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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.

Unlabelled: The process of aging has recently been shown to substantially affect the ability of cells to respond to inflammatory challenges. We demonstrate that aging leads to hepatic hyperresponsiveness to interleukin 1beta (IL-1beta), and we examine the factors that could be responsible for this phenomenon. IL-1beta-induced phosphorylation of c-jun N-terminal kinase (JNK) in hepatocytes isolated from aged rats was 3 times more potent than that in hepatocytes from young rats. Moreover, JNK was activated by substantially lower doses of IL-1beta. These age-related changes in JNK phosphorylation correlated with diminished IL-1beta-induced degradation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Expression levels of IL1beta receptor I, total JNK, IRAK-1, and transforming growth factor-beta-activated kinase-1 (TAK-1) were not affected by aging. However, increased neutral sphingomyelinase activity was observed in hepatocytes from old animals, which we show is caused by induction of the plasma membrane localized neutral sphingomyelinase-2 (NSMase-2). We provide evidence that NSMase-2 is both required and sufficient for the onset of IL-1beta hyperresponsiveness during aging. Overexpression of NSMase-2 in hepatocytes from young rats leads both to a reduction in IRAK-1 degradation and potentiation of JNK phosphorylation, mimicking that seen in hepatocytes from old animals. More importantly, inhibition of NSMase activity in hepatocytes from aged rats using either scyphostatin or short interfering ribonucleic acid (siRNA) leads to reversion to the "young" phenotype of IL-1beta response., Conclusion: These results show that the process of aging causes increased basal NSMase-2 activity in hepatocytes, which in turn leads to IRAK-1 stabilization, JNK potentiation, and ultimately IL-1beta hyperresponsiveness.

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is primarily produced in the liver during inflammation and regulates biological activities of IGF-I. Here we demonstrate that interleukin-1beta (IL-1beta) stimulates IGFBP-1 mRNA production in a dose-dependent manner in hepatocytes from Fisher 344 rats. Employment of c-Jun N-terminal kinase (JNK) inhibitor SP600125 resulted in 3-fold reduction of IGFBP-1 mRNA and protein levels, indicating that IL-1beta-induced IGFBP-1 production is mediated through JNK activation. We further show that hepatocytes from aged rats (20-22 mo), as compared to young (3-4 mo), exhibit up to 2-fold higher levels of IGFBP-1 in response to IL-1beta. IL-1beta-induced phosphorylation of JNK was also significantly higher in aged hepatocytes, and SP600125 treatment eliminated age-related differences in IGFBP-1 mRNA production. Moreover, glutathione depletion in hepatocytes from young rats potently activated JNK, as well as increased IL-1beta-induced IGFBP-1 mRNA levels, suggesting that age-related oxidative stress underlies the upregulated JNK activation and IGFBP-1 expression.