Acid sphingomyelinase plays important roles in ceramide homeostasis, which has been proposed to be linked to insulin resistance. To test this association in vivo, acid sphingomyelinase deletion (asm ؊/؊ ) was transferred to mice lacking the low density lipoprotein receptor (ldlr ؊/؊ ), and then offsprings were placed on control or modified (enriched in saturated fat and cholesterol) diets for 10 weeks. The modified diet caused hypercholesterolemia in all genotypes; however, in contrast to asm ؉/؉ /ldlr ؊/؊ , the acid sphingomyelinase-deficient littermates did not display hepatic triacylglyceride accumulation, although sphingomyelin and other sphingolipids were substantially elevated, and the liver was enlarged. asm ؊/؊ /ldlr ؊/؊ mice on a modified diet did not accumulate body fat and were protected against diet-induced hyperglycemia and insulin resistance. Experiments with hepatocytes revealed that acid sphingomyelinase regulates the partitioning of the major fatty acid in the modified diet, palmitate, into two competitive and inversely related pools, triacylglycerides and sphingolipids, apparently via modulation of serine palmitoyltransferase, a rate-limiting enzyme in de novo sphingolipid synthesis. These studies provide evidence that acid sphingomyelinase activity plays an essential role in the regulation of glucose metabolism by regulating the hepatic accumulation of triacylglycerides and sphingolipids during consumption of a diet rich in saturated fats.

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 ␣ (TNF␣) secretion in macrophages. ASMase-deficient (asm ؊/؊ ) mice and isolated peritoneal macrophages produce severalfold more TNF␣ than their wildtype (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 TNF␣-converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNF␣) to an active 17-kDa form (soluble (s)TNF␣). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNF␣ production. A substantial portion of the translated pro-TNF␣ was not processed to sTNF␣; instead, it was rapidly internalized and degraded in the lysosomes. Production of tumor necrosis factor ␣ (TNF␣), 3 the major mediator of the innate immune response, is tightly regulated by transcriptional, post-transcriptional, and post-translational mechanisms. Dysregulation of TNF␣ synthesis and/or turnover has been linked to various disease conditions, including rheumatoid arthritis, sepsis, and cancer (1-3). Lipopolysaccharide (LPS), a component of the bacterial cell wall, is a potent inducer of TNF␣ production and the underlying signaling mechanisms are well understood. LPS binding to its cognitive receptor, MD-2, induces dimerization of the signaling Toll-like receptor-4 (TLR-4) and activation of interleukin-1 receptor-associated kinase-1 (IRAK-1) in a myeloid differentiation factor 88 (MyD88)-dependent manner. Ultimately, the nuclear translocation of nuclear factor B (4, 5), AP-1, Ets, and Elk-1 (6 -8) transcription factors results in multifold induction of TNF␣ mRNA synthesis. LPS also regulates mRNA stability through AU-rich elements at the 3Ј-untranslated region of TNF␣ mRNA (9, 10).In mice, TNF␣ mRNA is translated into a 26-kDa precursor protein (pro-TNF␣), part of which is immediately N-glycosylated (11). pro-TNF␣ is selected as a cargo for the Golgin p230-positive vesicles (12) and transported to the plasma membrane via Rab11 recycling vesicles (13,14). pro-TNF␣ is integrated in the plasma membrane as a type II membrane protein (15); its ectodomain is cleaved by TNF␣-converting enzyme (TACE) (16) and released as a biologically active 17-kDa soluble form (sTNF␣). TACE is a member of the a disintegrin and metalloproteinase family of proteases. In addition to TNF␣, TACE also processes the two TNF␣ receptors (p55 and p75), transforming growth factor ␣, L-selectin, and other secretory proteins (17)(18)(19)(20).The regulation of TNF␣ mRNA transcription has been extensively studied, and it is well understood. In contrast, evidence for a regulatory role of TACE has emerged only recently. It was reported that a substantial portion of pro-TNF␣ is not immediately processed by TACE but is rapidly internalized a...

AimsSphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production.ResultsWe demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown.InnovationThese findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production.ConclusionOur data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity.

Aims Chronic heart failure (CHF) causes inspiratory (diaphragm) muscle weakness and fatigue that contributes to dyspnoea and limited physical capacity in patients. However, the mechanisms that lead to diaphragm dysfunction in CHF remain poorly understood. Cytokines and angiotensin II are elevated in CHF and stimulate the activity of the enzyme sphingomyelinase (SMase) and accumulation of its reaction product ceramide. In the diaphragm, SMase or ceramide exposure in vitro causes weakness and fatigue. Thus, elevated SMase activity and ceramide content have been proposed as mediators of diaphragm dysfunction in CHF. In the present study, we tested the hypotheses that diaphragm dysfunction was accompanied by increases in diaphragm SMase activity and ceramide content. Methods and results We used myocardial infarction to induce CHF in rats. We measured diaphragm isometric force, SMase activity by high-performance liquid chromatography, and ceramide subspecies and total ceramide using mass spectrometry. CHF depressed diaphragm force and accelerated fatigue. Diaphragm neutral SMase activity was increased by 20% in CHF, while acid SMase activity was unchanged. We also found that CHF increased the content of C18-, C20, and C24-ceramide subspecies and total ceramide. Downstream of ceramide degradation, diaphragm sphingosine was unchanged, and sphingosine-1-phosphate (S1P) was increased in CHF. Conclusion Our major novel finding was that diaphragm dysfunction in CHF rats was accompanied by higher diaphragm neutral SMase activity, which is expected to cause the observed increase in diaphragm ceramide content.

The propensity of LDLs (low-density lipoproteins) for aggregation and/or oxidation has been linked to their sphingolipid content, specifically the levels of SM (sphingomyelin) and ceramide. To investigate this association in vivo, ldlr (LDL receptor)-null mice (ldlr−/−) were fed on a modified (atherogenic) diet containing saturated fats and cholesterol. The diet led to significantly elevated SM content in all serum lipoproteins. In contrast, ceramide increased only in the LDL particles. MS-based analyses of the lipid acyl chain composition revealed a marked elevation in C16:0 fatty acid in SM and ceramide, consistent with the prevalence of palmitic acid in the modified diet. The diet also led to increased activity of the S-SMase [secretory SMase (sphingomyelinase)], a protein that is generated by ASMase (acid SMase) and acts on serum LDL. An increased macrophage secretion seemed to be responsible for the elevated S-SMase activity. ASMase-deficient mice (asm−/−/ldlr−/−) lacked S-SMase activity and were protected from diet-induced elevation in LDL ceramide. LDL from asm−/−/ldlr−/− mice fed on the modified diet were less aggregated and oxidized than LDL from asm+/+/ldlr−/− mice. When tested in vitro, the propensity for aggregation was dependent on the SM level: only LDL from animals on modified diet that have high SM content aggregated when treated with recombinant S-SMase. In conclusion, LDL-SM content and S-SMase activity are up-regulated in mice fed on an atherogenic diet. S-SMase mediates diet-induced changes in LDL ceramide content and aggregation. S-SMase effectiveness in inducing aggregation is dependent on diet-induced enrichment of LDL with SM, possibly through increased hepatic synthesis.