Fig. 5: GSH in the liver supports triglyceride levels in an NRF2-dependent manner.

A Schematic of inducible liver-specific Gclc deletion (Gclc L-KO), Nrf2 deletion (Nrf2 L-KO), and Gclc-Nrf2 deletion (L-DKO). Liver-specific knockout of genes was achieved using AAV-TBG-Cre. All mice were sacrificed 21 days post AAV-TBG-Cre treatment unless otherwise indicated. (B-D) Relative expression of (B) Gclc, (C) Nrf2 and (D) Nqo1 mRNA in the liver of WT (n = 4), Gclc L-KO (n = 4), Nrf2 L-KO (n = 4), and L-DKO (n = 6) mice following treatment with AAV-TBG-Cre. Expression levels were normalized to the expression of the reference gene Rps9. A one-way ANOVA with subsequent Dunnett’s multiple comparisons test (used in (B) and (C)) or Šidák’s multiple comparisons test (used in (D)) was used to determine statistical significance ((B): WT vs. Gclc L-KO P value < 0.0001, WT vs. Nrf2 L-KO P value = 0.0004, WT vs. L-DKO P value < 0.0001. C WT vs. Gclc L-KO P value = 0.7972, WT vs. Nrf2 L-KO P value < 0.0001, WT vs. L-DKO P value < 0.0001. D Gclc L-KO vs. L-DKO P value < 0.0001). E Representative immunoblot analysis of NRF2 in the liver of WT, Gclc L-KO, Nrf2 L-KO, and L-DKO mice following treatment with AAV-TBG-Cre. Data shown are representative of at least three replicates. F Relative expression of Scd1 mRNA in the liver of WT (n = 4), Gclc L-KO (n = 4), Nrf2 L-KO (n = 4), and L-DKO (n = 6) mice following treatment with AAV-TBG-Cre. Expression levels were normalized to the expression of the reference gene Rps9. A one-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (Gclc L-KO vs. L-DKO P value = 0.0866). G Serum triglyceride levels for WT (n = 12), Gclc L-KO (n = 14), Nrf2 L-KO (n = 6), and L-DKO (n = 8) mice 3 weeks post-treatment with AAV-TBG-Cre. A one-way ANOVA with subsequent Dunnett’s multiple comparisons test was used to determine statistical significance (Gclc L-KO vs. WT P value < 0.0001, Gclc L KO vs. Nrf2 L-KO P value = 0.0040, Gclc L-KO vs. L-DKO P value = 0.01421). H Relative expression of lipogenic genes mRNA in the liver of WT (n = 4), Gclc L-KO (n = 4), Nrf2 L-KO (n = 4), and L-DKO (n = 4) mice expressed as Log2 fold change. I Relative expression of proteins related to the lipogenic pathway in the liver of WT (n = 4), Gclc L-KO (n = 4), Nrf2 L-KO (n = 4) and L-DKO (n = 4) mice expressed as Log2 fold change. J Relative abundance of serum triglycerides in the liver of WT (n = 4), Gclc L-KO (n = 4), Nrf2 L-KO (n = 4) and L-DKO (n = 4) mice expressed as Log2 fold change. K Relative mRNA expression of Scd1 in the liver of WT mice following a 4-day treatment with either vehicle (n = 4) or CDDO-methyl (n = 8). Expression levels were normalized to the expression of the reference gene Rps9. An unpaired two-tailed t-test was used to determine statistical significance (Vehicle vs. CDDO-methyl P value = 0.0065). L Triglyceride levels in the serum of WT mice without AAV-TBG-Cre injection following a 4-day treatment with either vehicle (n = 4) or CDDO-methyl (n = 8). An unpaired two-tailed t test was used to determine statistical significance (Vehicle vs. CDDO-methyl P value = 0.0171). M Relative expression of Scd1 mRNA in the liver of WT (n = 4) and L-KO (n = 4) mice treated with either vehicle or T0901317 on days 17–20 post AAV-TBG-Cre injection. Expression levels were normalized to the expression of the reference gene Rps9. A one-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (vehicle vs. T0901317: WT P value < 0.0001, L-KO P value = 0.0462; WT/Vehicle vs. L-KO/T0901317 P value = 0.1254). N Triglyceride levels in the serum of WT (n = 4) and L-KO (n = 4) mice treated with either vehicle or T0901317 on days 17–20 post AAV-TBG-Cre injection. A one-way ANOVA with subsequent Šidák’s multiple comparisons test was used to determine statistical significance (vehicle vs. T0901317: WT P value = 0.9994, L-KO P value = 0.0560; WT/Vehicle vs. L-KO/T0901317 P value = 0.0155). 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.