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).