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Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

Lee JE, Oney M, Frizzell K, Phadnis N, Hollien J - G3 (Bethesda) (2015)

Bottom Line: The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh).Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress.Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, Utah 84112.

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Atf4 is necessary and sufficient for up-regulation of glycolytic genes and Ldh. (A−B) We incubated S2 cells with dsRNA targeting either GFP (as a negative control) or Atf4, allowed cells to recover, and incubated with and without either DTT (2 mM, 6 hr, A) or Tm (5 μg/mL, 16 hr, B). We then measured the relative RNA levels for the indicated genes by qPCR. *P < 0.05; **P < 0.005 vs. DTT or Tm-treated control cells (GFP RNAi), Student’s paired t-test. (C) Samples from (A) were analyzed for relative mRNA levels of TCA cycle and respiratory chain complex genes, by qPCR. (D) We stably transfected S2 cells with a plasmid expressing Atf4 under the control of a copper-inducible promoter. We incubated both untransfected and stable cell lines with and without copper (250 μM, 36 hr), then measured mRNA levels by qPCR. *P < 0.05; **P < 0.005 vs. untransfected control cells, student’s paired t-test. For all panels, data are presented as means ± SDs of 3 independent experiments. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin
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fig3: Atf4 is necessary and sufficient for up-regulation of glycolytic genes and Ldh. (A−B) We incubated S2 cells with dsRNA targeting either GFP (as a negative control) or Atf4, allowed cells to recover, and incubated with and without either DTT (2 mM, 6 hr, A) or Tm (5 μg/mL, 16 hr, B). We then measured the relative RNA levels for the indicated genes by qPCR. *P < 0.05; **P < 0.005 vs. DTT or Tm-treated control cells (GFP RNAi), Student’s paired t-test. (C) Samples from (A) were analyzed for relative mRNA levels of TCA cycle and respiratory chain complex genes, by qPCR. (D) We stably transfected S2 cells with a plasmid expressing Atf4 under the control of a copper-inducible promoter. We incubated both untransfected and stable cell lines with and without copper (250 μM, 36 hr), then measured mRNA levels by qPCR. *P < 0.05; **P < 0.005 vs. untransfected control cells, student’s paired t-test. For all panels, data are presented as means ± SDs of 3 independent experiments. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin

Mentions: We transfected S2 cells with 2 μg of plasmid using Cellfectin II (Invitrogen). For polyclonal stable cell lines (as in Figure 3D and Figure 5A), we cotransfected our expression plasmids (1.8 μg) with a hygromycin or puromycin resistance plasmid (0.2 μg) and selected for resistant cells. For Atf4 overexpression studies (Figure 3D), we induced expression with CuSO4 (250 μM, 36 hr) before collecting RNA samples. For promoter constructs, we treated control cells transfected with pMT-GFP with CuSO4 (250 μM, 16 hr) before inducing ER stress with DTT as described above. Finally, for transiently transfected cells (Figure 5B), we allowed cells to recover overnight before inducing ER stress.


Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

Lee JE, Oney M, Frizzell K, Phadnis N, Hollien J - G3 (Bethesda) (2015)

Atf4 is necessary and sufficient for up-regulation of glycolytic genes and Ldh. (A−B) We incubated S2 cells with dsRNA targeting either GFP (as a negative control) or Atf4, allowed cells to recover, and incubated with and without either DTT (2 mM, 6 hr, A) or Tm (5 μg/mL, 16 hr, B). We then measured the relative RNA levels for the indicated genes by qPCR. *P < 0.05; **P < 0.005 vs. DTT or Tm-treated control cells (GFP RNAi), Student’s paired t-test. (C) Samples from (A) were analyzed for relative mRNA levels of TCA cycle and respiratory chain complex genes, by qPCR. (D) We stably transfected S2 cells with a plasmid expressing Atf4 under the control of a copper-inducible promoter. We incubated both untransfected and stable cell lines with and without copper (250 μM, 36 hr), then measured mRNA levels by qPCR. *P < 0.05; **P < 0.005 vs. untransfected control cells, student’s paired t-test. For all panels, data are presented as means ± SDs of 3 independent experiments. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390581&req=5

fig3: Atf4 is necessary and sufficient for up-regulation of glycolytic genes and Ldh. (A−B) We incubated S2 cells with dsRNA targeting either GFP (as a negative control) or Atf4, allowed cells to recover, and incubated with and without either DTT (2 mM, 6 hr, A) or Tm (5 μg/mL, 16 hr, B). We then measured the relative RNA levels for the indicated genes by qPCR. *P < 0.05; **P < 0.005 vs. DTT or Tm-treated control cells (GFP RNAi), Student’s paired t-test. (C) Samples from (A) were analyzed for relative mRNA levels of TCA cycle and respiratory chain complex genes, by qPCR. (D) We stably transfected S2 cells with a plasmid expressing Atf4 under the control of a copper-inducible promoter. We incubated both untransfected and stable cell lines with and without copper (250 μM, 36 hr), then measured mRNA levels by qPCR. *P < 0.05; **P < 0.005 vs. untransfected control cells, student’s paired t-test. For all panels, data are presented as means ± SDs of 3 independent experiments. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin
Mentions: We transfected S2 cells with 2 μg of plasmid using Cellfectin II (Invitrogen). For polyclonal stable cell lines (as in Figure 3D and Figure 5A), we cotransfected our expression plasmids (1.8 μg) with a hygromycin or puromycin resistance plasmid (0.2 μg) and selected for resistant cells. For Atf4 overexpression studies (Figure 3D), we induced expression with CuSO4 (250 μM, 36 hr) before collecting RNA samples. For promoter constructs, we treated control cells transfected with pMT-GFP with CuSO4 (250 μM, 16 hr) before inducing ER stress with DTT as described above. Finally, for transiently transfected cells (Figure 5B), we allowed cells to recover overnight before inducing ER stress.

Bottom Line: The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh).Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress.Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, Utah 84112.

Show MeSH
Related in: MedlinePlus