Autophagy: resetting glutamine-dependent metabolism and oxygen consumption

Abstract

Autophagy is a catabolic process that functions in recycling and degrading cellular proteins, and is also induced as an adaptive response to the increased metabolic demand upon nutrient starvation. However, the prosurvival role of autophagy in response to metabolic stress due to deprivation of glutamine, the most abundant nutrient for mammalian cells, is not well understood. Here, we demonstrated that when extracellular glutamine was withdrawn, autophagy provided cells with sub-mM concentrations of glutamine, which played a critical role in fostering cell metabolism. Moreover, we uncovered a previously unknown connection between metabolic responses to ATG5 deficiency and glutamine deprivation, and revealed that WT and atg5 (-/-) MEFs utilized both common and distinct metabolic pathways over time during glutamine deprivation. Although the early response of WT MEFs to glutamine deficiency was similar in many respects to the baseline metabolism of atg5 (-/-) MEFs, there was a concomitant decrease in the levels of essential amino acids and branched chain amino acid catabolites in WT MEFs after 6 h of glutamine withdrawal that distinguished them from the atg5 (-/-) MEFs. Metabolomic profiling, oxygen consumption and pathway focused quantitative RT-PCR analyses revealed that autophagy and glutamine utilization were reciprocally regulated to couple metabolic and transcriptional reprogramming. These findings provide key insights into the critical prosurvival role of autophagy in maintaining mitochondrial oxidative phosphorylation and cell growth during metabolic stress caused by glutamine deprivation.

Figure 1. Metabolomics profiling of atg5^−/− MEFs in full medium is similar to that of WT MEFs after 6 h of glutamine deprivation. (A) Double dendrogram cluster analyses defining the effect of glutamine deprivation on metabolic intermediates by comparing biomolecule levels in sample sets shown in Figure S1. (B) Compendium of global metabolic profiling. Red ↑, increasing level; Green ↓, decreasing level; -, no difference. (C, upper panel) Hierarchical cluster analysis examining selected biomolecules selected from Figure S1. (Lower panel) Heatmap profile of putrescine, glutamine, reduced and oxidized glutathione, phosphoethanolamine, PRPP, spermine and spermidine in WT and atg5^−/− MEFs after glutamine withdrawal (6 and 24 h). (B and C) Signal fold change was normalized to 0 h WT (0 h) as 1. All data points were log₂ transformed and used to calculate the Self-Organizing Map. Transformed fold changes are shown in green (decreasing) and red (increasing).

Figure 2. Autophagy deficiency leads to higher intake of EAA and reduced EAA levels under glutamine depletion. WT and atg5^−/− MEFs were grown in serum-supplemented DMEM without glutamine for 0, 6 and 24 h in five replicates. Relative quantification of (A) histidine, (B) isoleucine, (C) leucine, (D) lysine, (E) methionine, (F) phenylalanine, (G) threonine, (H) tryptophan and (I) valine was performed, and the relative levels were determined by setting the levels in non-treated WT MEFs as 1. *p < 0.05; **p < 0.01; ***p < 0.001; Gln, glutamine; n = 15.

Figure 3. Glutamine withdrawal and autophagy deficiency increase BCAA catabolism. Cell preparation, relative quantification and data analyses were performed as described in Figure 2. Profiles of leucine metabolites: (A) 4-methyl-2-oxopentanoate and (B) isovalerylcarnitine; isoleucine metabolites: (C) 3-methyl-2-oxovalerate; valine metabolites: (D) isobutyrylcarnitine; (E) 3-methyl-2-oxobutyrate and (F) 2-methylbutyroylcarnitine. *p < 0.05; **p < 0.01; ***p < 0.001; Gln, glutamine; n = 15.

Figure 4. Glutamine depletion affects TCA cycle intermediates. Cell preparation, relative quantification and data analyses were performed as described in Figure 2. Profiles of (A) glutamine, (B) glutamate, (C) fumarate, (D) malate, (E) citrate, (F) pyruvate and (G) NAD are shown. *p < 0.05; **p < 0.01; ***p < 0.001; Gln, glutamine; n = 15.

Figure 5. Glutamine depletion inhibits cell proliferation and decreases intracellular ATP levels. (A) ATP levels were reduced by glutamine depletion. WT and atg5^−/− MEFs were seeded in complete medium to reach 50% confluence on the day of treatment, and then grown in serum-supplemented DMEM (without glutamine) in the presence or absence of glutamine (4 mM), 2-DG (10 mM) or methylpyruvate (10 mM) for the indicated time periods prior to harvesting. ATP assays were performed as described in Materials and Methods. Cell proliferation was inhibited by glutamine depletion and 2-DG treatment. WT and atg5^−/− MEFs were seeded in complete medium to reach 10% confluence on the day of treatment, and then grown in serum-supplemented DMEM in the presence and absence of glutamine (4 mM) or in the full medium with 2-DG (10 mM) for indicated time periods. Relative cell proliferation (determined by the cell proliferation assay described in methods) for each treatment was calculated by comparing to that at 0 h, set as 1. Results from three independent experiments are shown as mean ± SD. The Student’s t-test was performed to determine the difference between (a) control (24/48 h) and -Gln (24/48 h); (b) control (24/48 h) and 2-DG (24/48 h). (B) Both DM-2-KG and NAC rescued cell proliferation. WT and atg5^−/− MEFs were seeded in complete medium to reach 10% confluence on the day of treatment, and then grown in glutamine-depleted DMEM with DM-2-KG (7 mM), NAC (2 mM), or both. Cell proliferation was assessed by cell proliferation assay. Significance was calculated using the Student’s t-test between: WT MEFs in Gln(-) (24/48/72 h) and WT MEFs in DM-2-KG/NAC/combination of DM-2-KG and NAC (24/48/72 h); atg5^−/− MEFs in Gln(-) (24/48/72 h) and atg5^−/− MEFs in DM-2-KG/NAC/combination of DM-2-KG and NAC (24/48/72 h); WT MEFs in NAC (24 h) and atg5^−/− MEFs in NAC (24 h). (C) Supplementing with NAC rescues the proliferation of glutamine-deprived cells. Real-time cell proliferation was monitored using an RTCA DP Analyzer. The m5-7 cells were maintained in medium without or with Dox to turn off Atg5 expression (right panel). Glutamine was withdrawn in the presence and absence of NAC supplements and cell growth determined and recorded every 30 min. (D) Minimal glutamine concentration required to support the proliferation of WT and atg5^−/− MEFs. Cell proliferation was determined as described in Material and Methods. Briefly, 5000 cells were seeded into 96-well plate over night and incubated overnight prior to switching to the medium with indicated glutamine concentration (0, 0.5 and 4 mM), while 4 mM representing the complete medium. Cell viability was determined at 24, 48 and 72 h afterwards and the % relative cell proliferation was calculated as that of 72 h as 100%. Gln(-), grown in DMEM without glutamine; Gln(+), grown in DMEM with glutamine; DM-2-KG, dimethyl-2-ketoglutarate; 2-DG, 2-deoxyglucose; NAC, N-acetyl-cysteine; Dox, doxycycline. (A and B) Results are shown as mean ± SD from three independent experiments. *p < 0.05.

Figure 6. Comparable effect of autophagy deficiency and glutamine restriction on mitochondrial function. (A) Real-time oxygen consumption rate in WT and atg5^−/− MEFs maintained at different glutamine concentration (0.5 and 4 mM) was determined in the presence or absence of NAC (10 mM) using Seahorse Extracellular Flux (XF-24) analyzer. Oligomycin (5 μg/ml), FCCP (1 μM) and rotenone (1 μM) were added sequentially to determine mitochondrial function. The tracing exhibited the distinct basal OCR between WT MEFs and autophagy-compromised MEFs maintained at different glutamine concentrations (upper panel) and differential response to NAC addition (lower panel) after normalizing with cell numbers. (B) NAC stimulates OCR in atg5^−/− MEFs maintained at 0.5 mM glutamine. The bar graph represents mean ± SD from three independent experiments. A two-tailed Student’s t-test was used to calculate statistical significance. *p < 0.01

Figure 7. Glutamine and autophagy reprogram transcript levels. (A) Glutamine reduction regulates selected gene expression. WT and atg5^−/− MEFs were seeded in complete medium to reach 50% confluence on the day of treatment, and then grown in serum-supplemented DMEM with or without glutamine for the indicated time periods prior to RNA isolation. RNA isolation and pathway focused quantitative RT-PCR analyses were performed as described in Materials and Methods. The relative fold change was calculated as each transcript levels in WT MEFs (0 h), after normalizing to that of Gapdh, were set as 1. Results are shown as mean ± SD from three independent experiments. *p < 0.05. Aco1, aconitase 1; Cs, citrate synthase; Flnb, fumarate hydratase 1; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Gpt2, glutamic pyruvate transaminase; Idh1/2, isocitrate dehydrogenase 1/2; Mdh1, malate dehydrogenase 1; Me1/2, malic enzyme 1/2; Myc, myelocytomatosis oncogene; Ogdh, oxoglutarate dehydrogenase; Sdha, Sdhb, succinate dehydrogenase complex; Slc7a5, cationic amino acid transporter; Slc1a5, neutral amino acid transporter; Slc3a2, activators of dibasic and neutral amino acid transport; Slc7a5, cationic amino acid transporter; Suclg1, Suclg2, Sucla2, succinate-CoA ligase. (B) Our proposed model illustrating the crosstalk between autophagy, glutamine utilization and the TCA cycle. mRNAs encoding enzymes in the TCA cycle and glutamine metabolism, studied by quantitative RT-PCR analyses, are boxed. (C) Glutamine restriction induces Cdkn1a (p21) and Bbc3 (Puma) expression in Atg5^−/−, but not WT MEFs. Pairs of mRNAs from (A) were used to determine the transcript level of pro-cell cycle arrest gene (Cdkn1a) and pro-apoptotic regulators (Pmaip1 and Bbc3).

Figure 8. Glutamine depletion fails to induce autophagy. (A) Starvation, but not glutamine depletion, induces autophagy. WT and atg5^−/− MEFs were seeded in complete medium to reach 50% confluence on the day of experiment and then grown in EBSS, serum-supplemented DMEM without glutamine, or treated with 2-DG (10 mM), a glycolysis inhibitor, for 0, 6 and 24 h prior to harvesting. Equal amounts of whole cell lysates were analyzed by western blot using anti-MAP1LC3 (LC3), anti-SQSTM1 (p62) and anti-ACTB (actin) antibodies. (B) Glutamine depletion fails to reduce GFP-LC3 fluorescence intensity. (Upper panel) MEF/GFP-LC3 cells were treated with EBSS for 3 and 6 h and then analyzed by FACS. The relative levels of GFP-LC3 fluorescence intensity vs. cell counts are shown in a histogram from a representative experiment (left). MEF/GFP-LC3 cells were cultured in EBSS medium for 3 or 6 h, with or without a 1 h pretreatment of Bafilomycin A₁ (Baf A1; 100 nM). For the treated samples, Baf A₁ was added to the EBSS media for the additional 6 h incubation. The relative levels of GFP-LC3 intensity from at least three independent experiments are calculated as mean ± SD and shown as a percentage of fluorescence intensity, where control level was designated as 100% (right). (Lower panel) Cells were subjected to glutamine withdrawal and analyzed, without Baf A₁-treatment, as shown in (upper panel). (C) Rapamycin induces autophagy in the presence and absence of glutamine. WT and atg5^−/− MEFs were seeded in complete medium to reach 50% confluence on the day of experiment and then grown in serum-supplemented DMEM with or without glutamine and analyzed as described in (A). (D) Glutamine depletion induces PRKAA2 (AMPK) and MTOR activation (left panel) and suppresses Atg5 mRNA level (right panel). The phosphorylation status of PRKAA2, MTOR and its downstream RPS6KB2 (p70S6K) and EIF4EBP1 was determined to indicate the activation status of the PRKAA2 and MTOR pathways after glutamine depletion. The expression level of the Atg5 transcript was measured by quantitative RT-PCR analysis. Cells were treated and analyzed as described in (A) with the indicated antibodies and quantitative RT-PCR was performed as described in Figure 7. Gln, glutamine; 2-DG, 2-deoxyglucose; Rapa, rapamycin.