Effect of procysteine on aging-associated changes in hepatic GSH and SMase: evidence for transcriptional regulation of smpd3

Abstract

In hepatocytes, aging-associated decline in GSH has been linked to activation of neutral SMase (nSMase), accumulation of bioactive ceramide, and inflammation. In this study, we seek to test whether dietary supplementation with the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC), would correct the aging-associated differences in hepatic GSH, nSMase, and ceramide. Young and aged mice were placed on a diet that either lacked sulfur-containing amino acids (SAAs) or had 0.5% OTC for 4 weeks. Mice fed standard chow were used as an additional control. SAA-deficient mice exhibited significant aging-associated differences in hepatic GSH, GSH/GSSG, ceramide, and nSMase. C24:1 ceramide, the major ceramide species in liver, was affected the most by aging, followed by the less abundant C16:0 ceramide. OTC supplementation eliminated the aging-associated differences in hepatic GSH and GSH/GSSG ratio. Surprisingly, however, instead of decreasing, the nSMase activity and ceramide increased in the OTC-fed mice irrespective of their age. These effects were due to elevated nSMase-2 mRNA and protein and appeared to be direct. Similar increases were seen in HepG2 cells following treatment with OTC. The OTC-fed aged mice also exhibited hepatic steatosis and triacylglyceride accumulation. These results suggest that OTC is a potent stimulant of nSMase-2 expression and that there may be unanticipated complications of OTC supplementation.

Keywords: aging; ceramide; diet; neutral Sphingomyelinase-2; oxidative stress; reduced glutathione supplementation; sphingomyelinase.

Fig. 1.

Aging-associated changes in GSH/GSSG levels, nSMase activity, and ceramide species in liver. Young (5 months) and aged (23 months) C57Bl6 mice were randomly placed on either a Std chow or a SAA def. diet for 4 weeks. Twenty percent homogenates from livers were prepared and used for analyses. Hepatic GSH and GSSG (A, B, D, E) were measured in individual animals (n = 3) using a Bioxytech® GSH/GSSG-412™ assay kit as outlined in the Materials and Methods. nSMase activity (C, F) was measured in whole liver homogenates (40 μg of protein/assay) using a fluorescently labeled analog NBD-SM as a substrate. The mean values for each individual mouse are shown. Quantification of the different ceramide species in the liver of young and aged animals was done by HPLC-ESI/tandem mass spectrometry (G). Mean values ± SD are shown (n = 3), *P < 0.05, **P < 0.01 according to Student’s t-test.

Fig. 2.

Effect of dietary OTC supplementation on hepatic GSH content. Young and aged C57Bl6 mice were placed on either a 0.5% OTC or a SAA def. control diet for 4 weeks. Hepatic GSH (A) and GSH/GSSG (B) contents were measured in whole liver homogenates prepared from individual animals (n = 3–4 per group) using a Bioxytech® GSH/GSSG-412™ assay kit. Data are shown as mean values ± SD. Statistical analyses were done by two-way ANOVA. The main effects of the diet and age are indicated with an asterisk (*P < 0.05). No interaction between both factors was present.

Fig. 3.

Effect of dietary OTC supplementation on nSMase activity and ceramide levels in mouse livers. Young and aged C57Bl6 mice were placed on either a 0.5% OTC or a SAA def. control diet for 4 weeks. nSMase activity (A) was assessed in whole liver homogenates (40 μg of protein/assay) using a fluorescently labeled NBD-SM as a substrate. Total ceramide (B) and ceramide species with different FA length (C) were measured in total lipid extracts by HPLC-ESI/tandem mass spectrometry. Mean values ± SD are shown (n = 3–4 mice in each group). Statistical analysis was done by two-way ANOVA followed by Bonferroni posttest analyses. The statistical significance of the main effects (aging and diet) are shown with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). The statistical significance of the interaction effect is shown with number signs (##P < 0.001).

Fig. 4.

Effect of dietary OTC supplementation on nSMase-2 protein in mouse livers. Young and aged C57Bl6 mice were placed on either a 0.5% OTC or a SAA def. control diet for 4 weeks. nSMase-2 (A, B) and nSMase-1 (C, D) protein levels in whole liver lysates were determined by Western blot using specific rabbit polyclonal antibodies against each protein. Cyclophilin-A was used to control for equal loading and for normalization. Mean values ± SD (n = 3–4 animals per group) of the ratio between the respective nSMase protein and the loading control are shown. ASMase activity (E) was determined in whole liver homogenates as described in the Materials and Methods. nSMase-2 specific activity (F) was calculated as the total nSMase activity measured for each individual mouse divided by the nSMase-2 protein amount quantified by Western blotting. Data are shown as mean values ± SD (n = 3–4 animals per group). Statistical analyses were done according to two-way ANOVA and Bonferroni posttest analyses. The statistical significance of the main effect of diet and aging is shown by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001). No statistically significant interaction effects were present.

Fig. 5.

Effect of dietary OTC supplementation on nSMase-2 mRNA levels in liver. Young and aged C57Bl6 mice were placed on either a 0.5% OTC or a SAA def. control diet for 4 weeks. Levels of nSMase-2 mRNA in the liver were assessed by quantitative RT-PCR (A, B) and real-time PCR (C). The levels of β-actin mRNA were used for normalization. Data are shown as mean values ± SD (n = 3–4). Statistical analyses were done according to two-way ANOVA and Bonferroni posttest analyses. The statistical significance of the main effect of diet and aging is shown by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001). No statistically significant interaction effects were present.

Fig. 6.

nSMase-2 protein and mRNA levels in HepG2 cells supplemented with OTC. HepG2 cells were cultured in complete growth medium supplemented with OTC (2.5 mM) for the indicated time (A) or for 6 days with the indicated concentrations (B–E). The levels of nSMase-2 mRNA were analyzed by real-time PCR (A) or quantitative RT-PCR (B, D). The levels of nSMase-2 protein were analyzed by Western blotting using nSMase-2 specific rabbit polyclonal antibody (C). β-Actin was used for normalization and as a control for equal loading. nSMase activity was measured in whole cell homogenates (D). Data are shown as a percent of the activity measured in vehicle-treated cells. Data are shown as mean values ± SD (n = 3). One-way ANOVA with Dunnett’s multiple comparison test are shown (*P < 0.05, ** P < 0.01, ***P < 0.001). Results are representative of at least three independent experiments.

Fig. 7.

Liver histology and TAG levels. Young and aged C57Bl6 mice were placed on either a 0.5% OTC or a SAA def. control diet for 4 weeks. Freshly dissected liver portions were flash-frozen, sliced, and stained with H and E (A) or Oil Red-O (B). Slides are representative of all animals in each group. The scale bar corresponds to 50 μm. Hepatic TAG mass was measured in total lipid extracts using an L-Type Triglyceride M kit and normalized to the total phospholipid content (C). Data from an individual mouse are shown. Statistical analysis was done by two-way ANOVA followed by Bonferroni posttest analyses. The statistical significance of the main effects (aging and diet) is shown by asterisks (*P < 0.05, **P < 0.01). The statistical significance of the interaction effect is shown by a number sign (#P < 0.01).