Akt mediates insulin rescue from apoptosis in brown adipocytes: effect of ceramide

Growth Hormone & IGF Research 2000, 10, 256–266 doi:10.1054/ghir.2000.0165, available online at http://www.idealibrary.com on

Akt mediates insulin rescue from apoptosis in brown adipocytes: effect of ceramide Paloma Navarro1, Angela M. Valverde1, Jennifer L. Rohn2, Manuel Benito1 and Margarita Lorenzo1 1 Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain and 2Imperial Cancer Research Fund, 44 Lincoln Inn Fields, London WC2A 3PX, UK

Summary We have recently shown that insulin can rescue serum deprived adipocytes from apoptosis in a PI 3 kinase and MAP kinase dependent manner. This study investigated the contribution of Akt and p70S6-kinase in insulin rescue from two different apoptotic triggers, serum deprivation and ceramide treatment. Insulin rescued serum-deprived immortalized brown adipocytes from apoptosis through phosphatidylinositol (PI) 3-kinase and Akt pathways, but independently of p70S6-kinase, as demonstrated by the use of inhibitors such as LY294002 or Rapamycin, and transfection experiments with dominant-negative constructs of Akt or p85 subunit of PI 3-kinase. A constitutively active Akt construct mimicked the insulin survival effect, decreasing the percentage of hypodiploid cells, the percentage of apoptopic cells and precluding the formation of apoptotic nuclei. We propose that the insulin survival effect on immortalized brown adipocytes is mediated through activation of Akt. However, insulin and EGF failed to rescue brown adipocytes from ceramide-induced apoptosis, as determined by DNA laddering, hypodiploid cells and apoptotic nuclei. Ceramide treatment blunted Akt activity but not PI 3-kinase activity, and insulin and EGF were unable to activate Akt. Ceramide also caused apoptosis in cells transfected with a constitutively active Akt construct, since phosphorylation of Akt was impaired under these experimental conditions. This study suggests that activation of Akt may be an absolute requirement for the survival of brown adipocytes. © 2000 Harcourt Publishers Ltd Key words: apoptosis, insulin, ceramide, PKB/Akt, PI 3-kinase, p70S6-kinase.

INTRODUCTION Apoptosis is an evolutionary conserved form of cell death critical for tissue homeostasis1. Although most cells in culture undergo apoptosis following serum/growth factor deprivation, the supply of specific factors prevents the apoptotic process in a number of cell types2–5. We have recently found that immortalized brown adipocytes Received 2 March 2000 Revised 30 May 2000 Accepted 18 August 2000 Correspondence to: Margarita Lorenzo, Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain. Tel.: 34–91–3941858; Fax: 34–91–3941779; E-mail: [email protected]

1096–6374/00/050256+11 $35.00/0

respond to serum deprivation undergoing growth arrest and apoptosis by activation of caspases and this process is precluded by treatment with insulin or IGF-I6–7. Studies of the signalling pathways involved in the antiapoptotic effect induced by these factors have shown that the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin blocks the protective action of NGF, PDGF or IGF-I/insulin in serum-deprived PC-12 cells2,4, Rat-1 fibroblasts8, haematopoietic cells9, oligodendrocytes10 and brown adipocytes6. Downstream of PI 3-kinase various serine kinases can be involved in the survival effect. Several findings indicate that the lipid kinase PI 3-kinase is involved in the regulation of Akt/PKB11,12 since its lipid products PI(3,4,5)P3 and PI(3,4)P2 activate PKB by causing its translocation to the plasma membrane13. The © 2000 Harcourt Publishers Ltd

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activation of Akt/PKB by insulin or IGF-I results from its phosphorylation at two residues (Thr308 and Ser473)14, the phosphorylation of both residues being required for maximal activation of PKB. Thr308 is phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1) and Ser473 is phosphorylated by a distinct membraneassociated kinase, which has tentatively been called PDK215,16. The p70S6-kinase has been identified as another downstream effector of the PI 3-kinase signalling pathway, but the upstream kinases linking PI 3-kinase with p70S6-kinase have not been identified17. Activation of p70S6-kinase is associated with increased phosphorylation of eight residues18. Recently it has been shown that the co-expression of p70S6-kinase with PDK1 results in the strong activation of the p70S6-kinase in vivo through selective phosphorylation at the residue Thr229,18–19. Several lines of evidence support the role of these serine kinases in suppressing apoptosis in various cell types. Thus, overexpression of constitutively active forms of the PI 3-kinase target Akt/PKB has been shown to be sufficient to block apoptosis in fibroblasts and neuronal cells20–23. It has recently been shown that Akt/PKB has a role in the anti-apoptotic effect of the NGF receptor Trk A and that this is independent of the p70S6-kinase24. PI 3kinase, acting through Akt/PKB, also plays a critical role in anoikis and apoptosis induced by myc and UV irradiation23. Activation of Akt ultimately leads to inhibition of caspase activity and protection from apoptotic cell death25,26. Rohn et al have recently shown that activated Akt suppresses CD95 (APO/Fas)-induced apoptosis and that it exerts its activity at a point downstream of FADD but upstream of caspase-827. Moreover, Akt is particularly important for IGF-I-dependent neuronal survival, since it has been found that active forms of Akt phosphorylate BAD, a distant member of the Bcl-2 family that promotes cell death, both in vivo and in vitro28,29. Furthermore, p70S6-kinase seems to have survival and anti-apoptotic activity in NIH3T3 fibroblasts and haematopoietic cells30,31 Ceramide, the second messenger of the sphingomyelin pathway, has emerged as a mediator that regulates apoptosis in many systems32. The generation of endogenous ceramide is regulated by agonists such as TNF-α, which causes apoptosis in brown fat cells33,34. Cell-permeable ceramide analogues mimic these agents and induce apoptosis in different cell types, including PC-12, neuron cell lines and fibroblasts35,36. Accordingly, it has been proposed that ceramide may decrease survival pathways through a decrease in Akt kinase activity, as has been recently reported in the neuron cell line HMN136. In the present work we have investigated the contribution of Akt and p70S6-kinase in insulin rescue from two different apoptotic triggers: serum-deprivation and ceramide treatment. Our results show that insulin

rescued serum-deprived adipocytes from apoptosis in an Akt/PKB-dependent and p70S6-kinase-independent manner. However, insulin and EGF failed to rescue ceramide-treated adipocytes from apoptosis due to their inability to activate Akt under these apoptotic conditions.

MATERIALS AND METHODS Reagents Fetal calf serum (FCS), phosphate-buffered saline (PBS) and cell culture media were obtained from Imperial Laboratories (Hampshire, UK). LY29400, rapamycin, C2ceramide and C2-dihydroceramide were purchased from Calbiochem (Calbiochem-Novabiochem, La Jolla, CA). Insulin was from Sigma Chemical Co (St. Louis, MO). For anti-Tyr(P) immunoprecipitation, the monoclonal antibody (Py72) was a gift from the Imperial Cancer Research Fund (London, UK). The anti-IRS-1 and anti-IRS-2 antibodies were a gift from M. F. White (Boston, MA). The anti-phospho-specific Akt (Ser473) antibody, the antiphospho-specific p70S6 kinase (Thr421/Ser424) antibody and the anti-total Akt antibody were purchased by New England Biolabs (Beverly, MA). Cell culture SV40 large T antigen-immortalized brown adipocyte cell line (MB 4.9.2) was obtained as previously described37. Cells were plated at 1 × 106 cells/100-mm tissue culture plates and propagated in 10 mL of DMEM-10% FCS with antibiotics38. After splitting (1/3) and overnight culture under the conditions described above, cells were washed twice with PBS and serum-deprived for 6 h either in the absence or in the presence of insulin (100 nM) with or without LY294002 (10 µM) or rapamycin (25 ng/mL). In another set of experiments, cells growing in the presence of serum were treated for 6 h with different doses of C2ceramide or C2-dihydroceramide, either in the absence or in the presence of insulin (100 nM) or EGF (3.3 nM). Transfections The following constructs were used for transfection experiments: pSG5-PKBgag encodes a portion of Moloney murine leukaemia virus (MuLV) Gag protein fused in-frame to the bovine Akt protein, a configuration that confers a constitutively activated kinase activity11; pSG5-PKB-CAAX encodes an Akt protein with dominantnegative activity. This construction was a generous gift of B. M. Th. Burgering (Utrecht, The Netherlands)39. The two Akt constructs are expressed under the control of the early SV40 promoter for eukaryotic expression. The

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dominant-negative p85 construct (Srα-∆p85) was kindly provided by M. Kasuga (Kobe, Japan)40. PCDNA3.1/ His/lacZ (Invitrogen) encodes the Escherichia coli β-galactosidase gene under the control of the CMV promoter. For transient co-transfection apoptosis assay, MB 4.9.2 cells were seeded in 6-well plates (1 × 105/well) and the next day transiently transfected with DNA using SuperFect method (Quiagen GmbH, Germany). Transfection complex mixtures contained a total of 7 µg of DNA and were distributed evenly onto triplicate wells (2.3 µg/well). In those plates where we put only the reporter plasmid (PCDNA3.1/his/lacZ), the total amount of DNA was kept constant at 7 µg using relevant empty vector DNA. Five hours after transfection, cells were washed three times in PBS, then replaced in serum free medium with or without insulin or LY294002, and 18 h later, cells were washed in PBS, fixed in 4% paraformaldehyde in PBS for 5–10 min washed again, and stained with 1 × PBS, 2 mM MgCl2, 5 mM K3(CN)6FC, 5 mM K4(CN)6FC, and X-gal (1 mg/mL) overnight at 37ºC. During the washing and staining, plates were centrifuged at 2500 rpm prior to supernatant removal, and removal was performed slowly and carefully with low vacuum to maximize recovery of floating cells. Using phase-contrast microscopy, approximately 500 randomly chosen lacZ-positive cells per well were scored for apoptotic morphology (a rounded, condensed appearance).

Analysis of cellular DNA content by flow cytometry Cells spontaneously detached from the monolayer were collected by centrifugation, and combined with cells from the same dish detached from the monolayer by the addition of trypsin-EDTA. Aliquots of 2 × 105 cells were used for each determination. The ploidy determination of cellular DNA was estimated by flow cytometry DNA analysis in a FACScan flow cytometer (Becton Dickinson, San Jose, CA), using the Kinesis-cycle test DNA reagent (Bio-Rad, Richmond, CA) to stain DNA with propidium iodide, as previously described6. This analysis was performed using a double discriminator module and the multicycle software (Phoenix Systems, AR). Hypodiploid fraction represents the percentage of cells with a DNA content lower than 2C. Analysis of the hypodiploid fractions was performed after transient transfection experiments by the calcium phosphate-mediated protocol (Stratagene, La Jolla, CA) with the constructs mentioned above, as described previously41. DNA fragmentation A modified version of the method of Lyons et al42 was used to assess the fragmentation of brown adipocytes DNA, as previously described6,41. DNA was purified by

phenol-chloroform extraction, precipitated in ethanol, dissolved in TE buffer containing 30% glycerol, 1 mg/mL ethidium bromide and electrophoresed in a 1.5% agarose gel. Gel was visualized and photographed under transmitted UV light with a Polaroid camera. Immunoprecipitations and PI 3-kinase activity For immunoprecipitation with the monoclonal antibody anti-Tyr(P) (Py72), or with the polyclonal antibodies anti-IRS-1 or anti-IRS-2, cells were lysed as previously described43. After determination of protein content, equal amounts of protein (600 µg) were immunoprecipitated at 4°C with the corresponding antibody. The immune complexes were collected on protein A agarose or antimouse IgG-agarose beads. PI 3-kinase activity was measured by in vitro phosphorylation of phosphatidylinositol and analysed by thin-layer chromatography (TLC) as previously described43. Western blotting Cells were lysed as previously described44 and cellular proteins (50 µg) were submitted to SDS-PAGE, transferred to Immobilon membranes, blocked and incubated overnight with the anti-phospho-Akt (1:1000) or anti-Akt (1:1000) or anti-phospho-p70S6-kinase (1:1000) antibodies in 0.05% Tween-20, 1% non-fat dried milk in 10 mM Tris-HCl and 150 mM NaCl, pH 7.5. Immunoreactive bands were visualized using the enhanced chemiluminiscence (ECL-plus) Western blotting protocol (Amersham Pharmacia Biotech). Protein determination was performed by Bradford dye method45, using Bio-Rad reagent and BSA as the standard. Statistical analysis Results are means ± SEM from independent experiments. Statistical significance was tested with a one-way analysis of the variance followed by the protected least-significant difference test. P values less than 0.01 were considered significant. RESULTS PI 3-kinase and Akt, but not p70S6-kinase pathways, are necessary for insulin rescue from apoptosis by serum-deprivation. We tested the effect of LY294002, that binds to and inhibits the catalytic p110 kDa subunit of PI 3-kinase46, as well as rapamycin, a selective inhibitor of p70S6kinase activation47 on the insulin stimulation of PI 3kinase, Akt/PKB and p70S6-kinase. Cells were serum-starved for 6 h, then pre-incubated for 15 min in

Akt mediates insulin rescue from apoptosis 259

A PIP C

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Fig. 1 Insulin stimulate PI 3-kinase, Akt and p70S6-kinase activities in brown adipocytes: effect of inhibitors. Brown adipocytes were cultured for 6 h in a serum-free medium either in the absence or in presence of 10 mM LY294002 (LY) or 25 ng/mL rapamycin (R), and then cells were stimulated for 10 min with 100 nM insulin (I). Control cells (C) were cultured for 6 h in a serum-free medium. A) Cells were lysed and immunoprecipitated with the monoclonal antibody anti-Tyr(P)(Py72) for determination of PI 3-kinase activity. The conversion of PI to PI phosphate (PIP) in presence of (γ-32P)ATP was analysed by TLC. B) Cells were lysed and total protein (50 µg) was subjected to SDS-PAGE, blotted to nylon membrane and immunodetected with the anti-phospho-Akt or anti-total-Akt antibodies. C) cells were lysed and total protein (50 µg) was subjected to SDS-PAGE, blotted to nylon membrane and immunodetected with the anti-phospho-p70S6-kinase antibody. Representative experiments out of three are shown.

the absence or in the presence of 10 µM LY294002 or 25 ng/mL rapamycin and subsequently, stimulated with 100 nM insulin for 10 min. Control cells were cultured for 6 h in a serum-free medium. The whole cell lysates were subjected to immunoprecipitation with the antiTyr(P) antibody and the resulting immune complexes were assayed for PI 3-kinase activity (Fig. 1A). Treatment with LY294002 inhibited insulin stimulated PI 3-kinase activation in immortalized brown adipocytes but rapamycin did not show any effect on the insulin stimulation of PI 3-kinase. Under the same experimental conditions as described above, cells were lysed and subjected to Western blot analysis with the anti-phosphoSer473-Akt antibody (Fig. 1B). The results showed a strong phosphorylation of Akt/PKB by insulin in immortalized brown adipocytes, this phosphorylation being precluded by the LY294002 but not by rapamycin. The amount of total Akt protein remained unaltered by the different treatments (Fig. 1B). We also tested the phosphorylation of p70S6-kinase using an antiphosphoThr421Ser424 antibody. Insulin strongly stimulated the phosphorylation of p70S6-kinase. In this case, both LY294002 or rapamycin were capable of inhibiting the phosphorylation of p70S6kinase induced by insulin (Fig. 1C). These results indicate that downstream of PI 3-kinase insulin stimulates both Akt and p70S6-kinase in immortalized brown adipocytes. Whether Akt is upstream of p70S6-kinase or represented an alternative pathway remains to be established.

Since chemical inhibitors are available for p70S6kinase, such as rapamycin, we decided to evaluate this pathway in the insulin rescue from apoptosis, comparing with the known effect of the PI 3-kinase inhibitor LY2940026 (Fig. 2). Apoptosis is often accompanied by a rapid cleavage of the cellular DNA into multiples of 180 base pairs (bp) corresponding to internucleosomal spacing, and the ‘180-bp ladder’ can be seen upon electrophoresis of DNA from apoptotic cells48. Cells were cultured for 6 h in a serum-free medium in the absence or presence of 100 nM insulin either without or with 25 ng/mL rapamycin or 10 µM LY294002, and then, extranuclear DNA laddering was analysed (Fig. 2A). As expected, insulin prevented DNA laddering occurring under serum deprivation and LY294002 blunted the antiapoptotic effect of insulin. However, rapamycin treatment does not affect insulin rescue from apoptosis on DNA laddering (Fig. 2A). EGF (3.3 nM) also rescued serum-deprived adipocytes from apoptosis in a similar fashion to insulin at 100 nM, the optimal dose for insulin survival effects in this cell system, as previously shown6 (Fig. 2A). Furthermore, we collected cells under the different treatments described above, and subjected them to FACScan analysis of cellular DNA after staining with propidium iodide. The percentage of cells with a DNA content lower than 2C (hypodiploid cells), corresponding to apoptotic cells is shown in Figure 2B. In agreement with the results on DNA laddering, LY significantly increased the percentage of hypodiploid cells found under insulin treatment. Meanwhile rapamycin did not modify the effect of the indicating that insulin p70S6kinase pathway is not contributing to insulin survival effect. The protein kinase Akt/PKB is another downstream PI 3-kinase target, so we decided to study its implication in insulin survival effect. Since no chemical inhibitors of this pathway are thus far available, we blocked this pathway with an Akt dominant-negative construct, ∆PKB (pSG5-Akt/PKB-CAAX). An Akt constitutively activated construct, PKBgag (pSG5-Akt/PKBgag), was available to test the implication of this enzyme in the cell survival in the absence of insulin. The use of these constructs required transfection of immortalized brown adipocytes (clone MB 4.9.2). Accordingly, we used a different technique to determine apoptosis; co-transfection with a lacZ reported plasmid and visualization of lacZ-positive cells with apoptotic morphology (a rounded condensed appearance) using phase-contrast microscopy. Brown adipocytes were co-transfected with a lac-Z reporter plasmid plus combinations of an empty vector, or Akt mutant constructs (constitutively activated, PKBgag or dominant-negative, ∆PKB) or a dominant-negative form of the regulatory subunit of PI 3-kinase (∆p85). Five hours after the transfection cells were washed and serum-free

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Fig. 2 Inhibition of PI 3-kinase but not p70S6-kinase impairs insulin rescue from apoptosis by serum-deprivation. Brown adipocytes cultured in 10% FCS were serum-deprived for 6 h either in the absence or presence of 100 nM insulin (with or without 10 µM LY294002 or 25 ng/mL rapamycin) or 3.3 nM EGF. A) Cells were scraped and subjected to extranuclear DNA extraction. Purified DNA was electrophoresed in a 1.5% agarose gel, and visualized by UV fluorescence after staining with ethidium bromide. A representative experiment out of four is shown. B) Cells were harvested and analysed for cellular DNA content in the FACscan. Results represent the percentage of cells with a DNA content lower than 2C (hypodiploid cells), and are means ± SEM (n=8) from four independent experiments. Statistical significance was tested with a one-way analysis of the variance followed by the protected least-significant difference test, where differences between values under serum-deprivation vs 10% FCS are represented by (▲), between values in the presence of insulin or EGF vs serum-deprivation are represented by (*) and between values in the presence of insulin + inhibitor vs insulin alone are represented by (●); ▲, * and ●, P<0.01.

medium was added with or without 100 nM insulin. The next day, cells were fixed, stained with X-gal, and lac-Z positive transfected cells were scored for apoptotic phenotype, as described in Materials and Methods. The results (Fig. 3, upper panel) show that insulin significantly decreases the percentage of apoptotic death found in serum-deprived cells in an identical fashion as shown above on DNA laddering. A significant decrease in the percentage of apoptotic cells was also observed after transfection with PKBgag in the absence of insulin, indicating that overstimulation of Akt/PKB pathway is sufficient to preclude apoptotic cell death. Transfection with ∆PKB and the subsequent treatment with insulin, produced a significant increase in the apoptotic death observed in the presence of insulin, even higher than that produced by ∆p85 (Fig. 3, upper panel), demonstrating that both PI 3-kinase and Akt/PKB activities are involved in the insulin rescue from apoptosis by serumdeprivation. In the other set of experiments, cells were transiently transfected with constructs encoding PKBgag or ∆PKB or ∆p85 or an empty vector (pSG5) by the calcium phosphate method. After transfection, cells were

incubated for 12 h in 10% FCS, serum deprived for 6 h with or without insulin, harvested and analysed for DNA content in the FACScan (Fig. 3, lower panel). Insulin significantly decreased the percentage of hypodiploid cells observed under serum deprivation, meanwhile transfection with PKBgag in the absence of insulin also produced a significant decrease in the percentage of hypodiploid cells. However, transfection with ∆p85 or ∆PKB followed by treatment with insulin significantly increased the percentage of hypodiploid cells compared with the data obtained after insulin treatment. Apoptotic nuclei were visualized after transient transfection with PKBgag or with an empty vector (in the absence or presence of insulin) by direct staining of cells with propidium iodide and confocal microscopy (Fig. 4). Constitutively active Akt decreases the proportion of apoptotic nuclei observed after 6 h of serum deprivation in a similar fashion to insulin, although a few dying cells are still present, probably representing non-transfected cells. These data indicate that Akt/PKB activity is involved in the insulin rescue from apoptosis in serumdeprived immortalized brown adipocytes.

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Fig. 3 Constitutively active Akt rescue brown adipocytes from apoptosis. Upper panel: Cells were transiently co-transfected with CMV-driven lacZ reporter plasmid (Vector) in conjunction with constructs encoding a constitutively active Akt (pSG5PKBgag)(PKBgag) or a dominant-negative Akt (pSG5-PKBCAAX)(∆PKB) or a dominant-negative p85 subunit of PI 3-kinase (Srα-∆p85) (∆p85), using Superfect reagent. Other cells were transfected only with CMV-lacZ. After washing away the complexes, the cells were incubated for 12 h in serum free medium with or without insulin (100 nM). Cells were then fixed, and blue cells were scored for apoptotic death by morphological criteria under phase microscopy. Lower panel: Cells were transiently transfected with constructs encoding PKBgag or ∆PKB or ∆p85 or an empty vector by calcium phosphate method. Then, cells were incubated for 12 h in 10% FCS and serum deprived for 6 h with or without insulin. Cells were harvested and analysed for DNA content (hypodiploid cells are represented) in the FACScan. Results are means ± SEM (n=8) from four independent experiments. Statistical significance was tested with a one-way analysis of the variance followed by the protected least-significant difference test, where differences between values in the presence of insulin or PKBgag vs vector in the absence of insulin are presented by (*), and values in the presence of dominant-negative constructs plus insulin vs vector plus insulin are represented by (●); * and ●, P<0.01.

Insulin fails to rescue from ceramide induced apoptosis by its inability to stimulate AKT pathway Ceramide has been reported to induce apoptosis in a number of cell types, and TNF-α, a generator of endogenous ceramide, has been described as an inductor of

PKBgag

Fig. 4 Constitutively active Akt avoids the formation of apoptotic nuclei. Cells were transiently transfected with constructs encoding a constitutively active Akt (PKBgag) or an empty vector pSG5 by calcium phosphate method. Then, cells were incubated for 12 h in 10% FCS and serum deprived for 6 h without (C) or with insulin (I). Cells were directly stained in the dish with propidium iodide and observed under confocal microscope. White nuclei represent apoptotic cells. Bar: 20 µM.

apoptosis in brown adipocytes33–34. We decided to check if the cell permeable ceramide analogue, C2-ceramide, was able to induce apoptosis in immortalized brown adipocytes under growing conditions (10% FCS) (Fig. 5). Cells were cultured for 6 h in the absence or in the presence of increasing amounts of ceramide, and the

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Fig. 5 Ceramide induces brown adipocytes apoptosis; insulin, EGF and PKBgag fail to rescue. A) Brown adipocytes cultured in 10% FCS were treated for 6 h with different doses of C2-ceramide, and cells were harvested and analysed for cellular DNA content. Results represent the percentage of hypodiploid cells, and are means ± SEM (n=8) from four independent experiments. Statistical significance was tested with a one-way analysis of the variance followed by the protected least-significant difference test, where differences between values in the presence of C2-ceramide vs 10% FCS growing cells are represented by (●); ●, P<0.01. B) Brown adipocytes cultured in 10% FCS were treated for 6 h with 100 µM ceramide either in the absence or presence of 100 nM nM insulin (I) or 3.3 nM EGF (E). Cells were scraped and subjected to extranuclear DNA extraction. Purified DNA was electrophoresed in a 1.5% agarose gel, and visualized by UV fluorescence after staining with ethidium bromide. A representative experiment out of four is shown. C) Cells cultured in 10% FCS were treated for 6 h with 100 µM ceramide either in the absence or presence of 100 nM insulin (I) or 3.3 nM EGF (E), except for the last two conditions where cells were transfected with a constitutively activated Akt construct (PKBgag) or the empty vector (V) incubated for 12 h in 10% FCS and treated for 6 h with ceramide treatment. At the end of the culture time, hypodiploid cells were analysed as in A. Results are means ± SEM (n=8) from four independent experiments.

E +

percentage of hypodiploid cells was determined as previously described. C2-ceramide induced apoptosis in a dose-dependent manner, however, the dose of 100 µM was used in further experiments as it produced a similar rate of apoptosis to that reached after 6 h-serum deprivation (Fig. 5A). The biologically inactive ceramide analogue, C2-dihydroceramide, did not exhibit apoptotic effect at the time (6 h) or concentration (100 µM) tested (results not shown). Next, we checked if insulin was able to rescue brown adipocytes from apoptosis induced by C2-ceramide treatment, either by analysis of DNA laddering or percentage of hypodiploid cells. In parallel, EGF rescue effect was tested. As shown in Fig. 5B, ceramide treatment for 6 h produced DNA laddering compared to cells growing only in the presence of serum. Simultaneous treatment with insulin (100 nM) or EGF (3.3 nM) and C2-ceramide failed to rescue brown adipocytes from apoptosis either in DNA laddering (Fig. 5B) or in percentage of hypodiploid cells (Fig. 5C).

Furthermore, we tested the ability of a constitutively active Akt construct to rescue from ceramide-induced apoptosis. Cells were transiently transfected with PKBgag or with an empty vector by the calcium phosphate method, and after transfection, cells were incubated for 12 h in 10% FCS and then, incubated for 6 h in the presence of ceramide. Transfection with PKBgag did not decrease the percentage of hypodiploid cells observed under ceramide treatment (Fig. 5C). In Fig. 6, apoptosis was determined morphologically by direct staining of cells with propidium iodide and confocal microscopy in the same experimental conditions as described in Fig. 5C. A dramatic increase in the proportion of apoptotic nuclei under C2-ceramide treatment was observed as compared with untreated cells, this proportion remained high under addition of insulin or EGF. Moreover, transfection with a constitutively active Akt did not prevent the ceramide apoptotic effect, a similar proportion of apoptotic nuclei was detected in cells

Akt mediates insulin rescue from apoptosis 263

F

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Fig. 6 Insulin, EGF and PKBgag fail to prevent the formation by ceramide of apoptotic nuclei. Brown adipocytes cultured in 10% FCS (F) were treated for 6 h with 100 µM ceramide (Cer) either in the absence or presence of 100 nM insulin (Cer+I) or 3.3 nM EGF (Cer+E). Some cells were transfected with a constitutively active Akt construct (PKBgag) or the empty vector prior to ceramide treatment (Cer+PKBgag, Cer+V). Cells were directly stained in the dish with propidium iodide and observed under confocal microscope. White nuclei represent apoptotic cells. Bar: 15 µM.

transfected with PKBgag or with an empty vector under ceramide treatment (Fig. 6). The activation of Akt seems to be necessary for insulin rescue from apoptosis by serum-deprivation, consequently we determined PI 3-kinase and Akt activities under ceramide treatment either in the absence or in the presence of insulin. In parallel, cells were also treated with EGF. Cells were treated for 6 h with C2-ceramide and then, stimulated for 10 min with insulin (100 nM) or EGF (3.3 nM). Control cells were cultured for 6 h in a 10% FCS-medium. PI 3-kinase activity was determined in the immune complexes after immunoprecipitation with the anti-Tyr(P) antibody, or with the anti-IRS-1 or antiIRS-2 antibodies. As shown in Figure 7A, PI 3-kinase is active under growing conditions (10% FCS) but further stimulates (3-fold increase) after treatment with ceramide for 6 h or stimulation for 10 min with insulin or EGF. C2ceramide treatment slightly increased insulin-stimulated PI 3-kinase activation associated to Tyr(P) or IRS-1 or IRS-2, although C2-ceramide did not affect EGF-stimulated PI 3-kinase (Fig. 7A). In addition, phosphorylation

of the Ser/Thr kinase Akt/PKB was studied in cells cultured under the same experimental conditions. Cells were lysed and subjected to Western blot analysis with the anti-phosphoSer473-Akt antibody. The results showed a strong phosphorylation of Akt/PKB under growing conditions (10% FCS) or under 10 min stimulation with insulin or EGF in immortalized brown adipocytes, this phosphorylation being precluded by treatment with C2ceramide under all the experimental conditions tested. The amount of total Akt protein, however, remains unaltered by the different treatments (Fig. 7B). Finally, the phosphorylation of Akt in cells transfected with PKBgag in the absence or presence of ceramide was also determined (Fig. 7C). Two Akt bands were observed in PKBgag-transfected cells immunoblotted with the antitotal-Akt antibody, corresponding to the PKBgag fusion protein and endogenous Akt protein, respectively. Both bands appeared phosphorylated in cells growing under 10% FCS. However, treatment with ceramide in the presence of 10% FCS in PKBgag-transfected cells impaired Akt phosphorylation, the phosphorylation of both bands

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Akt endog. PKBgag PKBgag + Cer

Fig. 7 Ceramide blunted Akt but not PI-3-kinase activities. Brown adipocytes cultured in 10% FCS (F) were treated for 6 h with 100 µM C2-ceramide (Cer) and then cells were stimulated for 10 min with 100 nM insulin (I) or 3.3 nM EGF (E). A) Cells were lysed and immunoprecipitated with the monoclonal antibody anti-Tyr(P)(Py72) or with the anti-IRS-1 or anti-IRS-2 antibodies for determination of PI 3-kinase activity. The conversion of PI to PIP in presence of (γ-32P)ATP was analysed by TLC. B) Cells were lysed and total protein (50 µg) was subjected to SDS-PAGE, blotted to nylon membrane and immunodetected with the anti-phospho-Akt or anti-Akt antibodies. C) Cells were transfected with a constitutively active Akt construct (PKBgag), incubated for 12 h in 10% FCS and treated for 6 h in the absence or in the presence of C2-ceramide. At the end of the culture time cells were lysed and total protein (50 µg) was submitted to SDS-PAGE and blotted to nylon membrane. Both PKBgag fusion protein and endogenous Akt protein in their phosphorylated form were immunodetected with the anti-phospho-Akt antibody. Total PKBgag fusion protein and endogenous Akt protein were detected with an anti-Akt antibody. Representative experiments out of three are shown in A, B and C.

was dramatically decreased, but no changes were observed in the expression of the total Akt protein (Fig. 7C). DISCUSSION Serum-deprivation of immortalized brown adipocytes has been shown to result in apoptosis by activation of caspases and upregulation of Bcl-xS and downregulation of Bcl-27. Insulin has been shown to rescue brown adipocytes from apoptosis by serum-deprivation in a PI 3-kinase and MAP-kinase dependent manner6. The data reported in this work indicate that downstream of PI 3-kinase, Akt is mediating the insulin survival effects in immortalized brown adipocytes under serum-deprivation. A dominant-negative construct of Akt blunted the insulin rescue effect in a similar fashion to a dominantnegative construct of PI 3-kinase or a chemical inhibitor of PI 3-kinase. Furthermore, a constitutively active Akt construct transiently transfected to brown adipocytes mimicked insulin effects in decreasing the percentage of

hypodiploid cells and apoptotic cells, as well as precluding the formation of apoptotic nuclei. These results agreed with those described for fibroblasts and neuronal cells overexpressing constitutively active Akt20–23. Moreover, Akt has been involved in mediating Trk A receptor survival effect24. Whether Akt could directly inactivate a critical component of the cell-intrinsic death machinery in brown adipocytes as it does in neurons phosphorylating BAD, remains to be explored29. Although the activity of p70S6-kinase is stimulated by insulin in brown adipocytes, our results indicate that insulin rescue from serum-deprived apoptosis is independent of this pathway. This result agrees with that reported for NGF in PC-12 cells8 but contrasts with those proposing that a rapamycin-sensitive pathways implicated in the prevention of apoptosis in haematopoietic cells and NIH3T3 fibroblasts30–31. Ceramide analogues induce apoptosis in immortalized brown adipocytes under growing conditions, in agreement with its apoptotic effect in other cell types, including PC-12 cells, the neuron cell line HMN1 and

Akt mediates insulin rescue from apoptosis 265

fibroblasts35,36,49. Our data indicate that ceramide inhibits Akt kinase activity but not PI 3-kinase activity (as other groups reported36,49,50), and therefore decreases the activity of a major survival/antiapoptotic pathway, leading immortalized brown adipocytes growing in 10% FCS to apoptotic cell death. C2-ceramide also inhibited stimulation of Akt by EGF, an event that is IRS-1 and IRS-2 independent. Since neither insulin or EGF are able to stimulate Akt under ceramide treatment, both factors fail to rescue brown adipocytes from ceramide-induced apoptosis. The mechanism by which ceramide inhibits Akt phosphorylation remains unclear, although this inhibition has been previously described36,50. The regulation of Akt by C2-ceramide could occur upstream of Akt, and may involve intermediate regulators downstream of PI 3-kinase such as PDK115,16, or other PI 3-kinaseindependent pathways. Another possibility could be the activation of a ceramide-activated protein phosphatase involved in Akt dephosphorylation, as recently proposed for PC-12 cells51. Transient transfection with a constitutively active Akt construct failed to preclude ceramide induced apoptosis, remaining a similar percentage of hypodiploid cells and apoptotic nuclei than in the ceramide apoptotic cells transfected with an empty vector. Furthermore, ceramide impaired Akt phosphorylation under transfection with PKBgag construct. These data favour the implication of a phosphatase stimulated by ceramide that dephosphorylates and inactivates Akt, avoiding its survival effects. Whether this is a specific ceramide-activated protein phosphatase51 remains to be established. In summary, Akt mediates the insulin survival effect in brown adipocytes under serum-deprivation. However, a complete inhibition of Akt, independent of PI 3-kinase, was produced by ceramide treatment, leading to apoptosis in brown adipocytes. Under these conditions, insulin, EGF and a constitutively active Akt construct failed to rescue brain adipocytes from ceramide-induced apoptosis since Akt phosphorylation, but not PI 3-kinase activity, was impaired.

ACKNOWLEDGEMENTS This work was supported by grants PM98/0082 from the Direccion General de Enseñanza Superior Investigacion Cientifica (Spain) and from Fundacion Ramon Areces (Spain). P. Navarro was recipient of a fellowship from Ministerio de Educacion y Cultura (Spain). We are very grateful to Dr B. M. Th. Burgering (Utrecht, The Netherlands) for providing Akt constructs. We thank Dr A. Alvarez (Universidad Complutense, Madrid, Spain) for his expert technical assistance with the Flow Cytometer and Confocal Microscopy.

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