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FoxO1 and SIRT1 Regulate β-Cell Responses to Nitric Oxide*

Open AccessPublished:January 01, 2011DOI:https://doi.org/10.1074/jbc.M110.204768
      For many cell types, including pancreatic β-cells, nitric oxide is a mediator of cell death; paradoxically, nitric oxide can also activate pathways that promote the repair of cellular damage. In this report, a role for FoxO1-dependent transcriptional activation and its regulation by SIRT1 in determining the cellular response to nitric oxide is provided. In response to nitric oxide, FoxO1 translocates from the cytoplasm to the nucleus and stimulates the expression of the DNA repair gene GADD45α, resulting in FoxO1-dependent DNA repair. FoxO1-dependent gene expression appears to be regulated by the NAD+-dependent deacetylase SIRT1. In response to SIRT1 inhibitors, the FoxO1-dependent protective actions of nitric oxide (GADD45α expression and DNA repair) are attenuated, and FoxO1 activates a proapoptotic program that includes PUMA (p53-up-regulated mediator of apoptosis) mRNA accumulation and caspase-3 cleavage. These findings support primary roles for FoxO1 and SIRT1 in regulating the cellular responses of β-cells to nitric oxide.

      Introduction

      Nitric oxide plays a central role in regulating the response(s) of pancreatic β-cells to cytokine treatment. Cytokines such as IL-1 (rat) and a combination of IL-1 + IFN-γ (mouse and human) stimulate the expression of the inducible isoform of nitric-oxide synthase (NOS) and the production of micromolar levels of nitric oxide by β-cells (
      • Southern C.
      • Schulster D.
      • Green I.C.
      ,
      • Eizirik D.L.
      • Flodström M.
      • Karlsen A.E.
      • Welsh N.
      ,
      • Corbett J.A.
      • McDaniel M.L.
      ,
      • Corbett J.A.
      • Tilton R.G.
      • Chang K.
      • Hasan K.S.
      • Ido Y.
      • Wang J.L.
      • Sweetland M.A.
      • Lancaster Jr., J.R.
      • Williamson J.R.
      • McDaniel M.L.
      ). Nitric oxide attenuates insulin secretion by inhibiting the oxidation of glucose to CO2 and the activity of mitochondrial iron-sulfur center containing enzymes such as aconitase and complexes of the electron transport system (
      • Corbett J.A.
      • Wang J.L.
      • Sweetland M.A.
      • Lancaster Jr., J.R.
      • McDaniel M.L.
      ,
      • Welsh N.
      • Eizirik D.L.
      • Bendtzen K.
      • Sandler S.
      ). The result is a 4-fold reduction in cellular ATP concentration (
      • Eizirik D.L.
      • Flodström M.
      • Karlsen A.E.
      • Welsh N.
      ,
      • Corbett J.A.
      • Wang J.L.
      • Hughes J.H.
      • Wolf B.A.
      • Sweetland M.A.
      • Lancaster Jr., J.R.
      • McDaniel M.L.
      ) that leads to the inhibition of glucose-induced insulin secretion due to the inability to generate sufficient levels of ATP to close the ATP-sensitive K+ channels, an event required for β-cell depolarization and Ca2+-dependent exocytosis (
      • Koster J.C.
      • Marshall B.A.
      • Ensor N.
      • Corbett J.A.
      • Nichols C.G.
      ,
      • Heitmeier M.R.
      • Corbett J.A.
      ). In addition to the inhibition of β-cell function, nitric oxide induces DNA strand breaks and oxidative DNA damage (
      • Delaney C.A.
      • Green M.H.
      • Lowe J.E.
      • Green I.C.
      ,
      • Eizirik D.L.
      • Delaney C.A.
      • Green M.H.
      • Cunningham J.M.
      • Thorpe J.R.
      • Pipeleers D.G.
      • Hellerström C.
      • Green I.C.
      ).
      The inhibitory actions of IL-1 on β-cell function and DNA damage are reversible (
      • Palmer J.P.
      • Helqvist S.
      • Spinas G.A.
      • Mølvig J.
      • Mandrup-Poulsen T.
      • Andersen H.U.
      • Nerup J.
      ,
      • Comens P.G.
      • Wolf B.A.
      • Unanue E.R.
      • Lacy P.E.
      • McDaniel M.L.
      ). The addition of a NOS inhibitor to islets pretreated for 24 h with IL-1 (without removing IL-1) results in a time-dependent recovery of insulin secretion and mitochondrial function (
      • Corbett J.A.
      • McDaniel M.L.
      ) and the repair of damaged DNA (
      • Hughes K.J.
      • Chambers K.T.
      • Meares G.P.
      • Corbett J.A.
      ,
      • Rosales A.L.
      • Cunningham J.M.
      • Bone A.J.
      • Green I.C.
      • Green M.H.
      ). This recovery response requires new gene expression, the activation of JNK, and can be stimulated by nitric oxide (
      • Scarim A.L.
      • Heitmeier M.R.
      • Corbett J.A.
      ,
      • Scarim A.L.
      • Nishimoto S.Y.
      • Weber S.M.
      • Corbett J.A.
      ). The ability of β-cells to recover from cytokine- and nitric oxide-induced damage is temporally limited. Following a 36-h exposure to IL-1, β-cells are no longer capable of recovering metabolic and secretory function, and the islets are committed to death (
      • Hughes K.J.
      • Chambers K.T.
      • Meares G.P.
      • Corbett J.A.
      ,
      • Scarim A.L.
      • Heitmeier M.R.
      • Corbett J.A.
      ). Studies have shown that cytokines can kill β-cells by nitric oxide-dependent and -independent necrotic and apoptotic mechanisms (
      • Steer S.A.
      • Scarim A.L.
      • Chambers K.T.
      • Corbett J.A.
      ,
      • Collier J.J.
      • Fueger P.T.
      • Hohmeier H.E.
      • Newgard C.B.
      ,
      • Liu D.
      • Pavlovic D.
      • Chen M.C.
      • Flodström M.
      • Sandler S.
      • Eizirik D.L.
      ,
      • Mandrup-Poulsen T.
      ,
      • Oyadomari S.
      • Takeda K.
      • Takiguchi M.
      • Gotoh T.
      • Matsumoto M.
      • Wada I.
      • Akira S.
      • Araki E.
      • Mori M.
      ,
      • Grunnet L.G.
      • Aikin R.
      • Tonnesen M.F.
      • Paraskevas S.
      • Blaabjerg L.
      • Størling J.
      • Rosenberg L.
      • Billestrup N.
      • Maysinger D.
      • Mandrup-Poulsen T.
      ,
      • Thomas H.E.
      • McKenzie M.D.
      • Angstetra E.
      • Campbell P.D.
      • Kay T.W.
      ,
      • Gurzov E.N.
      • Ortis F.
      • Cunha D.A.
      • Gosset G.
      • Li M.
      • Cardozo A.K.
      • Eizirik D.L.
      ). This dichotomy in the type of cell death that has been observed appears to reflect the temporal changes in the metabolic responses and the extent of DNA damage caused by nitric oxide. Following short exposures to cytokines, under conditions in which nitric oxide-mediated damage is reversible, biochemical assays indicate that cell death is necrotic in nature. At points in which β-cells no longer have the capacity to recover from this damage, cytokine-mediated β-cell death shifts to an apoptotic process that is associated with irreversible DNA damage and caspase activation (
      • Hughes K.J.
      • Chambers K.T.
      • Meares G.P.
      • Corbett J.A.
      ). Similar to what has been observed in β-cells, nitric oxide has been implicated in the induction of both necrosis and apoptosis of multiple cell types (
      • Brüne B.
      ,
      • Brown G.C.
      • Borutaite V.
      ,
      • Calabrese V.
      • Cornelius C.
      • Rizzarelli E.
      • Owen J.B.
      • Dinkova-Kostova A.T.
      • Butterfield D.A.
      ).
      Although nitric oxide is a known activator of p53 (
      • Wang X.
      • Michael D.
      • de Murcia G.
      • Oren M.
      ,
      • McLaughlin L.M.
      • Demple B.
      ), we recently observed that the repair of nitric oxide-damaged DNA in β-cells occurs by a p53-independent but GADD45α-dependent process (
      • Hughes K.J.
      • Meares G.P.
      • Chambers K.T.
      • Corbett J.A.
      ). A number of studies have shown that GADD45α expression is regulated by p53 (
      • Carrier F.
      • Smith M.L.
      • Bae I.
      • Kilpatrick K.E.
      • Lansing T.J.
      • Chen C.Y.
      • Engelstein M.
      • Friend S.H.
      • Henner W.D.
      • Gilmer T.M.
      • et al.
      ). Our findings suggest that there is an alternative pathway because p53 knockdown does not modify GADD45α expression or GADD45α-dependent DNA repair in β-cells. Members of the Forkhead family of transcription factors are known to regulate genes involved in cell cycle, stress resistance, DNA repair, and apoptosis (
      • Giannakou M.E.
      • Partridge L.
      ,
      • Huang H.
      • Tindall D.J.
      ). FoxO1 is one member of this family whose activity is regulated by various external stimuli, including insulin, growth factors, and oxidative stress (
      • Frescas D.
      • Valenti L.
      • Accili D.
      ,
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). These stimuli can regulate FoxO1 activity by modifying its subcellular localization (cytoplasmic versus nuclear) and posttranslational modifications (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Qiang L.
      • Banks A.S.
      • Accili D.
      ). In response to insulin signaling, Akt phosphorylates FoxO1, resulting in its binding to 14-3-3 and sequestration in the cytoplasm where it is inactive (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). In contrast, stresses such as nutrient deprivation attenuate Akt-mediated phosphorylation and result in the nuclear translocation of FoxO1 (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). Once in the nucleus, the activity of FoxO1 is controlled by acetylation via the histone acetyltransferase p300/CBP and deacetylation by the NAD+-dependent deacetylase SIRT1 (
      • Brunet A.
      • Sweeney L.B.
      • Sturgill J.F.
      • Chua K.F.
      • Greer P.L.
      • Lin Y.
      • Tran H.
      • Ross S.E.
      • Mostoslavsky R.
      • Cohen H.Y.
      • Hu L.S.
      • Cheng H.L.
      • Jedrychowski M.P.
      • Gygi S.P.
      • Sinclair D.A.
      • Alt F.W.
      • Greenberg M.E.
      ,
      • Greer E.L.
      • Brunet A.
      ,
      • Kitamura Y.I.
      • Kitamura T.
      • Kruse J.P.
      • Raum J.C.
      • Stein R.
      • Gu W.
      • Accili D.
      ). Nuclear deacetylated FoxO1 promotes the transcription of genes involved in DNA repair and stress resistance, whereas acetylated FoxO1 promotes the expression of genes involved in apoptosis (
      • Brunet A.
      • Sweeney L.B.
      • Sturgill J.F.
      • Chua K.F.
      • Greer P.L.
      • Lin Y.
      • Tran H.
      • Ross S.E.
      • Mostoslavsky R.
      • Cohen H.Y.
      • Hu L.S.
      • Cheng H.L.
      • Jedrychowski M.P.
      • Gygi S.P.
      • Sinclair D.A.
      • Alt F.W.
      • Greenberg M.E.
      ,
      • Greer E.L.
      • Brunet A.
      ). Recently, evidence has suggested that acetylation may also direct nuclear localization of FoxO1 independent of phosphorylation (
      • Qiang L.
      • Banks A.S.
      • Accili D.
      ).
      In this study we provide evidence that FoxO1 and its regulation by SIRT1 play primary roles in regulating the cellular responses to nitric oxide. Nitric oxide, produced in response to IL-1, or supplied exogenously, stimulates the nuclear localization of FoxO1 and the FoxO1-dependent expression of GADD45α and repair of nitric oxide-damaged DNA in β-cells. This protective response is controlled by the activity of SIRT1. Activators of SIRT1 accelerate, whereas inhibitors attenuate, the repair of nitric oxide-damaged DNA. Further, inhibition of SIRT1 is associated with the induction of apoptosis that is characterized by enhanced expression of the proapoptotic gene p53-up-regulated mediator of apoptosis (PUMA)
      The abbreviations used are: PUMA
      p53-up-regulated mediator of apoptosis
      DEANO
      (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate
      NMMA
      l-NG-monomethyl arginine
      TSA
      trichostatin A
      AMPK
      AMP-activated protein kinase.
      and caspase activation. These findings suggest that the fate of β-cells in response to nitric oxide is controlled by the cellular localization of FoxO1 and activity of SIRT1 to either promote a protective response or induce β-cell death by apoptosis.

      DISCUSSION

      The mechanisms regulating the response of cells to high output (micromolar levels) nitric oxide have yet to be fully identified. It has been reported that cytokines stimulate nitric oxide-dependent cell death by necrosis or apoptosis and in many cases in the same cell type (
      • Calabrese V.
      • Cornelius C.
      • Rizzarelli E.
      • Owen J.B.
      • Dinkova-Kostova A.T.
      • Butterfield D.A.
      ). In pancreatic β-cells and insulinoma cell lines, cytokines have been shown to induce an early necrotic response that can be prevented by inhibitors of NOS (
      • Steer S.A.
      • Scarim A.L.
      • Chambers K.T.
      • Corbett J.A.
      ,
      • Collier J.J.
      • Fueger P.T.
      • Hohmeier H.E.
      • Newgard C.B.
      ). Others have shown that prolonged incubation with cytokines, such as IL-1, stimulates an apoptotic cascade that is associated with the induction of endoplasmic reticulum stress and can be prevented using inhibitors of NOS (
      • Mandrup-Poulsen T.
      ,
      • Oyadomari S.
      • Takeda K.
      • Takiguchi M.
      • Gotoh T.
      • Matsumoto M.
      • Wada I.
      • Akira S.
      • Araki E.
      • Mori M.
      ,
      • Grunnet L.G.
      • Aikin R.
      • Tonnesen M.F.
      • Paraskevas S.
      • Blaabjerg L.
      • Størling J.
      • Rosenberg L.
      • Billestrup N.
      • Maysinger D.
      • Mandrup-Poulsen T.
      ,
      • Thomas H.E.
      • McKenzie M.D.
      • Angstetra E.
      • Campbell P.D.
      • Kay T.W.
      ,
      • Gurzov E.N.
      • Ortis F.
      • Cunha D.A.
      • Gosset G.
      • Li M.
      • Cardozo A.K.
      • Eizirik D.L.
      ,
      • Oyadomari S.
      • Araki E.
      • Mori M.
      ). Prolonged exposures of islets for 7–9 days with cytokines have been reported to stimulate nitric oxide-independent apoptosis (
      • Liu D.
      • Pavlovic D.
      • Chen M.C.
      • Flodström M.
      • Sandler S.
      • Eizirik D.L.
      ). Complicating these divergent responses are reports that β-cells have the capacity to restore metabolic function and repair damaged DNA if the source of NO is removed and the cells are allowed a reasonable amount of time to recover (8 h for maximal recovery/repair) (
      • Corbett J.A.
      • McDaniel M.L.
      ,
      • Hughes K.J.
      • Meares G.P.
      • Chambers K.T.
      • Corbett J.A.
      ).
      The findings presented in this study and in two recent reports (
      • Hughes K.J.
      • Chambers K.T.
      • Meares G.P.
      • Corbett J.A.
      ,
      • Hughes K.J.
      • Meares G.P.
      • Chambers K.T.
      • Corbett J.A.
      ) provide evidence in support of a molecular mechanism that may explain these divergent cellular responses to nitric oxide. The central contributors to these responses appear to be the Forkhead transcription factor FoxO1 and the regulation of FoxO1-dependent transcription by SIRT1. FoxO1 is a transcription factor that is activated in response to various stress stimuli to alert the cell to damage. The cellular response is to initiate repair/protective pathways or, when the damage is too extensive, induce cell death by apoptosis. Growth factors and hormones, such as insulin, stimulate Ser-256 phosphorylation by Akt, inactivating FoxO1 by sequestering this transcription factor in the cytoplasm (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). In response to nitric oxide (either endogenously produced following cytokine treatment or exogenously applied using a donor), FoxO1 is dephosphorylated on Ser-256 and translocates to the nucleus (FIGURE 1, FIGURE 2). Once in the nucleus, deacetylated FoxO1 directs the expression of genes involved in stress resistance and repair (
      • Brunet A.
      • Sweeney L.B.
      • Sturgill J.F.
      • Chua K.F.
      • Greer P.L.
      • Lin Y.
      • Tran H.
      • Ross S.E.
      • Mostoslavsky R.
      • Cohen H.Y.
      • Hu L.S.
      • Cheng H.L.
      • Jedrychowski M.P.
      • Gygi S.P.
      • Sinclair D.A.
      • Alt F.W.
      • Greenberg M.E.
      ). Recently, we have shown that the repair of nitric oxide-damaged DNA in β-cells requires the expression of GADD45α by a mechanism that is at least partially dependent on JNK (
      • Hughes K.J.
      • Meares G.P.
      • Chambers K.T.
      • Corbett J.A.
      ). In this study, we now show that nitric oxide activates GADD45α expression in a FoxO1-dependent manner. We are currently examining the potential role of JNK in the regulation of FoxO1.
      The regulation of FoxO1 transcriptional activity is controlled by the NAD+-dependent deacetylase SIRT1 (
      • Giannakou M.E.
      • Partridge L.
      ,
      • Brunet A.
      • Sweeney L.B.
      • Sturgill J.F.
      • Chua K.F.
      • Greer P.L.
      • Lin Y.
      • Tran H.
      • Ross S.E.
      • Mostoslavsky R.
      • Cohen H.Y.
      • Hu L.S.
      • Cheng H.L.
      • Jedrychowski M.P.
      • Gygi S.P.
      • Sinclair D.A.
      • Alt F.W.
      • Greenberg M.E.
      ,
      • Guarente L.
      ). The role of SIRT1 in the regulation of stress responses has been described previously; however, studies have yet to examine its role in the response of cells to nitric oxide. When active, SIRT1 functions to deacetylate FoxO1 resulting in the expression of protective stress-resistant genes (
      • Brunet A.
      • Sweeney L.B.
      • Sturgill J.F.
      • Chua K.F.
      • Greer P.L.
      • Lin Y.
      • Tran H.
      • Ross S.E.
      • Mostoslavsky R.
      • Cohen H.Y.
      • Hu L.S.
      • Cheng H.L.
      • Jedrychowski M.P.
      • Gygi S.P.
      • Sinclair D.A.
      • Alt F.W.
      • Greenberg M.E.
      ,
      • Guarente L.
      ). Consistent with a role for FoxO1 deacetylation in the repair pathway, inhibitors of SIRT1, which increase FoxO1 acetylation, attenuate the repair of nitric oxide-damaged DNA. Under these conditions, the extent of DNA damage is enhanced above the levels induced by nitric oxide alone. In contrast, the SIRT1 activator resveratrol enhances the rate of DNA repair by 3-fold (Fig. 5). The specificity of resveratrol has been questioned because it has been shown to activate targets in addition to SIRT1 (e.g. AMPK) (
      • Baur J.A.
      ), and the activation of these nonselective targets may contribute to the enhancement in DNA repair (Fig. 5C). Further, we have recently shown that nitric oxide is an activator of AMPK and that AMPK participates in the recovery of islet metabolic function (
      • Meares G.P.
      • Hughes K.J.
      • Jaimes K.F.
      • Salvatori A.S.
      • Rhodes C.J.
      • Corbett J.A.
      ). In an attempt to control for these potential nonselective actions of resveratrol, the effects of SIRT1 inhibition and activation on the repair of DNA damage have been evaluated. In addition, we have examined the effects of siRNA knockdown of SIRT1. Although we have taken both molecular and pharmacological approaches, it is possible that additional SIRT1-dependent or -independent factors, such as AMPK activation, also contribute to this DNA repair process.
      In addition to activating this recovery response, SIRT1 protects β-cells from cytokine-mediated apoptosis. Consistent with previous studies (
      • Steer S.A.
      • Scarim A.L.
      • Chambers K.T.
      • Corbett J.A.
      ,
      • Collier J.J.
      • Fueger P.T.
      • Hohmeier H.E.
      • Newgard C.B.
      ), IL-1 fails to stimulate apoptosis following a 24-h incubation; however, when SIRT1 is inhibited, there is an accumulation of PUMA mRNA and the activation of caspases-3 and -9 by cleavage (Fig. 6). Under these conditions, the inhibition of SIRT1 does not modify IL-1 induced nitric oxide production, indicating that changes in nitric oxide levels are not responsible for the differential gene expression and caspase activation under conditions of SIRT1 inhibition. Rather, we favor changes in the activation state of SIRT1 as the mechanism governing whether FoxO1 activates a protective or apoptotic transcriptional response to nitric oxide. Recently, we have shown the induction of caspase-3 activity, caspase-3 cleavage, and PUMA mRNA accumulation when islets are cultured for 36 h with IL-1 (
      • Hughes K.J.
      • Chambers K.T.
      • Meares G.P.
      • Corbett J.A.
      ). These effects, which are not observed following a 24-h incubation with the cytokine, are prevented by inhibitors of NOS. It is likely that as damage induced by nitric oxide becomes more extensive, SIRT1 is inhibited, resulting in a shift in the FoxO1 transcriptional program from protection to the induction of apoptosis, as evidenced by PUMA expression and caspase-3 and -9 activation. Currently, the mechanisms responsible for inhibition of SIRT1 by nitric oxide are unknown. One potential target is the activation of polyADP-ribose polymerase, which uses NAD+ as a substrate (
      • Szabó C.
      ). Thus, overactivation of polyADP-ribose polymerase resulting from DNA damage could result in the reduction in cellular NAD+ levels (
      • Rosales A.L.
      • Cunningham J.M.
      • Bone A.J.
      • Green I.C.
      • Green M.H.
      ). Reductions in cellular NAD+ levels have been shown to inhibit the deacetylase activity of the NAD+-dependent enzyme, SIRT1 (
      • Pillai J.B.
      • Isbatan A.
      • Imai S.
      • Gupta M.P.
      ,
      • Zhang J.
      ). It is also possible that enhanced production of nitric oxide results in the direct modifications in SIRT1 through S-nitrosation or nitration of tyrosine residues, attenuating SIRT1 activity (
      • Kornberg M.D.
      • Sen N.
      • Hara M.R.
      • Juluri K.R.
      • Nguyen J.V.
      • Snowman A.M.
      • Law L.
      • Hester L.D.
      • Snyder S.H.
      ). We are currently examining these potential mechanisms responsible for the regulation of SIRT1 activity by nitric oxide.
      We are also exploring the mechanisms responsible for the activation of FoxO1 by nitric oxide. The regulation of FoxO1 is likely controlled by Akt. Nitric oxide has been shown to inhibit Akt activity (
      • Størling J.
      • Binzer J.
      • Andersson A.K.
      • Züllig R.A.
      • Tonnesen M.
      • Lehmann R.
      • Spinas G.A.
      • Sandler S.
      • Billestrup N.
      • Mandrup-Poulsen T.
      ,
      • Yasukawa T.
      • Tokunaga E.
      • Ota H.
      • Sugita H.
      • Martyn J.A.
      • Kaneki M.
      ), and we show that nitric oxide attenuates the phosphorylation of FoxO1 on the Akt-mediated site, Ser-256 (Fig. 1). JNK is also a known activator of FoxO-dependent gene expression and JNK participates in the GADD45α-dependent repair of nitric oxide-mediated DNA damage (
      • Hughes K.J.
      • Meares G.P.
      • Chambers K.T.
      • Corbett J.A.
      ,
      • Essers M.A.
      • Weijzen S.
      • de Vries-Smits A.M.
      • Saarloos I.
      • de Ruiter N.D.
      • Bos J.L.
      • Burgering B.M.
      ). Although we have yet to observe a direct effect of JNK on FoxO1 nuclear localization,
      K. J. Hughes and J. A. Corbett, unpublished observation.
      there is potential cross-talk of transcription factors that can be activated by JNK, such as ATF-2 (
      • Hayakawa J.
      • Depatie C.
      • Ohmichi M.
      • Mercola D.
      ), with FoxO1 in the regulation of expression of potential recovery factors such as GADD45α. Additional studies will be required to determine the mechanism of action of JNK in both the induction of a defense/repair response and the induction of apoptosis.
      Overall, this study provides evidence that FoxO1 regulates both DNA repair and the induction of β-cell apoptosis in response to nitric oxide. The proposed mechanism that controls these pathways is outlined in Fig. 7. Central to this regulation of FoxO1 is the activity of the NAD+-dependent deacetylase SIRT1. We hypothesize that when SIRT1 is active, it deacetylates FoxO1, thereby directing a transcriptional program that attenuates apoptosis and stimulates the expression of factors that protect β-cells from nitric oxide. In contrast, when SIRT1 is less active, FoxO1 becomes more acetylated and directs a proapoptotic transcriptional program resulting in apoptosis. These findings provide a working model to explain how it is possible that nitric oxide induces a protective response as well as cell death by either necrosis or apoptosis in the same cell type and in response to the same stimuli.
      Figure thumbnail gr7
      FIGURE 7Proposed mechanism that controls β-cell fate in response to nitric oxide. In response to nitric oxide (supplied exogenously using donor molecules or endogenously following cytokine treatment) there is the loss of Akt-dependent FoxO1 phosphorylation that correlates with FoxO1 nuclear localization. In response to SIRT1 inhibitors, a proapoptotic transcriptional program appears to be activated as indicated by enhanced PUMA mRNA accumulation. If SIRT1 is activated, a FoxO1-dependent DNA repair response is activated as indicated by GADD45 mRNA accumulation.

      Acknowledgments

      We thank Colleen Kelly Bratcher for expert technical assistance and Dr. Michael Moxley for helpful discussion related to these studies.

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