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TO THE EDITOR:

We appreciate the concerns shared by Dr.Sekhar regarding our recently published manuscript. With respect to the title of the letter to the Editor by Dr. Sekhar “Lack of evidence to support a beneficial role for glutathione depletion on body weight or glucose intolerance” it appears pertinent to first summarize the available literature that has addressed the role of glutathione depletion in obesity and metabolism. Abundant evidence has now revealed that mice genetically deficient for glutathione or glutathione peroxidase are resistant to weight gain and diet-induced obesity, are protected from glucose intolerance and insulin resistance, do not develop hepatic steatosis, and exhibit a higher metabolic rate (1–4). Therefore, there is indeed unequivocal evidence that glutathione depletion elicits a phenotype in mice that is beneficial for body weight regulation and glucose homeostasis. Moreover, our data using a well-established pharmacological model of glutathione depletion are entirely consistent with this evidence obtained from genetic models. Due to the nature of our article being a short communication and considering the extensive literature describing buthionine sulfoximine (BSO)-induced glutathione depletion in rodents (5, 6), we did not show glutathione reduction in our initial manuscript. However, we found a reduction of almost 70% in total glutathione levels in epididymal adipose tissue of BSO treated mice (P < 0.05). The extent of glutathione reduction in response to BSO in our studies is similar to that previously seen in pharmacologic or genetic models of glutathione depletion (2, 5, 7).

With respect to the second comment on the measurements of food intake, these experiments were performed following the guidelines set forth by the National Institutes of Health (NIH)-funded Mouse Metabolic Phenotyping Centers (8). Specifically, we employed a calorimetry system that provided investigator-independent automated feeding systems. Food intake was measured cumulatively during a 72 h time period and expressed as gram food consumption per day. Our observation that BSO-treatment did not affect food consumption is, again, in line with various previous publications demonstrating normal food intake in BSO-treated mice (5, 9) as well as with the above-described genetic models of glutathione depletion (2). Nonetheless, we agree with Dr. Sekhar that further comprehensive studies to define this intriguing phenotype are warranted, including particularly pair-feeding experiments.

Finally, Dr. Sekhar expresses concerns about possible toxic effects of BSO. We have elected to treat mice with a dose of 30 mM BSO because this dose has been previously demonstrated to effectively decrease glutathione in mice (7). Moreover, extensive characterization of various organ functions in mice treated with this dose, including liver function tests and cytochrome P-450 concentrations, revealed no toxic effects after prolonged treatment (7). In our study, high fat diet fed mice exhibited a significant increase in liver weight during the course of the study, an effect likely secondary to steatosis. In contrast, the development of this phenotype was prevented in BSO-treated mice (data not shown). This observation is consistent with data obtained in mice that are genetically deficient for glutathione and has been attributed to a specific beneficial effect of glutathione depletion on the expression of lipogenic genes (2, 3, 6). Consequently, it is not surprising that the authors of the study referred to by Dr. Sekhar (9) also noted an effect of BSO on liver weight. However, in this study Watanabe et al. (9) did not observe any avoidance of food/water intake, and the authors did not interpret 30 mM of BSO as hepatotoxic, considering that BSO did not increase alanine transaminase (ALT), alkaline phosphatase (ALP), or the expression levels of drug-metabolizing pathways (albeit a modest isolated increase in aspartate transaminase (AST) levels). Collectively, these studies do not support the assumption that the dose of 30 mM BSO used in our studies elicited toxic activities that contribute to the beneficial obesity and metabolism phenotype.

In summary, our manuscript supports a more complex role of glutathione in the regulation of energy balance and metabolism than previously anticipated. We acknowledge that our manuscript, collectively with the discussed literature, challenges the conventional hypothesis that depletion of glutathione may be detrimental for obesity and metabolism. Determining the molecular mechanisms underlying these phenotypes and potential pathological consequences constitute important avenues for future research.

References

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    Loh K, Deng H, Fukushima A, et al. Reactive oxygen species enhance insulin sensitivity. Cell Metab 2009; 10: 260-272.
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    Kendig EL, Chen Y, Krishan M, et al. Lipid metabolism and body composition in Gclm(−/−) mice. Toxicol Appl Pharmacol 2011; 257: 338-348.
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    Haque JA, McMahan RS, Campbell JS, et al. Attenuated progression of diet-induced steatohepatitis in glutathione-deficient mice. Lab Invest 2010; 90: 1704-1717.
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    Chen Y, Krishan M, Nebert DW, et al. Glutathione-deficient mice are susceptible to TCDD-induced hepatocellular toxicity but resistant to steatosis. Chem Res Toxicol 2012; 25: 94-100.
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    Cattan V, Mercier N, Gardner JP, et al. Chronic oxidative stress induces a tissue-specific reduction in telomere length in CAST/Ei mice. Free Radic Biol Med 2008; 44: 1592-1598.
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    Brandsch C, Schmidt T, Behn D, et al. Glutathione deficiency down-regulates hepatic lipogenesis in rats. Lipids Health Dis 2010; 9: 50.
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    Sun JD, Ragsdale SS, Benson JM, et al. Effects of the long-term depletion of reduced glutathione in mice administered L-buthionine-S,R-sulfoximine. Fundam Appl Toxicol 1985; 5: 913-919.
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    Ellacott KL, Morton GJ, Woods SC, et al. Assessment of feeding behavior in laboratory mice. Cell Metab 2010; 3: 156.
  • 9
    Watanabe T, Sagisaka H, Arakawa S, et al. A novel model of continuous depletion of glutathione in mice treated with L-buthionine (S,R)-sulfoximine. J Toxicol Sci 2003; 28: 455-469.