Fig 1: GDNF/RET signaling pathway promotes glucose absorption and subsequently activates SREBP-1 through HBP. U251 and U87 glioma cells cultured in DMEM complete medium with (A) different dose of GDNF for 48 h or with (B) 50 ng/ml GDNF at indicated times and (C) in the presence or absence of 20 μM RPI-1 (GDNF/RET inhibitor). Glucose uptake ability of glioma cells evaluated by fluorescent glucose 2-NBDG. (D and E) Western blot analysis of total cell lysates (RET/p-RET, ERK/p-RET) or nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM glucose-free medium, treated with 50 ng/ml GDNF, 10 mM glucose or in combination with 20 μM RPI-1 for 48 h. (F) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) from U251 glioma cells cultured in DMEM glucose-free medium and treated with 50 ng/ml GDNF or 25 mM glucose or in combination with 20 μM RPI-1 for 48 h. Quantitative evaluation of SREBP-1 nuclear intensity using ImageJ (n=20). Images were captured at x200 magnification. (G) RT-qPCR analysis of mRNA levels in U251 and U87 glioma cells cultured in DMEM glucose-free medium and treated with 50 ng/ml GDNF, 10 mM glucose, or 20 μM RPI-1 for 24 h. (H and I) Western blot analysis of nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM glucose-free medium with or without 10 mM glucose, 10 mM lactate, 10 mM pyruvate, or 20 mM HBP or in combination with 20 μM azaserine (HBP inhibitor) for 48 h. (J) Western blot analysis of total cell lysates (RET/p-RET) or nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM glucose-free medium, treated with or without GDNF or in combination with 20 μM RPI-1 or 20 mM HBP for 48 h. (K) RT-qPCR analysis of mRNA levels in U251 and U87 glioma cells cultured in DMEM complete medium and treated with 50 ng/ml GDNF and in the presence or absence of 20 μM azaserine for 24 h. (L) U251 and U87 glioma cells cultured in DMEM complete medium treated with 50 ng/ml GDNF and in combination with 20 μM azaserine or 20 mM HBP for 48 h. Relative viability of glioma cells detected by MTT assay. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS: not significant). GDNF, glial-derived neurotrophic factor; RET, rearranged during transfection; SREBP-1, sterol regulatory element-binding protein-1; HBP, N-acetylglucosamine; p-, phosphorylated; RT-qPCR, reverse transcription-quantitative PCR.
Fig 2: GDNF/RET signaling is upregulated in glioma and promote lipid metabolism. mRNA expression of GDNF normal brain tissues and glioma (A) RT-qPCR and (C) TCGA data analysis. (B) Association between GDNF expression and degree of malignancy (RT-qPCR). (D) Survival curves for patients with low and high GDNF mRNA expression levels in glioma (TCGA database). Western blot analysis of total cell lysates (FASN, SCD1, ACC) or nuclear extracts (nSREBP-1) from U251 and U87 glioma cells cultured in DMEM complete medium with (E) different dose of GDNF for 48 h or (F) with 50 ng/ml GDNF at indicated times. (G) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) from U251 glioma cells were cultured in DMEM complete medium and treated with 50 ng/ml GDNF for 48 h. Quantitative evaluation of SREBP-1 nuclear intensity with ImageJ (n=20). Images were captured at x200 magnification. (H) RT-qPCR analysis of mRNA levels in U251 and U87 glioma cells cultured in DMEM complete medium with or without GDNF (50 ng/ml) for 24 h. (I) SREBP-1 mRNA expression in normal brain tissues and glioma (TCGA data analysis). (J) Survival curves for patients with low and high SREBP-1 mRNA expression levels in glioma (TCGA database). (K) Western blot analysis of p-RET, p-ERK and nSREBP-1 levels in U251 and U87 glioma cells that were cultured in DMEM complete medium with or without 50 ng/ml GDNF in the presence or absence of 20 μM RPI-1 (GDNF/RET inhibitor) for 48 h. (L) U251 and U87 glioma cells cultured in DMEM complete medium with different dose of GDNF for 48 h or with 50 ng/ml GDNF at indicated times. Relative viability of glioma cells detected by MTT assay. (M) Relative viability of glioma cell cultured in DMEM complete medium with 50 ng/ml GDNF in the presence or absence of 20 μM RPI-1 for 48 h. Significance was determined by unpaired Student's t test (*P<0.01, ***P<0.001, ****P<0.0001). GDNF, glial-derived neurotrophic factor; RET, rearranged during transfection; RT-qPCR, reverse transcription-quantitative PCR; TCGA, The Cancer Genome Atlas; FASN, fatty acid synthase; SCD1, stearoylCoA desaturase-1; ACC, acetyl CoA carboxylase; SREBP-1, sterol regulatory element-binding protein-1; p-, phosphorylated.
Fig 3: GDNF/RET Signaling promotes glucose absorption by upregulating HIF-1. (A) Western blot analysis of p-RET, HIF-1 levels in U251 and U87 glioma cells that were cultured in DMEM complete medium with or without 50 ng/ml GDNF in the presence or absence of 20 μM RPI-1 (GDNF/RET inhibitor) for 48 h. U251 and U87 glioma cells were transfected with siHIF-1 for 48 h and cultured in DMEM complete medium with or without 50 ng/ml GDNF; (B) Glucose uptake ability of glioma cells was evaluated by fluorescent glucose 2-NBDG; (C) Protein expression determined by western blotting; (D) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) and quantitative evaluation of SREBP-1 nuclear intensity with ImageJ (n=20). Images were captured at x200 magnification. (E) Reverse transcription-quantitative PCR analysis of mRNA levels in U87 and U251 glioma cells following knock down of HIF-1 and cultured in DMEM complete medium with or without 50 ng/ml GDNF for 24 h. U251 and U87 glioma cells transfected with siHIF for 48 h and cultured in DMEM complete medium with 50 ng/ml GDNF or 20 mM HBP. (F) Protein expression was determined by western blotting. (G) Relative viability of glioma cells detected by MTT assay. Significance was determined by unpaired Student's t test (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS: not significant). GDNF, glial-derived neurotrophic factor; RET, rearranged during transfection; HIF-1, hypoxia-inducible factor 1; p-, phosphorylated; RET, rearranged during transfection; HBP, N-acetylglucosamine.
Fig 4: HBP regulates SREBP-1 activity by promoting SCAP N-glycosylation. (A) Western blot analysis of SCAP and nuclear SREBP-1 levels in U251 and U87 glioma cells after SCAP gene silenced by siRNA, cultured in DMEM complete medium treated with 50 ng/ml GDNF for 48 h. (B) NetNGlyc server showed that SCAP presents both N- and O-glycosylation. (C) Western blot analysis of nuclear extracts from U251 and U87 glioma cells cultured in DMEM glucose-free medium, treated with or without 50 ng/ml GDNF or in combination with tunicamycin (N-glycosylation inhibitor) (2 μg/ml) or 20 μM OSMI-1 (O-glycosylation inhibitor) for 48 h. (D) Immunofluorescence staining of SREBP-1 (red) and DAPI (blue) from U251 glioma cells cultured in DMEM complete medium, treated with 50 ng/ml GDNF or in combination with 2 μg/ml tunicamycin or 20 μM OSMI-1 for 48 h. Quantitative evaluation of SREBP-1 nuclear intensity using ImageJ (n=20). Images were captured at x200 magnification. (E) Western blot analysis of total cell lysates (SCAP N-glycosylation levels) or nuclear extracts (nSREBP-1) from U251 glioma cells cultured in DMEM glucose-free medium, treated with 50 ng/ml GDNF or/and 10 mM glucose for 48 h. Western blot analysis of total cell lysates (SCAP N-glycosylation levels) or nuclear extracts (nSREBP-1) from U251 glioma cells cultured in DMEM complete medium, treated with 50 ng/ml GDNF or in combination with (F) 20 mM RPI-1 (GDNF/RET inhibitor), (G) 20 μM azaserine (HBP inhibitor) and/or (H) 2 μg/ml tunicamycin (N-glycosylation inhibitor) for 48 h. Significance was determined by unpaired Student's t test (****P<0.0001; NS: not significant). HBP, N-acetylglucosamine; SREBP-1, sterol regulatory element-binding protein-1; SCAP, SREBP cleavage-activating protein; GDNF, glial-derived neurotrophic factor.
Fig 5: Quantification of Ret activation enhancement effect on inhibitory postsynaptic currents using XIB4035. (A) Inter-event interval (K-S sIPSCs p < 0.01 D = 0.068, mIPSCs p < 0.01 D = 0.088), (B) amplitude (K-S sIPSCs p < 0.01 D = 0.144, mIPSCs p < 0.01 D = 0.166) and (C) rise-time (K-S sIPSCs p < 0.01 D = 0.032; mIPSCs p < 0.01 D = 0.034) cumulative distribution plots of spontaneous (left) and miniature IPSCs (right) from control and GDNF-incubated slices (n = 511 events per cell). The line markers in the scatter plots depict the median of averages per cell. Mann–Whitney test for the averages–not significant, Kolmogorov–Smirnov test for distribution comparisons. p < 0.01.
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