Long-term supplementation with a cystine-based antioxidant delays loss of muscle mass in aging

Abstract

Oxidative stress increases with age and is postulated to be a major causal factor for sarcopenia in aging. Here, we examined whether the administration of a cystine-based antioxidant (F1) can alleviate/delay age-specific changes in skeletal muscles. C57BL6 male mice aged 17 months (middle aged) were fed with normal diet with or without supplementation of F1 (3 mg/kg food) for 6 months. Compared with young (5 months old) mice old mice exhibited increased markers of oxidative stress, inflammation, and muscle cell apoptosis and decreased muscle weight. These age-related changes were further associated with inactivation of adenosine-5'-monophosphate-activated protein kinase (AMPK), increased lipogenesis, activation of c-Jun NH2-terminal kinase, and decreased expression of Delta 1, phospho-Akt, and proliferating cell nuclear antigen in aged skeletal muscle. Such alterations were significantly prevented by F1. These results demonstrate the beneficial effects of F1 to attenuate loss of muscle mass associated with aging.

Keywords: Antioxidant; Mice; Muscle cell apoptosis; Oxidative stress; Sarcopenia..

Figure 1.

Dietary supplementation of F1 improves muscle morphology and suppresses oxidative stress, serum interleukin-6 levels, c-Jun NH₂-terminal kinase activation, and apoptosis in old mice. (A) Dietary supplementation of F1 to middle-aged mice for 6 months results in an apparent increase in muscle fiber size in old mice similar to the phenotype observed in young controls. Old mice exhibit higher oxidative stress in the skeletal muscle, as indicated by low muscle-reduced glutathione/muscle-oxidized glutathione ratio (B) and increased circulating interleukin-6 levels (C) compared with young mice. These age-related changes are significantly (p < .05) attenuated by antioxidant treatment. Values are mean ± standard error of the mean (SEM). Means with unlike superscripts are significantly (p < .05) different. Means with superscripts A are different from means with superscripts B. However, means with superscripts AB are not different from means with either superscripts A or B. (D) Western blots of muscle lysates from old, F1-supplemented old and young mice show suppression of age-related increase in phospho-c-Jun NH₂-terminal kinase levels by antioxidant treatment. The gels are representative of two mice belonging to each group from one of three separate experiments. Glyceraldehydes-3-phosphate dehydrogenase in the immunoblot is shown as a loading control. (E) Quantification of band intensities. Values are mean ± SEM. Means with unlike superscripts are significantly (p < .05) different. (F) Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling assay shows, compared with young muscles where no apoptosis is detected, a distinct increase in the incidence of muscle cell apoptosis in aged muscles, and that can be effectively prevented by F1 treatment. Scale bar = 25 µm. (G) Quantitation of muscle cell apoptosis in young, old, and F1-supplemented old mice. Apoptotic rate was expressed as the percentage of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive nuclei per total nuclei (apoptotic plus nonapoptotic nuclei) counted in a unit reference area. Antioxidant treatment significantly prevents age-related increase in muscle cell apoptosis. Values are mean ± SEM. Means with unlike superscripts are significantly (p < .001) different.

Figure 2.

(A) Immunohistochemical analysis of gastrocnemius muscle sections shows a marked suppression of age-related increase in 4-hydroxynonenal immunoreactivity in muscles from aged mice supplemented with F1. (B) Western blot analysis of muscle lysates from young old, and old mice supplemented with F1 (old + F1) shows complete suppression of age-related increase in 4-hydroxynonenal levels by F1. The gels are representative of two mice belonging to each group from one of three separate experiments. Glyceraldehydes-3-phosphate dehydrogenase in the immunoblot is shown as a loading control. (C) Quantification of band intensities. Values are mean ± SEM. Means with unlike superscripts are significantly (p < .05).

Figure 3.

(A) Western blot analysis of muscle lysates from young old, and old mice supplemented with F1 (old + F1) shows upregulation of SOD2 expression in aged muscles by F1. The gels are representative of two mice belonging to each group from one of three separate experiments. Glyceraldehydes-3-phosphate dehydrogenase in the immunoblot is shown as a loading control. (B) Quantification of band intensities. Values are mean ± SEM. Means with unlike superscripts are significantly (p < .05).

Figure 4.

(A) Western blot analysis shows a substantial reduction in phospho-AMPK levels (inactivation) in gastrocnemius muscles from old mice compared with that from young mice. Such age-related inactivation of AMPK is further associated with decreased phosphorylation and activation of acetyl-CoA-carboxylase and upregulation of fatty acid synthase, and decreased glucose-6-phosphate dehydrogenase levels in aged muscles compared with young muscles. Dietary supplementation of F1 effectively prevents AMPK inactivation and upregulation of fatty acid synthase in aged muscles but with no effect on phospho-acetyl-CoA-carboxylase expression. The gels are representative of two mice belonging to each group from one of three separate experiments. Glyceraldehydes-3-phosphate dehydrogenase in the immunoblot is shown as a loading control. (B) Quantification of band intensities. Values are mean ± SEM. Means with unlike superscripts are significantly (p < .05).

Figure 5.

(A) Western blots of muscle lysates show decreased levels of Delta 1, proliferating cell nuclear antigen, and phospho-Akt in aged muscles compared with young muscles. There is no change in the expression of Notch1 between young and aged muscles. These age-related changes are effectively prevented by dietary supplementation of F1. (B) Quantification of band intensities. Values are mean ± SEM. Means with unlike superscripts are significantly (p < .05) different.