Exploring the Lean Phenotype of Glutathione-Depleted Mice: Thiol, Amino Acid and Fatty Acid Profiles

<div><p>Background</p><p>Although reduced glutathione (rGSH) is decreased in obese mice and humans, block of GSH synthesis by buthionine sulfoximine (BSO) results in a lean, insulin-sensitive phenotype. Data is lacking about the effect of BSO on GSH precursors, cysteine and glutamate. Plasma total cysteine (tCys) is positively associated with stearoyl-coenzyme A desaturase (SCD) activity and adiposity in humans and animal models.</p><p>Objective</p><p>To explore the phenotype, amino acid and fatty acid profiles in BSO-treated mice.</p><p>Design</p><p>Male C3H/HeH mice aged 11 weeks were fed a high-fat diet with or without BSO in drinking water (30 mmol/L) for 8 weeks. Amino acid and fatty acid changes were assessed, as well as food consumption, energy expenditure, locomotor activity, body composition and liver vacuolation (steatosis).</p><p>Results</p><p>Despite higher food intake, BSO decreased particularly fat mass but also lean mass (both P<0.001), and prevented fatty liver vacuolation. Physical activity increased during the dark phase. BSO decreased plasma free fatty acids and enhanced insulin sensitivity. BSO did not alter liver rGSH, but decreased plasma total GSH (tGSH) and rGSH (by ~70%), and liver tGSH (by 82%). Glutamate accumulated in plasma and liver. Urine excretion of cysteine and its precursors was increased by BSO. tCys, rCys and cystine decreased in plasma (by 23–45%, P<0.001 for all), but were maintained in liver, at the expense of decreased taurine. Free and total plasma concentrations of the SCD products, oleic and palmitoleic acids were decreased (by 27–38%, P <0.001 for all).</p><p>Conclusion</p><p>Counterintuitively, block of GSH synthesis decreases circulating tCys, raising the question of whether the BSO-induced obesity-resistance is linked to cysteine depletion. Cysteine-supplementation of BSO-treated mice is warranted to dissect the effects of cysteine and GSH depletion on energy metabolism.</p></div

Effect of BSO on fatty acid precursors and products of stearoyl coenzyme A desaturase ¹.

<p>Effect of BSO on fatty acid precursors and products of stearoyl coenzyme A desaturase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163214#t002fn001" target="_blank">¹</a>.</p

Effect of BSO on glutathione status after 8 weeks of treatment.

<p>A-C: Plasma total glutathione (tGSH), reduced glutathione (rGSH) and rGSH/tGSH ratio. D-F: Liver tGSH, rGSH and rGSH/tGSH ratio. Data is presented as median, 25^th and 75^th percentiles, with individual data plotted; N = 20 (BSO) and N = 23 (controls). Groups were compared by Mann-Whitney <i>U</i> test.</p

Phenomaster cage data at week 2 in BSO-treated and control mice.

<p>A, B: Food intake in g/day, and food intake normalized to body weight. C, D: Physical activity measured as infrared beam breaks in the horizontal and vertical planes during the light and dark phases of a 24-h period. E: O₂ consumption (VO₂)/lean mass ratio and CO₂ production (VCO₂)/ lean mass ratio. F: Adjusted mean VO₂ and VCO₂ controlling for lean mass as a covariate (at a common lean mass of 23.6 g) using general linear modelling. G: Respiratory exchange ratio (RER). H: Water intake in the studied groups. Data is presented as mean ± SEM and compared by independent samples T-test. N = 16–18 (control) and N = 17–20 (BSO). *, ** P<0.05 and P<0.001 respectively vs controls.</p

Insulin sensitivity and SCD activity in BSO-treated and control mice.

<p>A, B: Plasma glucose and insulin measured after a 6h fast after 6 weeks of BSO treatment. C: Homeostatic model of insulin resistance index (HOMA-IR) calculated from the plasma glucose and insulin values. D: Plasma leptin after 6 weeks of BSO treatment. E-H: Stearoyl coenzyme A desaturase (SCD-16 and SCD-18) activity indices calculated from plasma free fatty acid profile (E, F) and from fatty acid profile in total plasma lipids (G, H) after 8 weeks of BSO treatment. Data is presented as median, 25^th and 75^th percentiles, with individual data plotted; N = 20 (BSO) and N = 18 (controls). Groups were compared by Mann-Whitney <i>U</i> test.</p

Effect of BSO on amino acid profile and related variables ¹.

<p>Effect of BSO on amino acid profile and related variables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163214#t003fn001" target="_blank">¹</a>.</p

Effect of BSO on cysteine after 8 weeks of treatment.

<p>A-D: Plasma total cysteine (tCys), reduced cysteine (rCys), cystine, and rCys/tCys ratio. E, F: Liver tCys and rCys. Data is presented as median, 25^th and 75^th percentiles, with individual data plotted; N = 20 (BSO) and N = 23 (controls). Groups were compared by Mann-Whitney <i>U</i> test.</p

Effect of BSO on urine excretion of glutathione and upstream sulfur amino acids.

<p>A-E: Urine concentration of total glutathione, methionine, total homocysteine, cystathionine and total cysteine, in nmol/μmol creatinine during the 2^nd and 4^th weeks of BSO treatment. Depicted P values are from repeated measures ANOVA. N = 6 BSO-treated mice, and 8 controls. Data is from a different cohort of mice of the same age and sex.</p

Plasma clinical biochemistry in BSO-treated mice after 8 weeks¹.

<p>Plasma clinical biochemistry in BSO-treated mice after 8 weeks<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163214#t001fn001" target="_blank">¹</a>.</p

Effect of BSO on body composition.

<p>A-D Body weight and body composition in BSO-treated and control mice at the start of BSO treatment (wk-0) and every two weeks till termination 8 weeks later (wk-8). Data represents mean ± SEM from N = 20 (BSO) and N = 24 (controls). Depicted <i>P</i> values are from repeated measures ANOVA. E Photomicrograph of liver sections stained with H&E, showing hepatoceullar vacuolation in control mice and absence of vacuolation in BSO mice. Scale bar represents 300 μm.</p