Title: FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells
Abstract: Article6 September 2013free access Source Data FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells Hyeonju Yeo Hyeonju Yeo Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Costas A Lyssiotis Costas A Lyssiotis Department of Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Yuqing Zhang Yuqing Zhang Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Haoqiang Ying Haoqiang Ying Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author John M Asara John M Asara Division of Medicine, Department of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA Search for more papers by this author Lewis C Cantley Lewis C Cantley Department of Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Ji-Hye Paik Corresponding Author Ji-Hye Paik Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Hyeonju Yeo Hyeonju Yeo Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Costas A Lyssiotis Costas A Lyssiotis Department of Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Yuqing Zhang Yuqing Zhang Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Haoqiang Ying Haoqiang Ying Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author John M Asara John M Asara Division of Medicine, Department of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA Search for more papers by this author Lewis C Cantley Lewis C Cantley Department of Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Ji-Hye Paik Corresponding Author Ji-Hye Paik Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, NY, USA Search for more papers by this author Author Information Hyeonju Yeo1, Costas A Lyssiotis2, Yuqing Zhang1, Haoqiang Ying3, John M Asara4, Lewis C Cantley2 and Ji-Hye Paik 1 1Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, NY, USA 2Department of Medicine, Weill Cornell Medical College, New York City, NY, USA 3Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA 4Division of Medicine, Department of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA *Corresponding author. Department of Pathology and Laboratory medicine, Cornell Weill Medical College, 1300 York Avenue, C-336, New York, NY 10065, USA. Tel.:+1 212 746 6151; Fax:+1 212 746 8302; E-mail: [email protected] The EMBO Journal (2013)32:2589-2602https://doi.org/10.1038/emboj.2013.186 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Forkhead Box O (FoxO) transcription factors act in adult stem cells to preserve their regenerative potential. Previously, we reported that FoxO maintains the long-term proliferative capacity of neural stem/progenitor cells (NPCs), and that this occurs, in part, through the maintenance of redox homeostasis. Herein, we demonstrate that among the FoxO3-regulated genes in NPCs are a host of enzymes in central carbon metabolism that act to combat reactive oxygen species (ROS) by directing the flow of glucose and glutamine carbon into defined metabolic pathways. Characterization of the metabolic circuit observed upon loss of FoxO3 revealed a drop in glutaminolysis and filling of the tricarboxylic acid (TCA) cycle. Additionally, we found that glucose uptake, glucose metabolism and oxidative pentose phosphate pathway activity were similarly repressed in the absence of FoxO3. Finally, we demonstrate that impaired glucose and glutamine metabolism compromises the proliferative potential of NPCs and that this is exacerbated following FoxO3 loss. Collectively, our findings show that a FoxO3-dependent metabolic programme supports redox balance and the neurogenic potential of NPCs. Introduction Stem cells maintain tissue homeostasis by replacing damaged or worn-out cells and the deterioration of stem-cell functions, including self-renewal capacity, is one of the key components of organismal ageing (Janzen et al, 2006; Molofsky et al, 2006; Rossi et al, 2007, 2008). Distinct metabolic programmes in stem cells are necessary to protect genomic stability and to generate precursors for macromolecular synthesis to facilitate continued self-renewal. Reactive oxygen species (ROS) may contribute to the functional decline in stem cells by inflicting chronic damage to cellular macromolecules, including genomic DNA, which accumulates during cellular division, ultimately leading to cytostasis or cytotoxicity (Rossi et al, 2008). In addition, excessive ROS drives stem cells out of quiescence and eventually lead to depletion of stem-cell reserves (Rossi et al, 2008). Animal models of precocious stem-cell depletion or dysfunction consistently emphasize the role of key molecules involved in oxidative defense in maintaining stem-cell reserves: Atm (Ito et al, 2004), Tsc1 (Chen et al, 2008), Prdm16 (Chuikov et al, 2010), and FoxO3 (Miyamoto et al, 2007; Yalcin et al, 2008; Paik et al, 2009; Renault et al, 2009). Furthermore, stem cells have intrinsic antioxidant and stress-resistance systems that maintain low levels of ROS (Ivanova et al, 2002; Ramalho-Santos et al, 2002). As a metabolic by-product, endogeneous ROS is intimately tied to cellular metabolic activity. Mitochondria are the primary source for ROS production through oxidative phosphorylation. While glucose is generally regarded as a major substrate of aerobic oxidation, recent studies indicate that other nutrients, including glutamine (Gln), are metabolized into intermediates of the tricarboxylic acid (TCA) cycle and therefore may drive mitochondrial oxidative phosphorylation and ROS production (DeBerardinis et al, 2007). On the other hand, metabolic programmes also tightly regulate cellular defense against oxidative stress. One such metabolism-dependent antioxidant defense is glutathione (GSH) production. While the availability of amino acids such as Gln, glutamate (Glu), and cysteine regulates the biosynthesis of cellular GSH, intracellular NADP(+)/NADPH level controls the oxidative state of GSH (Beatty and Reed, 1980; Whillier et al, 2011). Under physiological conditions, the oxidative pentose phosphate pathway (PPP) generates reducing potential in the form of NADPH using the glucose metabolite glucose-6-phosphate (G6P). As such, the shunting of glucose carbon into the PPP plays an important role in the maintenance of redox homeostasis (Pandolfi et al, 1995). In fact, metabolic anti-oxidant defense programmes respond to and are activated by cellular ROS levels. For example, a recent study demonstrated that ROS disrupts the active tetrameric state of pyruvate kinase M2 (PKM2), a rate-limiting glycolytic enzyme that catalyses the reaction generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP, through the direct oxidation of Cys358. The inactivation of PKM2 creates a bottleneck at the end of glycolysis thereby redirecting glycolytic metabolites into the PPP and forming a feedback redox balancing mechanism that generates reducing potential in the form of NADPH (Anastasiou et al, 2011). Among the many molecular determinants of ageing and oxidative stress responses, the PI3K-AKT-FoxO signalling pathway plays a central role. To date, studies from experimental model organisms have demonstrated primary roles of FoxO in dietary restriction-induced longevity and suppression of ROS (Kops et al, 2002; Nemoto and Finkel, 2002; Greer et al, 2007). The latter serves to maintain the homeostasis of adult tissue stem cells and partly explains the core mechanism of lifespan extension by activated FoxO (Miyamoto et al, 2007; Tothova et al, 2007). For example, haematopoietic stem cells (HSCs) deficient for multiple FoxO isoforms showed a decrease in the expression of ROS-detoxifying enzymes, such as catalase and MnSOD (Tothova et al, 2007). Furthermore, we demonstrated that loss of FoxO function led to a transient increase in proliferation followed by progressive loss of self-renewal in neural stem/progenitor cells (NPCs), a phenotype tightly associated with excessive ROS (Paik et al, 2009). However, the mechanisms through which FoxO controls metabolic programmes that maintain redox potential, and therefore sustains stem-cell reserves, remain to be determined. To understand FoxO-mediated metabolic regulation of redox homeostasis, we set out to dissect the metabolic alterations induced by the loss of FoxO3, the predominant FoxO isoform expressed in NPCs. By combining global analysis of metabolites with tracing experiments, we identified glycolysis and Gln metabolism as two major metabolic modules affected by FoxO3 deficiency. Impaired utilization of the Gln carbon skeleton contributes to oxidative stress that in turn downregulates PKM2 activity. At the same time, FoxO3 deficiency leads to downregulation of glucose uptake and depression of oxidative PPP activity. Collectively, these metabolic alterations contribute to a more oxidative cellular environment that may lead to the progressive accumulation of oxidative damage. In addition to previously characterized MnSOD or Catalase-dependent protective functions of FoxO, our study demonstrates an unexpected role of FoxO3 in the maintenance of metabolic homeostasis in NPCs that counteracts oxidative stress and preserves their long-term proliferative potential. Results Glutamine metabolism, glucose metabolism, and FoxO3 suppress ROS in NPCs We previously reported that FoxO-null NPCs (derived from FoxO1/3/4 combined KO, hGFAP-Cre+: FoxO1/3/4L/L mice) show an increase in intracellular ROS and a decrease in self-renewal potential relative to wild-type (WT) controls (Paik et al, 2009). In order to gain mechanistic insight, we examined associated changes in cellular functions and pathways. First, genes differentially regulated in FoxO-null NPCs were analysed using gene set enrichment analysis (GSEA). Metabolic gene sets for KEGG pathways (i.e., arginine and proline metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathways) were significantly enriched as functional categories (Supplementary Figure S1A–C). Independent Ingenuity Pathway Analysis identified glutamate and pyruvate metabolism among the most significantly affected pathways in FoxO-null NPCs, adding additional pathways to the list of FoxO-dependent metabolic alterations (Supplementary Figure S1D). On the basis of these results, we pursued the function of FoxO in NPC metabolism and focussed on the role of FoxO3, the most predominantly expressed FoxO isoform in NPCs (Paik et al, 2009). Consistent with previous reports FoxO3 KO NPCs exhibited increased mitochondrial abundance and respiration, presumably leading to the observed accumulation of mitochondrial superoxide (Supplementary Figure S2A–C) (Jensen et al, 2011; Ferber et al, 2012). Notably, the expression of MnSOD did not change and only a few ROS-detoxifying enzymes downregulated in FoxO3 KO NPCs (Supplementary Figure S2D). ROS accumulation is closely associated with increased production by mitochondria as well as with the rate of clearance that are mediated by metabolic and/or transcriptional programmes. As transcriptional MnSOD regulation was not altered despite the elevated mitochondrial superoxide level, we questioned whether metabolic ROS clearance is compromised in FoxO3 KO NPCs. First, we determined the contribution of glucose and Gln metabolism to ROS production at different time points after lowering glucose (25 mM to 1 mM) and/or Gln (2 mM to 0.2 mM). Depletion of Gln profoundly increased ROS production in both WT and FoxO3 KO NPCs. Additionally, both lowering glucose concentration and FoxO3 deficiency elevated ROS levels under all conditions (Figure 1A). These data suggest that Gln and glucose metabolism as well as FoxO3 expression is important for suppression of ROS. Of note, the cells used for in vitro analyses are referred to as NPCs, based on the heterogeneity resulting from 3D culture conditions (Reynolds and Rietze, 2005). Figure 1.Decreased glutaminolysis in FoxO3 KO NPCs. WT and FoxO3 KO NPCs were cultured with high (25 mM) or low (1 mM) glucose and high (2 mM) or low (0.2 mM) Gln containing media for the indicated times. (A) Intracellular ROS level was measured by DCF-DA staining and (J) the NADP/NADPH ratio was measured. Error bars represent ±s.d. values of the mean. 13C-Gln-derived metabolite pools (B) and total metabolite pools (C) in FoxO3 KO NPCs were measured using LC-MS/MS (n=3). Mean±s.d. values are shown. (D) GLS and (E) GLUD activity is assayed in WT and FoxO3 KO NPCs. Mean±s.d. values are shown. Gln-derived Glu tracing into GSH (13C-GSH) and GSSG (13C-GSSG) was analysed by targeted LC-MS/MS (F) as well as the level of GSH and the GSH/GSSG ratio (G) were measured in WT and FoxO3 KO NPCs. Mean±s.e. values are shown. (H) Intracellular ROS level was measured by DCF-DA staining in WT and FoxO3 KO NPCs cultured with Gln-free media and treated with Gln metabolites (2 mM Gln, 4 mM Glu, or 2 mM GSH ethyl ester) for 16 h. Mean±s.e. values are shown. (I) Intracellular ROS level was measured by DCF-DA staining in FoxO3 KO NPCs overexpressing GLS1 and GLUD1 or knocking down for GLS1. The expression of GLS1 and GLUD was confirmed by V5 immunoblotting and RT–qPCR, respectively. Error bars represent ±s.d. values of the mean, and comparison was made with one-way ANOVA. *P<0.05, **P<0.01 Download figure Download PowerPoint Decreased glutaminolysis in FoxO3 KO NPC Gln metabolism can control the redox balance through a number of mechanisms; among the most well characterized are its contribution as Glu to GSH biosynthesis and/or the generation of reducing potential in the form of NADPH from cytosolic isocitrate dehydrogenase 1 (IDH1) or malic enzyme 1 (ME1) activity (Ashcroft and Randle, 1970; MacDonald and Marshall, 2001; Son et al, 2013). Given the increase in oxidative stress observed in FoxO3 KO NPCs, which is exacerbated following Gln withdrawal, we investigated FoxO3-mediated changes in Gln metabolism. To trace Gln metabolism, we grew FoxO3 KO NPCs in growth media containing uniformly 13C-labelled Gln [U-13C5]-Gln and analysed the Gln metabolome by metabolomic profiling after steady-state labelling. Importantly, we used WT and FoxO3 KO NPCs with comparable growth kinetics (4–7 times passaged) and that retained >99% nestin expression to avoid an indirect consequence of compromised proliferation and aberrant differentiation, which can influence anaplerosis of Gln (DeBerardinis et al, 2007). Interestingly, FoxO3 KO NPCs exhibited decreased glutaminolysis as evidenced by decreased abundance of Gln-derived metabolic intermediates (Figure 1B). Consistently, the total metabolite pools for many of the TCA cycle intermediates were universally decreased (Figure 1C). This observation suggests that FoxO3 KO cells utilize less Gln for anaplerotic filling of the TCA cycle. Collectively, our results demonstrate that loss of FoxO3 impairs metabolism of imported Gln in NPCs. Importantly, we did not observe a difference in Gln uptake, expression levels of the Gln transporters ASCT2 and LAT1, or intracellular Gln abundance between WT and FoxO3 KO NPCs (Figure 1B and C; Supplementary Figure S3A). These data suggest that FoxO3 may function downstream of Gln uptake, where FoxO3 loss impairs glutaminolysis. To test this hypothesis, we examined the expression of glutaminase (GLS), which converts Gln into Glu, and glutamate dehydrogenase 1 (GLUD1), which converts Glu into alpha-ketoglutarate (α-KG). The expression levels of Gls1 and Gls2 were not altered, whereas that of Glud1 decreased in FoxO3 KO NPCs compared with WT control (Figure 1D and E; Supplementary Figure S3A). These results suggest that the decreased turnover of Gln to Glu may be due to decreased activity, rather than decreased expression, of GLS. In order to determine whether GLS activity is affected by FoxO deficiency, we examined its enzymatic activity in NPCs. Compared with WT NPCs, ablation of FoxO3 suppressed GLS activity (Figure 1D). Furthermore, GLUD1 activity is also downregulated, though this presumably results from decreased expression (Figures 1E and 5B). Elevated oxidative stress in FoxO3 KO NPCs Gln is a crucial metabolite in the defense against oxidative stress (Mates et al, 2002), as Gln-derived Glu can serve as a precursor for the biosynthesis of GSH (DeBerardinis and Cheng, 2010). Consistent with the decrease in glutaminolysis, we also observed less Gln-derived Glu in GSH in FoxO3 KO NPCs, as evidenced by [U-13C5] Gln tracing (Figure 1F). In steady state, total GSH was reduced whereas GSSG remained similar leading to reduced GSH/GSSG ratios in FoxO3 KO NPCs (Figure 1G). In order to determine whether ROS accumulation is due to decreased GSH level when Gln is depleted, we treated Gln-starved cells with a cell permeable analogue of GSH. This suppressed the Gln-deprivation induced ROS in both WT and FoxO3 KO NPCs, suggesting its major antioxidant role as a downstream metabolite of Gln (Figure 1H). In addition, supplementing the culture with the GSH precursors Gln or Glu suppressed the ROS level in WT NPCs. In FoxO3 KO NPCs, however, the addition of Gln was not as effective as Glu or GSH in suppressing ROS, consistent with suppressed glutaminolysis through decreased GLS activity (Figure 1D and H). In order to determine the importance of glutaminolysis in anti-oxidative metabolism in FoxO3 KO NPCs, we modulated GLS1 and GLUD1 expression. As shown in Figure 1I, further downregulation of GLS1 increased ROS compared with control FoxO3 KO NPCs, while ectopic expression of GLS1 or GLUD1 attenuated ROS accumulation. Collectively, our results suggest that decreased glutaminolysis contributes to exacerbated oxidative stress in FoxO3 KO NPCs, and that this may be through decreased Glu production and GSH biosynthesis. FoxO3 regulates glucose metabolism to maintain NADP/NADPH Next, we examined the ratio of NADP and NADPH, which is the major determinant of reduced GSH level. While Gln deprivation did not have an appreciable effect on the NADP/NADPH ratio, reducing glucose levels significantly increased NADP/NADPH. This effect was more significant in FoxO3 KO than in WT NPCs (Figure 1J), suggesting that FoxO3-dependent glucose, but not Gln metabolism, is critical for maintaining NADP/NAPDH levels. Indeed, one of the central pathways controlling the NADP/NADPH ratio under physiological conditions is the oxidative arm of the PPP. Together, these data suggest that the reduction in the GSH-to-GSSG ratio is due to both a decrease in the Gln-derived GSH and a decrease in glucose-dependent NADPH generation in FoxO3 KO NPCs. The role of FoxO3 in stem-cell glucose metabolism has not yet been defined. In order to determine how the lack of FoxO3 affects glucose utilization in NPCs, we measured key steps of glycolysis. Surprisingly, FoxO3 KO NPCs showed decreased glucose uptake as confirmed by 2-NBDG uptake analysis (Figure 2A). To understand the basis of glucose metabolic alterations induced by FoxO3 inactivation in NPCs, we performed global metabolite profiling of polar metabolites in FoxO3 WT and KO NPCs (Ying et al, 2012; Yuan et al, 2012). We observed a decrease in upstream glycolytic intermediates (i.e., G6P and F6P) and accumulation of downstream metabolites (i.e., BPG and 3PG) with a seven-fold accumulation of PEP, indicating inhibition of the rate-limiting glycolytic step (Figure 2B and C). Figure 2.Decreased glucose metabolism in FoxO3 KO NPCs. (A) Uptake of 2-NBDG for 2 h analysed by flow cytometry in WT and FoxO3 KO NPCs. Values are mean±s.d. (B) Schematic summary of changes observed in glucose metabolism in FoxO3 KO NPCs and (C) and the corresponding data were measured by LC-MS/MS. Error bars represent ±s.d. values of the mean. (D) The indicated protein expression in WT and FoxO3 KO NPCs was analysed by immunoblotting. Ratios of pAKT band intensities from a representative experiment are presented. (E, I) HK activity was assayed in WT and FoxO3 KO NPCs (E) and ca-FoxO3 adenovirus-infected FoxO3 KO NPCs (I). The values are mean±s.d. (F) Mitochondrial translocation of HK2 (green) stained with Mitotracker (red) was determined as percent co-localization (doubly green/red pixels) in WT and FoxO3 KO NPCs. Clotrimazole (CTZ) treatment was used to disrupt HK2 localization from the mitochondria. Error bars represent ±s.d. values of the mean. (G) mRNA expression of Pik3ca and Rictor was measured by RT–qPCR analysis in WT and FoxO3 KO NPCs and GFP or ca-FoxO3 adenovirus-infected FoxO3 KO NPCs. The values are mean±s.e. (H) GFP or ca-FoxO3 adenovirus-infected FoxO3 KO NPCs were treated with 10 μM BYL719 (BYL) and 3 μM BKM120 (BKM) for 3 h or with 100 nM Rapamycin (Rapa) for 24 h, and the expression of the indicated proteins was analysed by immunoblotting. (J, K) The uptake of 2-NBDG and HK activity was measured in either control GFP (Ad-GFP) or NPCs expressing dominant-negative AKT1 (DN-AKT) after adenoviral infection. Values are mean±s.d. of fold change. DN-AKT1 expression was confirmed by immunoblotting of HA epitope. *P<0.05, **P<0.01. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; B(1,3)PG, 1,3-bisphosphoglycerate; B(2,3)PG, 2,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2 [embj2013186-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint Next, we set out to determine the molecular basis of decreased glucose uptake. First, neither the expression of glucose transporters nor the rate-limiting enzyme responsible for phosphorylating and trapping glucose in the cell, hexokinase (HK) 1 or 2, was decreased in FoxO3 KO NPCs (Figure 2D; Supplementary Figure S3A). Rather, FoxO3 KO NPCs showed a decreased glucose metabolism resulting from lower HK activity (Figure 2E). Previous studies have shown that activation of the PI3K-AKT pathway promotes the translocation of HK2 to the outer mitochondrial membrane, thereby increasing its activity (Bustamante and Pedersen, 1977; Gottlob et al, 2001). Given the significant decrease in pAKT in FoxO3 KO NPCs, we examined the mitochondrial localization of HK2 in WT and FoxO3 KO NPCs by co-staining with Mitotracker. FoxO3 KO NPCs showed decreased HK2 translocation to mitochondria compared with WT NPCs consistent with the attenuation of HK2 activity (Figure 2F). To understand the mechanism how the loss of FoxO3 decreases AKT phosphorylation, we tested the role of Pik3ca (p110α) and Rictor, upstream activators of AKT. Expression of both Pik3ca and Rictor was decreased in FoxO3 KO NPCs (Figure 2G). Consistently, the expression of a constitutively active form of FoxO3 (ca-FoxO3) in FoxO3 KO NPCs robustly induced phosphorylation of AKT, which was accompanied by strong upregulation of both Pik3ca and Rictor expressions (Figure 2G and H). In order to determine the contribution of these signal transducers to AKT activation, we treated cells with PI3K inhibitors BYL719 (p110α specific) or BKM120 (pan) and inhibited mTORC2 with rapamycin (Sarbassov et al, 2006; Young et al, 2013). Inhibition of PI3K by BYL719 and BKM120 attenuated the increased pAKT observed in ca-FoxO3 expressing FoxO3 KO cells, whereas rapamycin did not (Figure 2H). Our results suggest that FoxO3 activates PI3K-AKT signalling as a feedback regulatory response that is mediated through PIK3CA. Next, NPCs expressing ca-FoxO3, and therefore activated AKT, showed partially restored HK activity compared with FoxO3 KO NPCs (Figure 2I). In agreement, dominant-negative AKT (DN-AKT) expression caused a decline in glucose uptake in WT NPCs (Figure 2J), and significantly reduced HK activity (Figure 2K). These data suggest that the decreased AKT and HK activity is at least partially responsible for the decreased glucose uptake in FoxO3 KO NPCs. ROS inhibits PKM2 activity in FoxO3 KO NPCs Notably, the level of PEP in FoxO3 KO NPCs was increased seven-fold, suggesting that the rate-limiting enzyme PK, which converts PEP into pyruvate, was inhibited. Indeed, PK activity was reduced to 70% in FoxO3 KO NPCs compared with WT controls, clearly suggesting the inhibition of this rate-limiting step of glycolysis (Figure 3A). Furthermore, expression of ca-FoxO3 partially restored the PK activity in FoxO3 KO NPCs, suggesting that FoxO3-dependent cellular changes are necessary to maintain PK activity in NPCs (Figure 3B). Figure 3.Increased ROS inhibits PK activity in FoxO3 KO NPCs. PK activity was assayed in WT and FoxO3 KO NPCs (A) and FoxO3 KO NPCs expressing ca-FoxO3. (B) Values are shown as mean±s.d. (C) mRNA expression of PKM1 and PKM2 was measured by RT-qPCR analysis, and protein levels of PKM2, total PKM, and PKLR were examined by immunoblotting. (D) PK enzyme activity was determined after treating cells with 250 μM diamide or 1 μM BSO (G) and 0.1 or 1 μM BPTES or 2.5 μM 968 in WT NPCs. Values represent mean±s.d. and statistical significance was determined by ANOVA. (E) PKM2-Flag expressing WT and FoxO3 KD 293T cell lysates were used for immunoprecipitation with Flag antibody. The interaction of endogeneous PKM2 with Flag-tagged exogeneous PKM2 was determined by immunoblotting with the PKM2 antibody. Ratio indicates density of endogeneous PKM2 co-immunoprecipitated over PKM2 in input. Increased ROS (F) and decreased PK activity (G) in WT NPCs treated with GLS inhibitors. *P<0.05, **P<0.01.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3 [embj2013186-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint PK exists as four isozymes (PK-L, PK-R, PK-M1, and PK-M2) encoded by two genes, PKLR and PKM. Among these, PKM2 is predominantly expressed in murine NPCs (Figure 3C). In particular, PKM2 is sensitive to oxidizing agents and prone to oxidative modification preventing the formation of active tetramers (Anastasiou et al, 2011). In agreement, pretreatment with diamide (thiol oxidizing) or glutathione-depleting buthionine sulfoximine (BSO) oxidants suppressed PKM2 activity, confirming that PKM2 is inhibited by an oxidizing environment in NPCs (Figure 3D). Importantly, PKM2 activity was reduced in FoxO3 KO NPCs, while its protein and mRNA expression were maintained (Figure 3A and C). Thus, we examined PKM2 multimer formation in FoxO3 knock-down (KD) 293T cells. Importantly, FoxO3 KO in this system manifests increased ROS accumulation (Supplementary Figure S2B). Our results showed decreased interaction between endogeneous PKM2 and Flag-tagged PKM2 subunits in FoxO3 KD (Figure 3E). These results form the basis for our hypothesis that accumulated ROS inhibits PKM2 multimerization and activity in FoxO3 KO NPCs. Given that our data indicate that deficiency in Gln metabolism leads to increased ROS, we queried the role of glutaminolysis-dependent anti-oxidant capacity in maintaining PK activity in NPCs. In order to examine the contribution of GLS1 activity in sustaining PKM2 activity, we evaluated the effect of 968 and BPTES, potent and selective allosteric GLS1 inhibitors (Robinson et al, 2007; Wang et al, 2010). Inhibition of GLS1 with 2.5 μM 968 as well as 0.1 and 1 μM BPTES increased ROS (Figure 3F). In these cells, PK activity was significantly inhibited, suggesting that glutaminolysis-dependent maintenance of redox potential is necessary to maintain PKM2 activity (Figure 3G). Taken together, these results strongly illustrate that the suppression of glutaminolysis in FoxO3 KO NPCs leads to increased ROS that impairs PKM2 activity. The oxidative arm of the PPP is impaired in FoxO3 KO NPCs Inhibition of PKM2 has been shown to promote the redirection of glucose into the oxidative arm of PPP in a feedback regulatory loop that suppresses ROS by generating reducing power in the form of NADPH (Anastasiou et al, 2011; Gruning et al, 2011). Despite having higher ROS, the abundance of PPP metabolites was surprisingly lower in FoxO3 KO NPCs than in control NPCs. These data suggest that ROS-mediated activation of the PPP is impaired in this context (Figure 4A and B). To measure the activity of the oxidative arm of the PPP, we monitored the production of 14CO2 from [1-14C]-glucose. The carbon at the 1-position of glucose is selectively lost during metabolism in the oxidative arm of the PPP. As previously reported (Anastasiou et al, 2011), diamide treatment increased 14CO2 production (Figure 4C). To determine whether ROS induced by FoxO3 deficiency or Gln depletion enhances glucose flux into the oxidative arm of the PPP, both WT and FoxO3 KO NPCs were grown in Gln-free