna treatment on PI3K-C2 protein levels in MIN6 cells treated or not with insulin. MIN6 cells were transfected with either control sirna (ctrl) or sirna against PI3K-C2 (C2 2), and protein levels were analyzed by Western blotting (WB) 96 h after transfection in cells treated ( ) ornot ( ) with insulin. Protein levels of PI3K-C2 are shown in relation to GAPDH protein levels. A representative blot out of 3 independent experiments is shown. E) Effect of PI3K-C2 overexpression on basal activity of PKB, PKB, and PKB. HIT cells were transfected with either control plasmid (mock), wild-type PI3K-C2 (C2 -WT), or kinase-dead PI3K-C2 (C2 -R1251P) in combination with and without HA-tagged PKB and PKB. PKB activity was measured in immunoprecipitates of PKB and PKB or HA immunoprecipitates, as indicated, obtained 48 h after transfection. PKB activity is percentage of PKB activity of mock-transfected control (given as 100%). Data are means se (n 3). *P 0.05, **P 0.01, ***P vs. respective control. PI3K-C2 MEDIATED INSULIN SECRETION 1829

7 PI3K-C2 -mediated activation of PKB is required for glucose-stimulated insulin secretion Since our data revealed that knockdown of PI3K-C2 reduces the expression levels of GK (Fig. 1F, G), i.e., the glucose sensor of the pancreatic -cell (37), we wanted to test whether changing expression levels of PI3K-C2 affects glucose-stimulated insulin release. While overexpression of PI3K-C2 had no effect on insulin secretion (Supplemental Fig. 2), sirna-mediated knockdown of PI3K-C2 expression resulted in a reduction by 25% of the first phase of glucose-stimulated insulin release but had no effect on basal insulin release (Fig. 3A, B). Noteworthy, reduction of PI3K-C2 levels to 20% did not affect K -stimulated insulin release (Fig. 3D). In agreement with the selective effect of PI3K-C2 on PKB activity (Fig. 2C E) as well as the selective effect of PKB -knockdown on GK expression levels (Fig. 2A, B), only the reduction of PKB levels but not the levels of PKB or PKB by sirna affected glucose-stimulated insulin secretion (Fig. 3C). Because the effect of knocking-down PI3K-C2, leading to a reduction of GK levels to 75%, on glucose-stimulated insulin secretion was equivalent to the effect achieved by a reduction of GK levels to 35% (Fig. 3A, B), factors in addition to GK are likely to be involved in the PI3K-C2 /PKB -mediated action on insulin release. One potential factor that is a downstream effector of PKB that has been reported to be involved in insulindependent GLUT4 cell-surface translocation in muscle and adipocytes (38) and in glucose-stimulated insulin secretion in the pancreatic -cell is the Rab GTPaseactivating protein AS160 (39). Interestingly, sirnamediated knockdown of PI3K-C2 led to a 1.4-fold increase in AS160 protein levels. However, the ratio of phosphorylated AS160 to total AS160 was not different from that of the control. Moreover, knockdown of PI3K-C2 abolished the insulin-mediated increase in phosphorylation of AS160 (Fig. 3E). PI3K-C2 contributes to the formation of PI(3,4)P 2 in living cells Activation of PKB via PI3K-C2 would require the formation of either PI(3,4)P 2 or PI(3,4,5)P 3 in vivo (40 42). Although it has been shown that PI3K-C2 can produce at least PI(3,4)P 2 in vitro, the only previously reported in vivo product of this enzyme in response to insulin is PI(3)P (19, 43), which is not an activator of PKB. We therefore analyzed the effect of sirna-mediated knockdown of PI3K-C2 on the PI-lipid profile in insulin-producing cells (Fig. 4; Supplemental Fig. 3). To minimize the contribution by class Ia PI3K (shown Figure 3. Role of PI3K-C2 and PKB isoforms in insulin secretion. A C) Effect of sirna-mediated knockdown of PI3K-C2, glucokinase and PKB isoforms on glucose-stimulated insulin secretion. MIN6 cells were transfected with either control sirna (control), sirna against PI3K-C2 (C2 2), glucokinase ( GK), or PKB isoforms (PKB, PKB, and PKB ). Cells were perifused with KRB containing 0.5 mm glucose for 10 min and then 10 min with KRB containing 11.1 mm glucose, followed by 10 min KRB containing 0.5 mm glucose. After the experiment, cells were lysed. Secretion profiles (A) and compiled data of perifusion and secretion phases (B, C). Data are means se (n 3). D) Effect of sirna-mediated knockdown of PI3K-C2 and glucokinase on KCl-stimulated insulin secretion. MIN6 cells were transfected with either control sirna (control), sirna against PI3K-C2 (C2 2), or sirna against glucokinase ( GK). Cells were perifused with KRB containing 0.5 mm glucose for 5 min and then 3 min with KRB containing 0.5 mm glucose and 30 mm KCl, followed by 5 min KRB containing 0.5 mm glucose. Secretion profiles are presented as mean values se (n 3). E) Effect of PI3K-C2 knockdown on insulin-dependent Thr642-phosphorylation of AS160. MIN6 cells were transfected with either control sirna (control) or sirna against PI3K-C2 (C2 2). Cells were stimulated 4 d after transfection for 30 min with 5 mu/ml insulin and lysed. Phosphorylation of AS160 was analyzed by Western blotting. Data are means se (n 3). A representative blot is shown Vol. 24 June 2010 The FASEB Journal LEIBIGER ET AL.

8 Figure 4. Effect of PI3K-C2 knockdown on in vivo PI-lipid profile. Effect of sirna-mediated knockdown of PI3K-C2 expression on the 3-PI lipids. MIN6 cells were transfected with either control sirna (ctrl) or sirna against PI3K-C2 (C2 2). Levels of PI(3)P, PI(3,4)P 2, and PI(3,4,5)P 3 were analyzed in cells treated or not with insulin in the presence of 25 M LY Data are normalized to combined PI(4)P and PI(4,5)P 2 levels and are the average se of 3 separate experiments. *P 0.05, **P 0.01; 1-way ANOVA with Fukeys posttest for multiple comparisons. in Supplemental Fig. 3), we treated control and PI3K- C2 -sirna-transfected cells with 25 M LY before and during insulin stimulation, a concentration that in these cells allowed insulin-stimulated GK transcription but inhibited PI3K class Ia-dependent insulin transcription (22). Noteworthy, during the whole experiment cells were incubated in fully supplemented culture medium including 10% FCS. As shown in Fig. 4, under our experimental conditions, we did not observe significant changes in PI(3)P levels. Insulin stimulation led to a small but significant increase in both PI(3,4)P 2 and PI(3,4,5)P 3 levels. While sirna-mediated knockdown of PI3K-C2 abolished the increase in PI(3,4)P 2, it did not affect the levels of PI(3)P and PI(3,4,5)P 3. PI3K activity that is associated with IR-B shows a wortmannin sensitivity profile that is similar to that of PI3K-C2 Our previous data showed that insulin activates GK transcription by signaling via the IR-B isoform (22). In contrast to IR-B-mediated activation of the c-fos gene, which requires endocytosis of the receptor (25), IR-Bmediated activation of the GK gene occurs from cell-surface standing receptor complexes (24, 25). While insulin activates its own gene via IR-A and class Ia PI3K from receptor complexes that are also plasma membrane-standing, our data showed that IR-A and IR-B signal from different membrane microdomains (24). This would provide a mechanistic explanation to why the 2 IR isoforms can have access to different pools of adapter/effector proteins, including different forms of PI3K, to initiate different signaling cascades. Consequently, we wanted to know whether cell-surface-standing IR-A and IR-B associate with different members of PI3K. We therefore analyzed the wortmannin sensitivity profiles of PI3K activities that were associated with GFP-tagged IR isoforms. We compared these profiles with the wortmannin sensitivity profile of PI3K-C2 and that of class Ia PI3K. In these studies, we used the GFP-tagged truncated version of IR-A and IR-B, i.e., 23. The 23 variants of IR-A and IR-B are able to activate the insulin and the GK promoter, respectively, in response to insulin (22, 24) but lack the C-terminal YTHM motif. The YTHM motif has been shown to bind PI3K Ia (25, 44 46) and therefore could contaminate the PI3K activity responsible for the activation of the insulin and the GK promoter. As expected, the wortmannin sensitivity profile of PI3K that was associated with GFP-tagged IR-A was very similar to that obtained with class Ia adapter protein p85 immunoprecipitates (Fig. 5Aa, b). In contrast, the PI3K activity associated with IR-B-GFP showed a similar wortmannin sensitivity profile to that obtained with PI3K-C2 immunoprecipitates (Fig. 5Ac, d). While the PI3K activity in p85 and IR-A immunoprecipitates was inhibited by wortmannin in the lower nanomolar range, the PI3K activity in the PI3K-C2 and IR-B immunoprecipitates was only inhibited at higher concentrations, which is typical for PI3K-C2. Although these data at first glance imply that IR-A preferentially associates with PI3K class Ia and IR-B with PI3K-C2, a potential additional class Ia activity in the IR-B immunoprecipitates could simply be masked by the less wortmannin-sensitive PI3K-C2 portion. PI3K-C2 and PKB are codistributed with IR-B at the plasma membrane To test whether IR isoforms preferentially associate with one or the other PI3K isoform at the plasma membrane, we performed codistribution analysis of fluorescently tagged IR isoforms and PI3K isoforms by TIRF microscopy in living cells. The ICQ obtained from the analyzed TIRF images ranges between 0.5 (high degree of codistribution), 0.0 (random distribution), and 0.5 (exclusion of the 2 signals). To calibrate the system for signals that are codistributed with IR-B, we used coexpression of IR-B-Tomato and IR-B-GFP as a control for high codistribution (ICQ: 0.406) and IR-B-Tomato with GFP as a control for random distribution (ICQ: 0.06; Fig. 5Ba, b; C). Coexpression of IR-B-Tomato and GFP-PI3K-C2 showed a high degree of codistribution in the plasma membrane (ICQ: 0.374; Fig. 5Bc, C). Interestingly, codistribution of IR-B-Tomato and GFP-tagged class Ia adapter p85 (47) was low as indicated by an ICQ of (Fig. 5Bd, C). Codistribution of IR-A and PI3K-C2 was also low (ICQ: 0.12; Fig. 5Be, C), while codistribution of IR-A and p85 was high (ICQ: 0.354; Fig. 5Bf, C). These data indeed imply a preferential codistribution of IR-A with class Ia and IR-B with PI3K-C2 at the plasma membrane. Next, we tested a possible interaction between the 2 molecules by FRET (Fig. 5D). By using a protocol of FRET between IR isoforms and PI3K-C2 by acceptor photobleaching (35), we observed FRET between Cerulean-PI3K-C2 and IR-B-Venus as well as between positive controls, such as combinations IR-B-Cerulean/ IR-B-Venus (24) and Cerulean-DEVD-Venus (26). No PI3K-C2 MEDIATED INSULIN SECRETION 1831

9 α α Figure 5. IR-isoform-dependent association with class Ia PI3K and PI3K-C2. A) Effect of wortmannin on PI3K activities coimmunoprecipitated with IR-A or IR-B compared with PI3K activities of class Ia or PI3K-C2. PI3K activity was measured in GFP immunoprecipitates obtained from insulin-stimulated HIT cells expressing either IR-A 23 -GFP (b) or IR-B 23 -GFP (c) and in p85 (a) or PI3K-C2 immunoprecipitates (d) of nontransfected HIT cells. Amount of wortmannin included in the in vitro assay is indicated. Data are means se (n 3). B) Distribution of class Ia, PI3K-C2, and IR isoforms in live MIN6 cells. a) Analysis of IR-B-Tomato with IR-B-GFP as a control for a high degree of codistribution. b) Analysis of IR-B-Tomato with GFP as a control for a random codistribution. c f) Codistribution of IR-A-Tomato or IR-B-Tomato with either GFP-PI3K-C2 or GFP-p85. MIN6 cells were cotransfected with either IR-A-Tomato (IR-A-Tom) (e, f) or IR-B-Tomato (IR-B-Tom) (c, d) in combination with either GFP-PI3K-C2 (GFP-C2 )(c, e) or GFP-p85 (d, f). Codistribution observed by TIRF microscopy is shown as PDM; ICQ was quantified by ICA (see Materials and Methods). Data are means se. C) Same data as in B, but presented as a bar graph. D, E) FRET analysis between IR variants and PI3K-C2 (D) and between mcerulean-pi3k-c2 and IR-B-mVenus at different culture conditions (E). Interaction between potential FRET partners was analyzed using a protocol for acceptor photobleaching (see Materials and Methods); mcerulean served as FRET donor, and mvenus served as FRET acceptor. FRET did occur if the value for E F was significantly higher than the value for C F, which was caused by the fact that photobleaching of the FRET acceptor mvenus leads to an increase in fluorescence of the FRET donor mcerulean. Data are means se. F) Western blot analysis of coimmunoprecipitated IR-B 23 -GFP and myc-pi3k-c2. MIN6 cells were transfected with control plasmid (mock), expression construct for myc-pi3k-c2 alone, or the combination IR-B 23 -GFP myc-pi3k-c2. Immunoprecipitates from 2 mg lysate of cells (IP), obtained with an anti-gfp antibody, and lysates from transfected cells were probed with an anti-myc antibody. A representative blot out of 3 independent experiments is shown. FRET was observed between IR-A-Venus and Cerulean- PI3K-C2 as well as between negative control combinations Cerulean-PI3K-C2 /Venus, IR-B-Venus alone, or Cerulean-PI3K-C2 alone. As shown in Fig. 5E, the efficiency of FRET between IR-B and PI3K-C2 was dependent on insulin. Efficiency of FRET was high at 11 mm glucose, i.e., when cells secrete insulin, and when cells at low glucose concentration (2 mm) were stimulated with either 5 mu/ml insulin or high glucose (16 mm). At low glucose concentration (2 mm), a 1832 Vol. 24 June 2010 The FASEB Journal LEIBIGER ET AL.

10 condition that allows only basal insulin release, the FRET efficiency was low. Because coimmunoprecipitation experiments combined with Western blot analysis were not sensitive enough to detect endogenous PI3K-C2 in IR-B immunoprecipitates and vice versa, we overexpressed tagged IR-B and PI3K-C2 in MIN6 cells. As shown in Fig. 5F, immunoprecipitation of IR-B 23 -GFP with an anti-gfp antibody allowed coimmunoprecipitation of coexpressed myc-pi3k-c2, as identified by an anti-myc antibody. Next, we analyzed the codistribution of IR isoforms and PKB isoforms at the plasma membrane (Fig. 6A; Supplemental Fig. 4). Coexpression of IR-B-Tomato and GFP-PKB showed a high degree of codistribution at the plasma membrane (ICQ: 0.38). Codistribution of IR-A-Tomato and GFP-PI3K-C2 was low and of random character (ICQ: 0.049). Interestingly, codistribution of IR-B/PKB (ICQ: 0.201) was less strong than that of IR-A/PKB (ICQ: 0.379). Finally, codistribution of PKB was low with IR-A (ICQ: 0.145) but high with IR-B (ICQ: 0.389). Although the latter observation shows that both PKB and PKB show a highly similar codistribution pattern with IR-B, interestingly, codistribution of PKB and PKB at the plasma Figure 6. Distribution of PI3K-C2, PKB isoforms, and IR isoforms in live MIN6 cells. A) Distribution of PKB isoforms and IR isoforms in live MIN6 cells. Analyses of IR-B-Tomato with IR-B-GFP as a control for a high degree of codistribution and of IR-B-Tomato with GFP as a control for a random codistribution are given as controls. Codistribution of IR-A-Tomato or IR-B-Tomato with either GFP-PKB, GFP-PKB, or GFP-PKB was analyzed applying TIRF microscopy images of MIN6 cells that were cotransfected with either IR-A-Tomato (IR-A-Tom) or IR-B-Tomato (IR-B-Tom) in combination with either GFP-PKB, GFP-PKB, or GFP-PKB. ICQ was quantified by by ICA (see Materials and Methods). Data are means se. B) Effect of the IR-B NPEY motif on the codistribution of PI3K-C2 with PKB in live MIN6 cells. Codistribution of IR-B-Tomato or IR-B NPEA -Tomato with either GFP-PI3K-C2 or GFP-PKB was analyzed from TIRF microscopy images of MIN6 cells that were cotransfected with either IR-B-Tomato or IR-B NPEA -Tomato in combination with either GFP-PI3K-C2 or GFP-PKB. ICQ was quantified by by ICA (see Materials and Methods). Data are mean se. C) Distribution of GFP-tagged PI3K-C2 and PI3K-C2 -CAAX. MIN6 cells were transfected with either GFP-PI3K-C2 (a) or GFP-PI3K-C2 -CAAX (b). Distribution in live MIN6 cells was analyzed by laser scanning confocal microscopy. Images of 1 representative cell for each expression construct are shown (n 15). D) Effect of expressed wild-type (C2 -WT) and plasma-membrane confined (C2 -CAAX) PI3K-C2 variants on basal insulin-promoter (solid bars) and GK-promoter (open bars) activities. MIN6 cells were cotransfected with either rins1.luc2neo/cmv.hrluccp or GK.Luc2neo/CMV.hRLucCP in combination with either empty vector (mock), wild-type (C2 -WT), and plasma-membrane confined (C2 -CAAX) PI3K-C2 variants, and ratio of Luc/hRLuc expression was obtained. Data are means se (n 3). **P 0.01 vs. mock-transfected control. E) Representative Western blot out of 3 experiments illustrates expression levels of PI3K-C2. PI3K-C2 MEDIATED INSULIN SECRETION 1833

11 membrane is low and almost random as indicated by an ICQ of 0.111, indicating that IR-B/PKB and IR-B/ PKB complexes are located within different subdomains at the plasma membrane. Our previous data (25) showed that IR-B-mediated activation of GK transcription required the integrity of the NPEY motif in the juxta-membrane region of IR-B and that mutating the IR-B NPEY motif to either NPEF or NPEA abolishes insulin-mediated activation of the GK promoter. The latter data are in agreement with a former observation by Urso et al. (16), suggesting the requirement of the juxta-membrane NPEY motif of the IR for insulin-dependent PI3K-C2 action. We therefore wanted to know whether mutating the NPEY motif would affect the interaction between IR-B and PI3K-C2 and the codistribution of IR-B with PI3K-C2 and PKB (Fig. 6B and Supplemental Fig. 5). Indeed, mutation of IR-B-NPEY to IR-B-NPEF led to a loss in FRET between IR-B and PI3K-C2 (Fig. 5D) and resulted in a loss of codistribution of IR-B-NPEF/PI3K-C2 (ICQ: 0.109) and IR-B-NPEF/PKB (ICQ: 0.141). PI3K-C2 has been implicated in different biological activities at different subcellular localizations, including the trans-golgi-network, endosomes, nucleus, and plasma membrane (36, 48 50). To test whether confining PI3K-C2 activity to the plasma membrane is sufficient for activation of GK, we targeted PI3K-C2 to the plasma membrane by adding the Ki-ras CAAX motif (51, 52) to the C terminus, thus generating PI3K-C2 -CAAX. Confocal microscopy on cells expressing a GFP-tagged variant, i.e., GFP-PI3K-C2 -CAAX, showed its pronounced targeting to the plasma membrane (Fig. 6C). As demonstrated in Fig. 6D, expression of the plasma membrane-associated PI3K-C2 -CAAX led to an elevation in basal GKpromoter activity to the same levels as PI3K-C2, thus suggesting that excluding PI3K-C2 activity from other subcellular compartments did not affect the increase in GK promoter activity. The data obtained by our PI-lipid profiling reflects the situation of the whole cell. To get further evidence for PI3K-C2 activity at the plasma membrane, we used fluorescent-tagged PI-lipid-binding motifs, i.e., PH domain of PKB for PI(3,4,5)P 3 and PI(3,4)P 2 (29, 53) and the Hrs-FYVE domain for PI(3)P (27) as indicators for PI lipids in living cells in confocal and TIRF microscopy. At insulin-stimulated conditions, cells expressing GFP-FYVE showed very low abundance of this PI(3)P probe in the plasma membrane as shown by confocal microscopy (Fig. 7Aa) and random codistribution with IR-B as judged by an ICQ of in TIRF microscopy (Fig. 7Ba). This is in agreement with our data on PI-lipid profiling in Fig. 4, i.e., showing no changes in PI(3)P levels under any shown experimental α α Figure 7. Distribution of probes for 3-PI lipids in live MIN6 cells. A) Colocalization of tagged IR-B with either a probe for PI(3)P (GFP-FYVE) (a) or PI(3,4)P 2 / PI(3,4,5)P 3 (GFP-PH-PKB ) (b) analyzed by laser scanning confocal microscopy in live MIN6 cells. MIN6 cells were cotransfected with either IR-B-Tomato (IR-B-Tom) and GFP-2xFYVE Hrs (GFP-FYVE) or with IR-B-Tomato and EGFP- PH Akt (GFP-PH-PKB ). Green is used as digital pseudo-color for fluorescence emitted from GFP; red is used as digital pseudo-color for fluorescence emitted from Tomato. Yellow, obtained after overlaying the GFP- and Tomato signals, indicates colocalization of the expressed proteins. Representative images out of 15 are shown. B) Codistribution of tagged IR-B with either a probe for PI(3)P (GFP-FYVE) (a) or PI(3,4)P 2 /PI(3,4,5)P 3 (GFP-PH-PKB ) (b) analyzed by TIRF microscopy. MIN6 cells were cotransfected with IR-B-Tomato (IR-B-Tom) and either EGFP-PH Akt (GFP-PH-PKB ) or GFP-2xFYVE Hrs (GFP-FYVE). Codistribution observed by TIRF microscopy is shown as PDM and the ICQ was quantified α by ICA (see Materials and Methods). Data are means sd. C) Effect of PI3K-C2 expression levels on the abundance of PI(3,4)P 2 /PI(3,4,5)P 3 in plasma membrane of life MIN6 cells. MIN6 cells were cotransfected with the probe for PI(3,4)P 2 /PI(3,4,5)P 3 (GFP-PH-PKB ) in combinantion with either control sirna, sirna for PI3K-C2 (C2 sirna2), empty plasmid (mock), or wild-type PI3K-C2. Cells were analyzed by laser scanning confocal microscopy; 100 cells were evaluated. Data are percentage of cells with GFP-PH-PKB in the plasma membrane. Data are means se (n 100). Representative Western blot out of 3 experiments illustrates expression levels of PI3K-C2 (overexpression is 4-fold, knockdown by sirna to 10%). α α α 1834 Vol. 24 June 2010 The FASEB Journal LEIBIGER ET AL.

12 conditions. On the other hand, like the full-length PKB, the PH domain of PKB also showed a high degree of codistribution with IR-B (ICQ: 0.391; Fig. 7Bb). Moreover, overexpression of PI3K-C2 increased the amount of cells exhibiting a pronounced GFP-PH- PKB expression in the plasma membrane, while sirna-mediated knockdown of PI3K-C2 led to a reduction of this cell population as shown by the analysis of confocal microscopy images (Fig. 7C). This indicates a role of PI3K-C2 in regulating the levels of PI(3,4)P 2 and/or PI(3,4,5)P 3 in the plasma membrane of insulinproducing cells. DISCUSSION PI3Ks and their lipid products in general, and PI(3,4)P 2 / PI(3,4,5)P 3 -activated PKB/Akt in particular, have been shown to be involved in multiple biological processes including proliferation, differentiation, cell survival, membrane trafficking, cell migration, glucose uptake, and metabolism (3, 5, 54). Dysregulation of 3-PI-lipid levels has been reported to contribute to the development of cardiovascular problems, cancer, allergy, chronic inflammation, and last but not least metabolic disorders such as type 2 diabetes mellitus (3, 54). Although progress has been made in identifying enzymes involved in the metabolism of 3-PI lipids and their pharmacological targeting (55), one of the remaining challenges in the function of different PI3Ks is to decipher signaling specificity. One possibility to gain selectivity in insulin signaling is signal transduction through the 2 isoforms of the IR, i.e., IR-A and IR-B. While in pancreatic -cells insulin activates its own gene by signaling via IR-A and PI3K class Ia, insulin up-regulates GK gene transcription via IR-B and a PI3K activity distinct from class Ia (22). Moreover, our previous observations (24) showed that IR-A and IR-B are localized in different plasma membrane microdomains and that selective signaling via the 2 receptor isoforms is differently sensitive toward cholesterol depletion. This observation would provide a mechanistic explanation to how the 2 IR isoforms could recruit different adapter and effector proteins, including different PI3K family members. We now demonstrate that the PI3K class II member PI3K-C2 is involved in glucose-stimulated insulin secretion, an effect mediated at least in part by PKB dependent regulation of GK gene expression and the activity of the PKB effector Rab GTPase AS160. Reduction of GK expression or its malfunction has been shown to impair glucose-stimulated insulin release and to cause the MODY2-type of diabetes mellitus (37). Interestingly, although knockdown of PI3K-C2 led to a slight increase in AS160 protein levels, the pas160/ AS160 ratio did not change when compared with the control conditions. Moreover, knockdown of PI3K-C2 abolished the increase in AS160 phosphorylation by PKB in response to insulin stimulation. Because knockdown of PI3K-C2 did not affect basal activity of PKB but abolished an insulin-dependent increase in PKB activity, AS160 can still be a substrate for PKB at basal conditions but will not be further phosphorylated on insulin stimulation. Although PI3K-C2 has been reported to exert its activity at various intracellular sites, e.g., trans-golginetwork, endosomes, nucleus, and plasma membrane (36, 48 50), our data suggest that its involvement in IR-B-mediated activation of GK expression takes place at the plasma membrane upstream of PKB. Our observations that a PI3K-C2 -like activity coimmunoprecipitates with IR-B but not IR-A, that PI3K-C2 codistributes in live -cells to a high degree with IR-B but not with IR-A, and that FRET is achieved between PI3K-C2 and IR-B but not with IR-A collectively support the concept that PI3K-C2 cosegregates with IR-B in IR-Aexcluded plasma membrane microdomains. We now show that modulation of the expression levels of PI3K-C2 by either overexpression or sirnamediated knockdown is directly reflected in the activity of PKB in response to insulin. The fact that expression levels of PI3K-C2 did not affect insulin-dependent activities of either PKB or PKB in the same cell preparations implies that PI3K-C2 activates a specific pool of PKB and thus contributes to the selectivity in insulin signaling. More interestingly, activation of PKB by PI3K-C2 requires the generation of PI(3,4)P 2 and/or PI(3,4,5)P 3. This is to our knowledge the first demonstration that PI3K-C2 in response to insulin generates lipid products that activate a selective PKB pool, here PKB. So far the only reported in vivo product of PI3K-C2 in response to insulin is PI(3)P generated in L6 myotubes (19, 43), a PI lipid that does not activate PKB. By combining classical whole-cell PI-lipid profiling with plasma membrane-selective PI-lipid analysis in living cells at the single-cell level using confocal and TIRF microscopy, we identified PI(3,4)P 2 as the lipid product of PI3K-C2 in response to insulin, which serves to activate the -isoform of PKB. This does not only provide the first evidence that PI(3,4)P 2 can be directly produced by PI3K-C2 in response to insulin and thus is not just a 5-phosphatase-mediated breakdown product of insulin-stimulated PI(3,4,5)P 3,but our findings also identify a new pathway whereby insulin can activate one of the most central players in insulin action, i.e., PKB /Akt1. Because in addition to insulin secretion, PI3K and PKB activity have been discussed to be involved in the regulation of -cell mass by modulating proliferation, cell size and survival, and gene expression (reviewed in refs ), future work will have to show to what extent these cellular functions require the involvement of PI3K-C2. Class Ia PI3K associate with the IR at 2 sites. The C-terminal HTMY motif represents a direct binding site, when Tyr-phosphorylated, for the p85-adapter via its SH2 domain. The second site is the juxtamembrane NPEY motif, which, when Tyr-phosphorylated, associates with IRS. Tyr-phosphorylated IRS allows binding with class Ia adapter p85 via its SH2 domain. The mode of how PI3K-C2 associates with IR is not known. Earlier studies by Urso et al. (16) suggest the involvement of the NPEY motif, which was supported by our earlier functional data showing that mutation of this motif to NPEA or NPEF abolishes insulin-stimulated PI3K-C2 MEDIATED INSULIN SECRETION 1835

13 GK expression (22, 25). Our data now show that PI3K-C2 coimmunoprecipitates with IR-B, that PI3K- C2 codistributes with IR-B but not with IR-B-NPEF, and finally, that FRET is occurring between PI3K-C2 and IR-B, but not with NPEY-mutated IR-B or with IR-A. While FRET does not necessarily mean that the 2 candidate molecules have to physically interact with each other, it demonstrates that the 2 proteins are in close proximity, i.e., 5 nm, allowing direct or indirect interaction. The finding that HTMY-deleted IR-A 23 and IR-B 23 show a clear difference in the codistribution pattern for class Ia vs. PI3K-C2 in our TIRF microscopy studies indeed implies a preferential association of the 2 IR-isoforms via the NPEY motif with different PI3K members at the plasma membrane and the formation of distinct signaling platforms. CONCLUSIONS Our data demonstrate that different members of the PI3K family contribute to selectivity in insulin signal transduction in the pancreatic -cell. Here we identify the class II member PI3K-C2 to be involved in glucose-stimulated insulin release. This is achieved by selectively activating PKB in response to insulin and, at least in part, by regulating GK expression and AS160 activity. This signaling pathway involves the generation of PI(3,4)P 2 by PI3K- C2, which allows the selective activation of PKB. Our data thus identify PI3K-C2 as a novel activator of PKB/ Akt in response to insulin. Because PKB has been discussed to be involved in the regulation of -cell mass, insulin secretion, and gene expression, impaired function of this pathway will consequently lead to -cell dysfunction, as shown here for insulin release. Hence, our data do not only describe a novel pathway for the activation of one of the central players in insulin action but also disclose the PI3K class II member PI3K-C2 as a potential drugable target in type 2 diabetes mellitus. The authors thank F. Saupe for technical assistance, and Dr. Dario R. Alessi (MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK), Dr. Harald A. Stenmark (Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway), Dr. Boudewijn M. T. Burgering (University Medical Center Utrecht, Utrecht, The Netherlands), Dr. Roger Y. Tsien (University of California-San Diego, La Jolla, CA, USA), Dr. Anders Tengholm (Uppsala University Biomedical Centre, Uppsala, Sweden), Dr. Georges Bismuth (Institut Cochin, Université Paris Decartes, Paris, France), and Dr. Morris J. Birnbaum (Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, PA, USA) for sharing expression constructs. This work was supported by funds from Karolinska Institutet and by grants from the Swedish Diabetes Association, the Swedish Research Council, the Novo Nordisk Foundation, Eurodia (FP ), the Berth von Kantzow Foundation, the Juvenile Diabetes Research Foundation, and the Family Erling-Persson Foundation. 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Metab. 283, E1113 E1122 Received for publication October 7, Accepted for publication December 17, PI3K-C2 MEDIATED INSULIN SECRETION 1837