Ammonia Derived from Glutaminolysis Is a Diffusible Regulator of Autophagy

Center for Integrative Biology and Biotherapeutics, Pfizer, Pearl River, NY 10965, USA.
Science Signaling (Impact Factor: 6.28). 04/2010; 3(119):ra31. DOI: 10.1126/scisignal.2000911
Source: PubMed


Autophagy is a tightly regulated catabolic process that plays key roles in normal cellular homeostasis and survival during periods of extracellular nutrient limitation and stress. The environmental signals that regulate autophagic activity are only partially understood. Here, we report a direct link between glutamine (Gln) metabolism and autophagic activity in both transformed and nontransformed human cells. Cells cultured for more than 2 days in Gln-containing medium showed increases in autophagy that were not attributable to nutrient depletion or to inhibition of mammalian target of rapamycin. Conditioned medium from these cells contained a volatile factor that triggered autophagy in secondary cell cultures. We identified this factor as ammonia derived from the deamination of Gln by glutaminolysis. Gln-dependent ammonia production supported basal autophagy and protected cells from tumor necrosis factor-alpha (TNF-alpha)-induced cell death. Thus, Gln metabolism not only fuels cell growth but also generates an autocrine- and paracrine-acting regulator of autophagic flux in proliferating cells.

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    • "They then treated cells with 20 mmol/L NH 4 Cl for different time points and observed increased LC3BII within 30 min (Fig. S2a). However, as NH 4 Cl is also an autophagic flux inducer[9], the observed LC3BII increase maybe just a result of NH 4 Cl-induced autophagy. Hence, it is necessary to confirm these results with the well-accepted autophagic flux inhibitors, such as bafilomycin A1 and chloroquine. "

    Full-text · Article · Dec 2015
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    • "H9c2 cells (CRL-1446) cells were purchased from American Type Culture Collection (Manassas, VA). U2OS cells stably expressing GFP-LC3 (U2OS-GFP-LC3 cells) [16] were a generous gift of Dr. Christina Eng (Pfizer Inc, Pearl River). These cells were maintained in high glucose Dulbecco’s Modification of Eagle’s Medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 10% (v/v) heat inactivated fetal bovine serum, 100 units/ml penicillin/streptomycin, and 2 mM L-glutamine (Life Technologies, Carlsbad, CA) at 37°C in a humidified air atmosphere at 5% CO2. "
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    ABSTRACT: Autophagy refers to the catabolic process in eukaryotic cells that delivers cytoplasmic material to lysosomes for degradation. This highly conserved process is involved in the clearance of long-lived proteins and damaged organelles. Consequently, autophagy is important in providing nutrients to maintain cellular function under starvation, maintaining cellular homeostasis, and promoting cell survival under certain conditions. Several pathways, including mTOR, have been shown to regulate autophagy. However, the impact of lysosomal function impairment on the autophagy process has not been fully explored. Basic lipophilic compounds can accumulate in lysosomes via pH partitioning leading to perturbation of lysosomal function. Our hypothesis is that these types of compounds can disturb the autophagy process. Eleven drugs previously shown to accumulate in lysosomes were selected and evaluated for their effects on cytotoxicity and autophagy using ATP depletion and LC3 assessment, respectively. All eleven drugs induced increased staining of endogenous LC3 and exogenous GFP-LC3, even at non toxic dose levels. In addition, an increase in the abundance of SQSTM1/p62 by all tested compounds denotes that the increase in LC3 is due to autophagy perturbation rather than enhancement. Furthermore, the gene expression profile resulting from in vitro treatment with these drugs revealed the suppression of plentiful long-lived proteins, including structural cytoskeletal and associated proteins, and extracellular matrix proteins. This finding indicates a retardation of protein turnover which further supports the notion of autophagy inhibition. Interestingly, upregulation of genes containing antioxidant response elements, e.g. glutathione S transferase and NAD(P)H dehydrogenase quinone 1 was observed, suggesting activation of Nrf2 transcription factor. These gene expression changes could be related to an increase in SQSTM1/p62 resulting from autophagy deficiency. In summary, our data indicate that lysosomal accumulation due to the basic lipophilic nature of xenobiotics could be a general mechanism contributing to the perturbation of the autophagy process.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "NH3 plays an additional role in virulence of H. pylori by enhancing host cell apoptosis through its modulation of endocytic vesicle trafficking [63]. Furthermore, NH3 is increasingly recognized as an important signaling molecule in cellular responses to stress [64], [65]. In the context of inflammation, elevated NH3 levels inhibit neutrophil chemotaxis [66], phagocytosis, and degranulation while also stimulating spontaneous oxidative bursts [67]. "
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    ABSTRACT: The intracellular enzyme urea amidolyase (Dur1,2p) enables C. albicans to utilize urea as a sole nitrogen source. Because deletion of the DUR1,2 gene reduces survival of C. albicans co-cultured with a murine macrophage cell line, we investigated the role of Dur1,2p in pathogenesis using a mouse model of disseminated candidiasis. A dur1,2Δ/dur1,2Δ strain was significantly less virulent than the wild-type strain, showing significantly higher survival rate, better renal function, and decreased and less sustained fungal colonization in kidney and brain. Complementation of the mutant restored virulence. DUR1,2 deletion resulted in a milder host inflammatory reaction. Immunohistochemistry, flow cytometry, and magnetic resonance imaging showed decreased phagocytic infiltration into infected kidneys. Systemic cytokine levels of wild-type mice infected with the dur1,2 mutant showed a more balanced systemic pro-inflammatory cytokine response. Host gene expression and protein analysis in infected kidneys revealed parallel changes in the local immune response. Significant differences were observed in the kidney IL-1 inflammatory pathway, IL-15 signaling, MAP kinase signaling, and the alternative complement pathway. We conclude that Dur1,2p is important for kidney colonization during disseminated candidiasis and contributes to an unbalanced host inflammatory response and subsequent renal failure. Therefore, this Candida-specific enzyme may represent a useful drug target to protect the host from kidney damage associated with disseminated candidiasis.
    Full-text · Article · Oct 2012 · PLoS ONE
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