Search

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.

FoxO1 transcription factor controls the glucose and lipid metabolism, as well as cell proliferation and stress response. Akt, activated by insulin and other growth factors, phosphorylates FoxO1 causing its nuclear export and activity suppression. In this manuscript, we show that IL-1β, a pro-inflammatory cytokine, has the opposite effects on FoxO1. IL-1β stimulation of primary rat hepatocytes and HEK293 cells overexpressing the IL-1β receptor (293-IL-1RI) results in increased nuclear and cytosolic FoxO1 protein but not mRNA levels. IL-1β stimulation also elevates the levels of a mutant FoxO1 that is resistant to Akt phosphorylation. This suggests that an Akt-independent mechanism is involved. Co-stimulation with insulin does not affect the IL-1β induction of FoxO1. The IL-1β effects on FoxO1 are counteracted, however, by the silencing or inhibition of neutral sphingomyelinase 2 (nSMase-2) using shRNAi, scyphostatin, or GW4869, as well as by the pharmacological inhibition of JNK and ERK. Reversely, the overexpression of nSMase-2 through adenovirus-mediated gene transfer potentiates, in a JNK- and ERK-dependent manner, the IL-1β effects. We also show that transcription of insulin-like growth factor-binding protein-1 mRNA, which requires active FoxO1, is stimulated by IL-1β and is suppressed by the inhibition of nSMase-2 and JNK. In conclusion, we propose that IL-1β regulates FoxO1 activity through a novel nSMase-2-dependent pathway.

Palmitic acid is a saturated fat found in foods that lead to obesity, cardiovascular disease, and Type II diabetes. It is linked to the development of resistance to insulin stimulation in muscle, liver and other organs involved in glucose metabolism, which, in turn, underlines the onset of Type II diabetes. The cellular and molecular mechanisms of this insulin resistance are complex and not completely understood. This article is focused on the role of palmitic acid as a precursor in the synthesis of sphingolipids, a class of lipid molecules that participate in cellular stress response. Recent evidence had indicated that increased dietary supply of palmitate can stimulate the rate of sphingolipid synthesis in "lean" tissues and generate excessive amounts of sphingolipid metabolites that have a negative effect on the insulin signaling cascade. Many experimental results point to the existence of a causative link between sphingolipid synthesis, insulin response, and hyperglycemia. It is not yet clear, however whether ceramides or glycosphingolipids are involved as both have been implicated to be inhibitors of the insulin signaling cascade. Evidence for a coordinated regulation of sphingolipid and tri/diacylglycerol metabolism complicates further the delineation of a single mechanism of sphingolipid effect on glucose homeostasis., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Acid sphingomyelinase (ASMase) has been proposed to mediate lipopolysaccharide (LPS) signaling in various cell types. This study shows that ASMase is a negative regulator of LPS-induced tumor necrosis factor alpha (TNFalpha) secretion in macrophages. ASMase-deficient (asm(-/-)) mice and isolated peritoneal macrophages produce severalfold more TNFalpha than their wild-type (asm(+/+)) counterparts when stimulated with LPS, whereas the addition of exogenous ceramides or sphingomyelinase reduces the differences. The underlying mechanism for these effects is not transcriptional but post-translational. The TNFalpha-converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNFalpha) to an active 17-kDa form (soluble (s)TNFalpha). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNFalpha production. A substantial portion of the translated pro-TNFalpha was not processed to sTNFalpha; instead, it was rapidly internalized and degraded in the lysosomes. TACE activity was 2-3-fold higher in asm(-/-) macrophages as compared with asm(+/+) macrophages and was suppressed when cells were treated with exogenous ceramide and sphingomyelinase. Indirect immunofluorescence analyses revealed distinct TNFalpha-positive structures in the close vicinity of the plasma membrane in asm(-/-) but not in asm(+/+) macrophages. asm(-/-) cells also had a higher number of early endosomal antigen 1-positive early endosomes. Experiments that involved inhibitors of TACE, endocytosis, and lysosomal proteolysis suggest that in the asm(-/-) cells a significant portion of pro-TNFalpha was sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis, it was recycled to the plasma membrane and processed to sTNFalpha.

Evidence has consistently indicated that activation of sphingomyelinases and/or ceramide synthases and the resulting accumulation of ceramide mediate cellular responses to stressors such as lipopolysaccharide, interleukin 1beta, tumor necrosis factor alpha, serum deprivation, irradiation and various antitumor treatments. Recent studies had identified the genes encoding most of the enzymes responsible for the generation of ceramide and ongoing research is aimed at characterizing their individual functions in cellular response to stress. This chapter discusses the seminal and more recent discoveries in regards to the pathways responsible for the accumulation of ceramide during stress and the mechanisms by which ceramide affects cell functions. The former group includes the roles of neutral sphingomyelinase 2, serine palmitoyltransferase, ceramide synthases, as well as the secretory and endosomal/lysosomal forms of acid sphingomyelinase. The latter summarizes the mechanisms by which ceramide activate its direct targets, PKCzeta, PP2A and cathepsin D. The ability of ceramide to affect membrane organization is discussed in the light of its relevance to cell signaling. Emerging evidence to support the previously assumed notion that ceramide acts in a strictly structure-specific manner are also included. These findings are described in the context of several physiological and pathophysiological conditions, namely septic shock, obesity-induced insulin resistance, aging and apoptosis of tumor cells in response to radiation and chemotherapy.

Interleukin-1beta (IL-1beta) is a major inducer of liver acute-phase protein expression in response to infection. Several transcription factors, including CCAAT/enhancer binding protein (C/EBP), are known mediators in this process, although the mechanisms by which they modulate IL-1beta's action are not completely understood. Activation of sphingomyelinase (SMase) and the subsequent generation of ceramide are early steps in the IL-1beta signaling cascade. In this study, we investigate the role of ceramide in the IL-1beta regulation of C/EBP in primary hepatocytes. The C/EBP DNA binding activity was found to increase in a dose-dependent manner after stimulation with IL-1beta and exogenous addition of C2-ceramide or treatment with SMase. These changes were accompanied by an increase in the nuclear content of C/EBPbeta. Both IL-1beta and ceramide led to extracellular signal-regulated kinase 1/2 (ERK1/2) activation as early as 15 min after treatment. Furthermore, the increase of cellular ceramide content resulted in increased phosphorylation of C/EBPbeta at serine 105 at later time points. Concurrently, the cytosolic levels of C/EBPbeta decreased, suggesting that IL-1beta and ceramide induced nuclear translocation of C/EBPbeta. Ceramide-induced C/EBPbeta phosphorylation, translocation, and DNA binding were suppressed by the addition of PD98059, an inhibitor of ERK1/2 phosphorylation. These results suggest that ceramide and ERK mediate a pathway in the IL-1beta signaling cascade, which results in rapid posttranslational activation of C/EBPbeta.