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. 2011 Dec 9;286(49):42483-42493.
doi: 10.1074/jbc.M111.304592. Epub 2011 Oct 23.

Altered glutathione homeostasis in heart augments cardiac lipotoxicity associated with diet-induced obesity in mice

Sanjoy Ghosh  1 , Dian C Sulistyoningrum  1 , Melissa B Glier  1 , C Bruce Verchere  1 , Angela M Devlin  2
Affiliations

Altered glutathione homeostasis in heart augments cardiac lipotoxicity associated with diet-induced obesity in mice

Sanjoy Ghosh et al. J Biol Chem. .

Abstract

Obesity-related cardiac lipid accumulation is associated with increased myocardial oxidative stress. The role of the antioxidant glutathione in cardiac lipotoxicity is unclear. Cystathionine β-synthase (Cbs) catalyzes the first step in the trans-sulfuration of homocysteine to cysteine, which is estimated to provide ∼50% of cysteine for hepatic glutathione biosynthesis. As cardiac glutathione is a reflection of the liver glutathione pool, we hypothesize that mice heterozygous for targeted disruption of Cbs (Cbs(+/-)) are more susceptible to obesity-related cardiolipotoxicity because of impaired liver glutathione synthesis. Cbs(+/+) and Cbs(+/-) mice were fed a high fat diet (60% energy) from weaning for 13 weeks to induce obesity and had similar increases in body weight and body fat. This was accompanied by increased hepatic triglyceride but no differences in hepatic glutathione levels compared with mice fed chow. However, Cbs(+/-) mice with diet-induced obesity had greater glucose intolerance and lower total and reduced glutathione levels in the heart, accompanied by lower plasma cysteine levels compared with Cbs(+/+) mice. Higher triglyceride concentrations, increased oxidative stress, and increased markers of apoptosis were also observed in heart from Cbs(+/-) mice with diet-induced obesity compared with Cbs(+/+) mice. This study suggests a novel role for Cbs in maintaining the cardiac glutathione pool and protecting against cardiac lipid accumulation and oxidative stress during diet-induced obesity in mice.

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Figures

FIGURE 1.
FIGURE 1.
Weight gain and adiposity in Cbs+/+ and Cbs+/− mice fed HFD from weaning for 13 weeks to induce obesity. A, body weight gain from weaning for 13 weeks. B, terminal body weight at 13 weeks. C, abdominal (gonadal), retroperitoneal, and subcutaneous (inguinal) fat pad weight at the end of the 13-week feeding period. Data are presented as means ± S.E., n = 9–20 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Significance was determined by two-way ANOVA.
FIGURE 2.
FIGURE 2.
Impaired glucose tolerance in Cbs+/− mice with diet-induced obesity. Fasting base-line plasma glucose (A) and insulin (B) are shown. C, plasma glucose concentrations following an i.p. injection of 2 g/kg glucose. D, area under the curve for plasma glucose levels during the 2-h intraperitoneal glucose tolerance test. Data are presented as means ± S.E., n = 5–6 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Significance was determined by two-way ANOVA.
FIGURE 3.
FIGURE 3.
Lipid accumulation in heart from Cbs+/− mice with diet-induced obesity. Triglyceride (A), cholesteryl ester (B), total phospholipids (C), and free cholesterol (D) concentrations in heart from Cbs+/− and Cbs+/− mice fed chow or the HFD. Data is presented as means ± S.E., n = 5–6 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Significance was determined by two-way ANOVA.
FIGURE 4.
FIGURE 4.
Enhanced apoptosis and oxidative stress in heart from Cbs+/− mice with diet-induced obesity. Markers of apoptosis were assessed by quantifying expression levels of cleaved caspase-3 (A) and XIAP (B). Indicators of oxidative stress were assessed by quantifying expression of nitrotyrosine (C) and lipid hydroperoxides (D). Data are presented as means ± S.E., n = 5–6 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Inset depicts representative immunoblots for analyzed proteins in comparison with β-actin bands from the same blot. Significance was determined by two-way ANOVA.
FIGURE 5.
FIGURE 5.
Mitochondrial dysfunction in heart from Cbs+/− mice with diet-induced obesity. Mitochondrial dysfunction was assessed by quantifying the mitochondrion-encoded cytochrome b gene (mt-Cytb) copy number relative to the nucleus-encoded β-actin gene (Actb) copy number (A) and by assessing expression of Pgc-1α (B). C, expression of Acox-1. Data are presented as means ± S.E., n = 5–6 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Inset depicts representative immunoblots for analyzed proteins in comparison with β-actin bands from the same blot. Significance was determined by two-way ANOVA.
FIGURE 6.
FIGURE 6.
Disturbances in glutathione metabolism in heart from Cbs+/− mice with diet-induced obesity. Glutathione metabolism in heart was assessed by quantifying levels of total glutathione (A), GSH (B), GSSG (C), Gclc expression (D), glutathione reductase activity (E), and Gpx-1 expression (F). Data are presented as means ± S.E., n = 5 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Inset depicts representative immunoblots for analyzed proteins in comparison with β-actin bands from the same blot. Significance was determined by two-way ANOVA.
FIGURE 7.
FIGURE 7.
NF-κB signaling in heart from Cbs+/− mice with diet-induced obesity. NF-κB signaling was assessed by quantifying levels of the NF-κB p65 subunit (A), IκB (B), phosphorylated-IκB (C), and the ratio of phosphorylated-IκB/IκB (D) in heart. Data are presented as means ± S.E., n = 5 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Inset depicts representative immunoblots for analyzed proteins in comparison with β-actin bands from the same blot. Significance was determined by two-way ANOVA.
FIGURE 8.
FIGURE 8.
No disturbances in glutathione metabolites in liver from Cbs+/− mice with diet-induced obesity. Total glutathione, GSSG, and GSH levels were quantified in liver. Data are presented as means ± S.E., n = 7–11 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Significance was determined by two-way ANOVA.
FIGURE 9.
FIGURE 9.
Decreased plasma cysteine in Cbs+/− mice with diet-induced obesity. Plasma total cysteine (A) and its related metabolites total homocysteine (B), methionine (C), total glutathione (D), and triglyceride (E) levels were quantified in plasma. Data are presented as means ± S.E., n = 5–9 mice per diet/genotype group. Open bars, Cbs+/+ mice; filled bars, Cbs+/− mice. Significance was determined by two-way ANOVA.
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