Glutathione supports lipid abundance in vivo

Abstract

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.

Keywords: NRF2; glutathione; lipids; liver; oxidative stress.

Figure 1.. GSH synthesis is required for the survival of adult animals.

(A) Schematic of the whole-body inducible Gclc knockout mouse model. (B) Relative expression of Gclc mRNA in tissues from the KO (n=4–5) compared to WT (n=5) mice 12–15 days following tamoxifen administration. Expression levels were normalized to the expression of the reference gene Rps9. (C) Representative immunoblot analysis of GCLC protein in the liver, kidney, and lung of WT and KO mice 12–15 days following tamoxifen administration. (D) Relative GSH abundance in the liver, kidney, and lungs of WT (n = 4) and KO (n = 4) mice 12–15 days following tamoxifen administration. (E) Percent survival of the WT (n = 9) and KO (n = 11) mice following tamoxifen treatment. Loss of greater than 20% body weight resulted in a humane endpoint for mice. (F) Percent change in body weight in WT (n=18) and KO (n=17) mice at death. Initial weight measurements were collected on Day 0 of tamoxifen administration, and final weight measurements were collected at endpoints. (G) Consumption of food by WT (n=7) and KO (n=5) mice over time. (H-I) Lean mass (H) and fat mass (I) in WT (n=13) and KO (n=7) mice as determined by dual-energy X-ray absorptiometry (DEXA) analysis on Day 14 post-tamoxifen injection. (J) Epididymal white adipose tissue (eWAT) mass normalized to body mass for WT (n=13) and KO (n=7) mice at endpoints. (K) Representative images from hematoxylin-eosin (H&E) staining of the epididymal fat from WT and KO mice. Tissues were collected 12–15 days following the tamoxifen injection (scale bars = 200 µm). (L) Adipocyte area measurements for WT and KO mice as quantified from representative H&E images with the Adiposoft plugin in ImageJ. N = 10 images for each genotype. Data are shown as mean ±SEM. A one-way ANOVA with subsequent Tukey’s multiple comparisons test was used in (D) and (L), and an unpaired two-tailed t-test was used in (F-J) to determine statistical significance. ns = not significant, * P value < 0.05, ** P value < 0.01, *** P value < 0.001, **** P value < 0.0001.

Figure 2.. Liver tissue from Gclc KO mice has induced NRF2 target genes and repressed lipogenic gene expression.

(A) GSH quantity (normalized to mg tissue weight) in the liver, kidney, lung, and brain from Gclc WT mice (n=4). (B) Representative H&E-stained histochemical images of the liver in WT and KO mice. Scale bars = 500 µm. (C) Relative abundance of GSH precursors and their related metabolites in the serum of WT (n=4) and KO (n=4) mice. (D) Gene Set Enrichment Analysis (GSEA) of oxidative stress-related pathways in the liver of KO (n=4) compared to WT (n=4) mice. (E) Proteomic analysis of upregulated liver proteins in KO (n=3) compared to WT (n=3) mice. Black data points = proteins with a p <0.05 and log2 fold change > 2. Red data points = annotated NRF2 target proteins. (F) Relative mRNA expression of annotated NRF2 target genes in the liver of WT (n=4) and KO (n=4) mice. Expression levels were normalized to the expression of the reference gene Rps9. (G) Representative immunoblot analysis of Nqo1 in the liver of WT and KO mice. (H) Representative immunofluorescence images of the liver from WT and KO mice stained with an antibody against NQO1. Scale bars = 200 µm. (I) Schematic of the proposed mechanism of NRF2-dependent repression of lipogenic gene expression. Data are shown as mean ±SEM. A one-way ANOVA with subsequent Tukey’s multiple comparisons test was used in (D) and (L), and an unpaired two-tailed t-test was used in (F-J) to determine statistical significance. ns = not significant, * P value < 0.05, ** P value < 0.01, *** P value < 0.001, **** P value < 0.0001.

Figure 3:. The abundance of triglycerides is lower following GSH depletion in mice.

(A-C) Lipidomic analysis of (A) adipose tissue, (B) liver tissue, and (C) serum in KO (n=4) compared to WT (n=4) mice. Black data points = lipid species with a p <0.05 and log2 fold change > 2. Red data points = triglycerides. (D) Triglyceride levels in the serum of WT (n=4) and KO (n=4) mice (E) Serum triglyceride levels for WT (n=4) and KO (n=4) mice fed with either normal chow or a High Fat Diet (HFD) for 14 days post-treatment with tamoxifen. (F) Percent change in weight for WT (n=7) and KO mice fed with either normal chow (n= 7) or a high-fat diet (HFD; n=9) 14 days post-treatment with tamoxifen. (G-H) Relative abundance of select (G) fatty acids in the liver and (H) triglyceride species in the serum from WT (n=4) and KO (n=4) mice. 16:0 = palmitic acid; 16:1;2O= oxidized palmitoleic acid; 18:0 = stearic acid; 18:1 = oleic acid. Data are shown as mean ±SEM. An unpaired two-tailed t-test was used in (A-D), and a one-way ANOVA with subsequent Tukey’s multiple comparisons test was used in (E-H) to determine statistical significance. ns = not significant, * P value < 0.05, ** P value < 0.01, *** P value < 0.001, **** P value < 0.0001.

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

(A) Schematic of inducible liver-specific Gclc deletion (L-KO). (B) Relative abundance of Gclc mRNA (top) and GCLC protein (bottom) in the liver of WT (n=4–8) and L-KO (n=4–7) mice following treatment with AAV-TBG-Cre. Expression levels of mRNA were normalized to the expression of the reference gene Rps9. (C) Relative abundance of GSH in liver tissue of WT (n=4) and L-KO (n=4) mice following treatment with AAV-TBG-Cre. (D) Percent survival of WT (n=6) and L-KO (n=6) mice following treatment with AAV-TBG-Cre. (E) Percent change in body weight of WT (n=8) mice and L-KO (n=10) mice at day 20 following treatment with AAV-TBG-Cre. (F) Epididymal white adipose tissue (eWAT) mass normalized to body mass in WT (n=16) and L-KO (n=17) mice. (G) Representative H&E-stained histochemical images of the liver of female WT and L-KO mice three weeks following treatment with AAV-TBG-Cre. Scale bars = 200 µm. (H-I) Relative mRNA levels of (H) Nqo1 and (I) Scd1 in WT (n=4–8) and L-KO (n=4–7) following treatment with AAV-TBG-Cre. Expression levels were normalized to the expression of the reference gene Rps9. (J) Triglyceride levels in the serum of WT (n=4) and L-KO (n=4) mice following treatment with AAV-TBG-Cre. (K) Triglyceride levels in the serum L-KO mice fed normal chow (n=4) and an HFD (n=7) 3 weeks following treatment with AAV-TBG-Cre. Data are shown as mean ±SEM. A one-way ANOVA with subsequent Tukey’s multiple comparisons test was used for (B-C) and (H-J), and an unpaired two-tailed t-test was used for (E-F) and (K) to determine statistical significance. ns = not significant, * P value < 0.05, ** P value < 0.01, *** P value < 0.001, **** P value < 0.0001.

Figure 5:. Nrf2 deletion in the liver rescues triglyceride levels and lipid synthesis in liver-specific Gclc KO mice.

(A) Schematic of inducible liver-specific Gclc deletion (Gclc L-KO), Nrf2 deletion (Nrf2 L-KO), and Gclc-Nrf2 deletion (L-DKO). (B-E) Relative expression of (B) Gclc, (C) Nrf2, (D) Nqo1, and (E) 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 three weeks following treatment with AAV-TBG-Cre. Expression levels were normalized to the expression of the reference gene Rps9. (F) Lipidomic analysis of the serum of WT (n=4), Gclc L-KO (n=4), Nrf2 L-KO (n=4) and L-DKO (n=6) mice. Data are shown as normalized peak intensities. (G) Serum triglyceride levels for WT (n=6), Gclc L-KO (n=6), Nrf2 L-KO (n=6), and L-DKO (n=8) mice three weeks post-treatment with AAV-TBG-Cre. (H) Percent survival of WT, Gclc L-KO, Nrf2 L-KO & L-DKO following treatment with AAV-TBG-Cre. (I) Percent change in weight of WT (n=4), Gclc L-KO (n=4), Nrf2 L-KO (n=4), and L-DKO (n=6) mice three weeks following treatment with AAV-TBG-Cre. (J) Representative H&E-stained histochemical images of the liver of female WT, Gclc L-KO, Nrf2 L-KO, and L-DKO mice three weeks following treatment with AAV-TBG-Cre. Scale bars = 200 µm. Data are shown as mean ±SEM. A one-way ANOVA with subsequent Tukey’s multiple comparisons test was used for (B-C) and (I), and an unpaired two-tailed t-test was used for (D-E) and (G) to determine statistical significance. ns = not significant, * P value < 0.05, ** P value < 0.01, *** P value < 0.001, **** P value < 0.0001.

Figure 6:. Prolonged Gclc deletion in the liver lowers adipose tissue mass.

(A) Schematic for extended monitoring of WT and L-KO mice. (B) Percent survival for WT and L-KO mice following treatment with AAV-TBG-Cre. (C) Percent change in body weight at the endpoint of WT (n=6) and L-KO (n=6) mice. (D) Representative H&E-stained histochemical images of the liver from female WT and L-KO mice ten weeks following treatment with AAV-TBG-Cre. Scale bars = 200 µm. (E) Serum triglyceride concentration of WT (n=6) and L-KO (n=5) mice ten weeks following treatment with AAV-TBG-Cre. (F) Epididymal fat adipose tissue (eWAT) mass normalized to body mass from WT (n=6) and L-KO (n=6) mice ten weeks post-treatment with AAV-TBG-Cre. Data are shown as mean ±SEM. To determine statistical significance, an unpaired two-tailed t-test was used for (C) and (E-F). ns = not significant, * P value < 0.05, ** P value < 0.01, *** P value < 0.001, **** P value < 0.0001.