A Western diet induced NAFLD in LDLR(-/)(-) mice is associated with reduced hepatic glutathione synthesis

Abstract

Oxidative stress plays a key role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Glutathione is the major anti-oxidant involved in cellular oxidative defense, however there are currently no simple non-invasive methods for assessing hepatic glutathione metabolism in patients with NAFLD. As a primary source of plasma glutathione, liver plays an important role in interorgan glutathione homeostasis. In this study, we have tested the hypothesis that measurements of plasma glutathione turnover could be used to assess the hepatic glutathione metabolism in LDLR(-/)(-) mice, a mouse model of diet-induced NAFLD. Mice were fed a standard low fat diet (LFD) or a high fat diet containing cholesterol (a Western type diet (WD)). The kinetics of hepatic and plasma glutathione were quantified using the (2)H2O metabolic labeling approach. Our results show that a WD leads to reduced fractional synthesis rates (FSR) of hepatic (25%/h in LFD vs. 18%/h in WD, P<0.05) and plasma glutathione (43%/h in LFD vs. 21%/h in WD, P<0.05), without any significant effect on their absolute production rates (PRs). WD-induced concordant changes in both hepatic and plasma glutathione turnover suggest that the plasma glutathione turnover measurements could be used to assess hepatic glutathione metabolism. The safety, simplicity, and low cost of the (2)H2O-based glutathione turnover approach suggest that this method has the potential for non-invasive probing of hepatic glutathione metabolism in patients with NAFLD and other diseases.

Keywords: Flux; Glutathione; Heavy water; Mass spectrometer; NAFLD; NASH; Oxidative stress; Western Diet.

Fig. 1

Metabolic labeling of glutathione in mice with ²H₂O. Panel A: Study design. Eight to ten week old LDLR^−/− mice (18 mice/group) were fed either a standard low fat diet (LFD) or a high fat diet containing cholesterol (a Western type diet (WD)) for 12 weeks. After euthanizing 3 mice from each group, the remaining animals were loaded with bolus injection of ²H₂O (22 μL/g body weight) and exposed to 5% ²H₂O in their drinking water for different duration. Three mice from each group were euthanized at 4, 8, 24 and 72 h and blood and liver tissue were collected for future assays. Panel B: ²H₂O rapidly equilibrates with total body water and ²H incorporates into amino acids including glutamic acid, cysteine, and glycine. The first step in glutathione synthesis involves glutamic acid and cysteine with the production of γ-glutamylcysteine dipeptide via γ-glutamylcysteine synthetase (GCS). In the second step glutathione synthase (GS) catalyzes coupling of γ-glutamylcysteine with glycine. ²H-glutamic acid, ²H-cysteine and ²H-glycine along with their unlabeled analogs are incorporated into glutathione.

Fig. 2

Glutathione analysis by LC–MS/MS. Panel A: Carbamidomethylated glutathione derivative is eluted at 4.2 min. Panel B: MS1 spectrum of precursor ion of carbamidomethylated glutathione with m/z 365.15 in positive ion mode. Panel C: MS1 zoom scan of precursor ion monitored between 362 m/z and 370 m/z. Panel D: Collision-indiced dissociation of derivatized glutathione yields abundant cysteine(carbamidomethyl)glycine ion at m/z 236.04 along with other fragment ions. Panel E: The zoom scanned spectra of cysteine(cabamidomethyl)glycine fragment ion before and after (4 h and 8 h) of ²H₂O treatment. The dashed lines are added to aid in visualizing differences in the isotopic abundance.

Fig. 3

Effect of a Western diet on hepatic glutathione redox ratio in LDLR^−/− mice. Hepatic content of reduced glutathione (GSH) (Panel A), total glutathione (GSH+GSSGR) (presented in Table 1) was analyzed by mass spectrometry as described in Section 2. Hepatic oxidized glutathione (GSSR) content (Panel B) was calculated as the difference of total and reduced glutathione. Glutathione redox ratio (GSH/GSSR) in the liver (Panel C) was calculated based on hepatic content of reduced and oxidized glutathione. Data are presented as mean ± SD (n = 6/group). *P < 0.05.

Fig. 4

Glutathione turnover assessed using ²H₂O-metabolic labeling approach. After intraperitoneal bolus load, ²H₂O was administered in drinking water (5%) for up to 72 h. Effect of a Western diet on the turnover rate constant of hepatic (Panel A) and plasma (Panel B) glutathione LDLR^−/− mice fed with a standard low fat diet (black) and a Western diet (red). *P < 0.05.

Fig. 5

A Western diet-induced alterations in hepatic glutathione metabolism. A Western diet activates glutathione peroxidase but deactivates glutathione reductase that result in accumulation of oxidized glutathione without any significant changes in lipid peroxidation products, including GSH–4-HNE. Impaired glutathione recycling increases the total glutathione pool that leads to reduced FSR of glutathione without significant changes in GSH levels. Impaired GSSG reduction to GSH would accumulate NADPH that could be channeled to fatty acid synthesis. (1) superoxide dismutase, (2) glutathione peroxidase, (3) glutamate cysteine ligase, (4) glutathione synthetase, (5) glutathione reductase, (6) glutathione-S transferase, (7) fatty acid synthethase. Red and blue colored arrows show up-regulated and down-regulated reactions or pathways, respectively.