A novel autoregulatory loop between the Gcn2-Atf4 pathway and L-Proline metabolism controls stem cell identity

D’Aniello, Cristina; Fico, Annalisa; Casalino, Laura; Guardiola, Ombretta; Napoli, Gabriele Di; Cermola, Federica; Cesare, Dario De; Tatè, Rosarita; Cobellis, Gilda; Patriarca, Eduardo J.; Minchiotti, Gabriella

doi:10.1038/cdd.2015.24

Cited by 54 publications

(92 citation statements)

References 48 publications

(72 reference statements)

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“…We thus performed a nuclear magnetic resonance (NMR)-based global metabolomic profiling of ESCs ± l -Pro and found strong differences in their metabolite content (Figure 3G and Table S4). In particular, PiCs showed increased levels of l -Pro and reduced levels of different amino acids ( l -Gln, l -Tyr, l -Gly, l -Ala l -Val, l -Leu, l -Ser) (Table S4), in line with our previous findings that l -Pro supplementation inactivates the amino acid stress response pathway in ESCs (D'Aniello et al., 2015). Remarkably, lactate levels significantly increased (Figures 3G, 3H, and S3F; Table S4) and PiCs were more susceptible to the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) compared with ESCs (Figures 3H, 3I, and S3G), suggesting a metabolic shift to glycolysis.…”

Section: Resultssupporting

confidence: 90%

“…Recent evidence from our laboratory demonstrates that ESCs suffer a highly specific intrinsic shortage of the nonessential amino acid l -Pro, which induces the amino acid stress response pathway (D'Aniello et al., 2015). Exogenously provided l -Pro alleviates this stress condition and converts round-shaped ESCs into flat-shaped pluripotent stem cells.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, it has now become evident that metabolites, including amino acids, act as key regulators of pluripotent stem cell plasticity and behavior. For instance, it has been shown that ESC self-renewal depends on l -threonine (Wang et al., 2009), while ESC identity is regulated by l -proline ( l -Pro) availability (Casalino et al., 2011, Comes et al., 2013, D'Aniello et al., 2015, Washington et al., 2010). Moreover, several metabolites act as epigenetic signals (Blaschke et al., 2013, Comes et al., 2013, Shyh-Chang et al., 2012), thus defining a regulatory network among metabolism, epigenetic modification, and pluripotency, knowledge of which is still limited (Harvey et al., 2016).…”

Section: Introductionmentioning

confidence: 99%

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Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum

et al. 2017

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SummaryMetabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor β signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum

et al. 2017

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“…A second pathway that was labeled equally strongly in both cell lines was proline biosynthesis. This pathway has recently been recognized as a critical pathway for cancer cells and embryonic stem cells (30,31) (Fig. 3F).…”

Section: Resultsmentioning

confidence: 99%

Deletion of Amino Acid Transporter ASCT2 (SLC1A5) Reveals an Essential Role for Transporters SNAT1 (SLC38A1) and SNAT2 (SLC38A2) to Sustain Glutaminolysis in Cancer Cells

Bröer

Rahimi

Bröer

2016

Journal of Biological Chemistry

199

231

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Many cancer cells depend on glutamine as they use the glutaminolysis pathway to generate building blocks and energy for anabolic purposes. As a result, glutamine transporters are essential for cancer growth and are potential targets for cancer chemotherapy with ASCT2 (SLC1A5) being investigated most intensively. Here we show that HeLa epithelial cervical cancer cells and 143B osteosarcoma cells express a set of glutamine transporters including SNAT1 (SLC38A1), SNAT2 (SLC38A2), SNAT4 (SLC38A4), LAT1 (SLC7A5), and ASCT2 (SLC1A5). Net glutamine uptake did not depend on ASCT2 but required expression of SNAT1 and SNAT2. Deletion of ASCT2 did not reduce cell growth but caused an amino acid starvation response and up-regulation of SNAT1 to replace ASCT2 functionally. Silencing of GCN2 in the ASCT2(؊/؊) background reduced cell growth, showing that a combined targeted approach would inhibit growth of glutamine-dependent cancer cells.Differentiated, non-dividing cells generate energy by metabolizing nutrients via the TCA 2 cycle and respiratory chain, resulting in the production of carbon dioxide and water (1). Synthesis of macromolecules is limited to essential turnover of proteins and polynucleotides. Rapidly dividing cells, by contrast, synthesize significant amounts of protein, DNA, RNA, and membrane lipids de novo. Synthesis of these compounds withdraws large amounts of metabolites from metabolic pathways. Cyclic pathways, such as the TCA cycle, are very sensitive to withdrawal of metabolites because depletion of TCA cycle intermediates will render the cycle non-functional and compromise the capacity to generate energy (2). Linear pathways, by contrast, increase the flow-through to adapt to high metabolic demand. Converting the TCA cycle into a linear pathway is of considerable advantage to rapidly dividing cells, such as cancer cells, activated lymphocytes, and stem cells. This linearized version of the TCA cycle has been termed glutaminolysis, allowing generation of energy and metabolic building blocks (2, 3). Glutamine, the starting substrate of this pathway, is imported by the cell and deaminated to glutamate. Glutamate is converted into 2-oxoglutarate by glutamate dehydrogenase or by a transaminase reaction. After going through several steps of the TCA cycle, malate or oxaloacetate is produced. Oxaloacetate can be converted into aspartate, which is a major precursor for nucleotide biosynthesis. Both oxaloacetate and malate can also be converted into pyruvate, which can be used to generate alanine, lactate, or acetyl-CoA. As a result, many cancer cells depend on glutamine unless they express glutamine synthetase (4). It is worth noting that glutaminolysis through the conversion of 2-oxoglutarate to oxaloacetate produces two NADH, one FADH, and one GTP and therefore provides the cell with significant energy. Activation of the glutaminolysis pathway in cancer cells is accompanied by a reduced entry of pyruvate into the TCA cycle (5).The adaptation of metabolism to cellular growth is associated with transcription fa...

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“…The uptake and metabolism of L-proline induces the differentiation of mouse embryonic stem (mES) cells into a later-stage pluripotent cell (early primitive ectoderm-like (EPL) cells) (Washington et al 2010, Tan et al 2011, and the regulation of intracellular L-proline concentrations has been linked to the maintenance of mES cells (D'Aniello et al 2015). mES and EPL cells differ in their differentiation kinetics, with EPL cells upregulating mesendoderm markers earlier than ES cells (Washington et al 2010).…”

Section: Amino Acid Utilization: Linking Metabolism To Epigeneticsmentioning

confidence: 99%

Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen

Lees¹,

Rathjen²,

Sheedy³

et al. 2015

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Oxygen is a powerful regulator of cell function and embryonic development. It has previously been determined that oxygen regulates human embryonic stem (hES) cell glycolytic and amino acid metabolism, but the effects on mitochondria are as yet unknown. Two hES cell lines (MEL1, MEL2) were analyzed to determine the role of 5% (physiological) and 20% (atmospheric) oxygen in regulating mitochondrial activity. In response to extended physiological oxygen culture, MEL2 hES cells displayed reduced mtDNA content, mitochondrial mass and expression of metabolic genes TFAM, NRF1, PPARa and MT-ND4. Furthermore, MEL2 hES cell glucose consumption, lactate production and amino acid turnover were elevated under physiological oxygen. In stark contrast, MEL1 hES cell amino acid and carbohydrate use and mitochondrial function were relatively unaltered in response to oxygen. Furthermore, differentiation kinetics were delayed in the MEL1 hES cell line following BMP4 treatment. Here we report the first incidence of metabolic dysfunction in a hES cell population, defined as a failure to respond to oxygen concentration through the modulation of metabolism, demonstrating that hES cells can be perturbed during culture despite exhibiting the defining characteristics of pluripotent cells. Collectively, these data reveal a central role for oxygen in the regulation of hES cell metabolism and mitochondrial function, whereby physiological oxygen promotes glucose flux and suppresses mitochondrial biogenesis and gene expression.

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A novel autoregulatory loop between the Gcn2-Atf4 pathway and L-Proline metabolism controls stem cell identity

Cited by 54 publications

References 48 publications

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum

Deletion of Amino Acid Transporter ASCT2 (SLC1A5) Reveals an Essential Role for Transporters SNAT1 (SLC38A1) and SNAT2 (SLC38A2) to Sustain Glutaminolysis in Cancer Cells

Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen

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