Fig. 4: Liver-specific GCLC expression sustains lipid synthesis and represses NRF2 activation.

A Schematic of inducible liver-specific Gclc deletion (L-KO). Liver-specific Gclc knockout was induced using the AAV-TBG-Cre. All mice were sacrificed 21 days post AAV-TBG-Cre injection unless otherwise indicated. B (Top) Relative abundance of Gclc mRNA in the liver of WT (n = 4) and L-KO (n = 4) mice, 1–3 weeks post-treatment with AAV-TBG-Cre. Expression levels of mRNA were normalized to the expression of the reference gene Rps9. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. L-KO: 1 week P value = 0.0002, 2 weeks P value < 0.0001, 3 weeks P value < 0.0001). (Bottom) Immunoblot analysis of GCLC protein in the liver of WT and L-KO mice 1–3 weeks post-treatment with AAV-TBG-Cre. C Relative abundance of GSH in liver tissue of WT (n = 4) and L-KO (n = 4) mice in 1–3 weeks following AAV-TBG-Cre treatment. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. L-KO: 1 week P value = 0.0424, 2 weeks P value < 0.0001, 3 weeks P value < 0.0001). D GSEA of oxidative stress-related pathways in the liver of L-KO (n = 4) compared to WT (n = 4) mice following treatment with AAV-TBG-Cre. Enrichment p values were calculated using an adaptive multi-level split Monte Carlo scheme and were corrected for multiple testing using Benjamini and Hochberg false discovery rate. E Representative immunoblot analysis of NRF2 in the liver of WT and L-KO mice following treatment with AAV-TBG-Cre. Data shown are representative of at least three replicates. F Relative mRNA levels of Nqo1 in WT (n = 8 for 1 and 2-week timepoints and n = 4 for 3-week timepoint) and L-KO (n = 6 for 1-week timepoint, n = 7 for 2-week timepoint and n = 4 for 3-week timepoint) mice,1–3 weeks following treatment with AAV-TBG-Cre. Expression levels were normalized to the expression of the reference gene Rps9. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. L-KO: 1 week P value = 0.9998, 2 weeks P value = 0.6378, 3 weeks P value < 0.0001). G GSEA of lipogenic-related pathways in the liver of KO (n = 4) compared to WT (n = 4) mice following treatment with AAV-TBG-Cre. Enrichment p values were calculated using an adaptive multi-level split Monte Carlo scheme and were corrected for multiple testing using Benjamini and Hochberg false discovery rate. H Relative mRNA levels of Scd1 in WT (n = 8 for 1 and 2-week timepoints and n = 4 for 3-week timepoint) and L-KO (n = 6 for 1-week timepoint, n = 7 for 2-week timepoint and n = 4 for 3-week timepoint) mice, 1–3 weeks following treatment with AAV-TBG-Cre. Expression levels were normalized to the expression of the reference gene Rps9. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. L-KO: 1 week P value = 0.0192, 2 weeks P value = 0.0222, 3 weeks P value = 0.0005). I Triglyceride levels in the serum of WT (n = 4) and L-KO (n = 4) mice in 1–3 weeks following treatment with AAV-TBG-Cre. A two-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (WT vs. L-KO: 1 week P value = 0.9995, 2 weeks P value = 0.0038, 3 weeks P value = 0.0069). J Triglyceride levels in the serum of L-KO mice fed normal chow (n = 4) and HFD (n = 7) 3 weeks following treatment with AAV-TBG-Cre. An unpaired two-tailed t-test was used to determine statistical significance (L-KO Normal Chow vs. L-KO High-fat Diet P value = 0.0004). Relative mRNA levels of (K) Nqo1 and (L) Scd1 in Gclc L-KO mice (n = 4) following treatment with GSH-ee (0, 50, 100, 500, and 1000 mg/kg) from days 17–20 post-AAV-TBG-Cre injection. mRNA levels were normalized to the expression of the reference gene Rps9. A one-way ANOVA with subsequent Dunnett’s multiple comparisons test was used to determine statistical significance (K): 0 mg/kg GSH-ee vs. 50 mg/kg GSH-ee P value = 0.9933, 0 mg/kg GSH-ee vs. 100 mg/kg GSH-ee P value = 0.9996, 0 mg/kg GSH-ee vs. 500 mg/kg GSH-ee P value = 0.2648, 0 mg/kg GSH-ee vs. 1000 mg/kg GSH-ee P value = 0.0454. L: 0 mg/kg GSH-ee vs. 50 mg/kg GSH-ee P value = 0.9816, 0 mg/kg GSH-ee vs. 100 mg/kg GSH-ee P value = 0.9664, 0 mg/kg GSH-ee vs. 500 mg/kg GSH-ee P value > 0.999, 0 mg/kg GSH-ee vs. 1000 mg/kg GSH-ee P value = 0.4731. M Triglyceride levels in the serum of Gclc L-KO mice (n = 4) following treatment with GSH-ee (0, 50, 100, 500, and 1000 mg/kg) from days 17–20 post-AAV-TBG-Cre injection. A one-way ANOVA with subsequent Dunnett’s multiple comparisons test was used to determine statistical significance (0 mg/kg GSH-ee vs. 50 mg/kg GSH-ee P value = 0.9663, 0 mg/kg GSH-ee vs. 100 mg/kg GSH-ee P value = 0.9879, 0 mg/kg GSH-ee vs. 500 mg/kg GSH-ee P value = 0.7552, 0 mg/kg GSH-ee vs. 1000 mg/kg GSH-ee P value = 0.9991). Indicated n values represent biologically independent samples from mice. Data are shown as mean ± SEM. ns = not significant, *P value < 0.05, **P value < 0.01, ***P value < 0.001, ****P value < 0.0001. (A) created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.