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Eukaryotic organisms as well as some prokaryotes and viruses contain sphingolipids, which are defined by a common structural feature, i.e. , a "sphingoid base" backbone such as D-erythro-1,3-dihydroxy, 2-aminooctadec-4-ene (sphingosine). The sphingolipids of mammalian tissues, lipoproteins, and milk include ceramides, sphingomyelins, cerebrosides, gangliosides and sulfatides; plants, fungi and yeast have mainly cerebrosides and phosphoinositides. The total amounts of sphingolipids in food vary considerably, from a few micromoles per kilogram (fruits) to several millimoles per kilogram in rich sources such as dairy products, eggs and soybeans. With the use of the limited data available, per capita sphingolipid consumption in the United States can be estimated to be on the order of 150-180 mmol (approximately 115-140 g) per year, or 0.3-0.4 g/d. There is no known nutritional requirement for sphingolipids; nonetheless, they are hydrolyzed throughout the gastrointestinal tract to the same categories of metabolites (ceramides and sphingoid bases) that are used by cells to regulate growth, differentiation, apoptosis and other cellular functions. Studies with experimental animals have shown that feeding sphingolipids inhibits colon carcinogenesis, reduces serum LDL cholesterol and elevates HDL, suggesting that sphingolipids represent a "functional" constituent of food. Sphingolipid metabolism can also be modified by constituents of the diet, such as cholesterol, fatty acids and mycotoxins (fumonisins), with consequences for cell regulation and disease. Additional associations among diet, sphingolipids and health are certain to emerge as more is learned about these compounds. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are shed from the plasma membrane, but the regulation and function of these EVs remain unclear. We found that oxidative stress induced by H 2 O 2 in Hela cells stimulated filopodia formation and the secretion of EVs. EVs were small (150 nm) and labeled for CD44, indicating that they were derived from filopodia. Filopodia-derived small EVs (sEVs) were enriched with the sphingolipid ceramide, consistent with increased ceramide in the plasma membrane of filopodia. Ceramide was colocalized with neutral sphingomyelinase 2 (nSMase2) and acid sphingomyelinase (ASM), two sphingomyelinases generating ceramide at the plasma membrane. Inhibition of nSMase2 and ASM prevented oxidative stress-induced sEV shedding but only nSMase2 inhibition prevented filopodia formation. nSMase2 was S-palmitoylated and interacted with ASM in filopodia to generate ceramide for sEV shedding. sEVs contained nSMase2 and ASM and decreased the level of these two enzymes in oxidatively stressed Hela cells. A novel metabolic labeling technique for EVs showed that oxidative stress induced secretion of fluorescent sEVs labeled with NBD-ceramide. NBD-ceramide-labeled sEVs transported ceramide to mitochondria, ultimately inducing cell death in a proportion of neuronal (N2a) cells. In conclusion, using Hela cells we provide evidence that oxidative stress induces interaction of nSMase2 and ASM at filopodia, which leads to shedding of ceramide-rich sEVs that target mitochondria and propagate cell death., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

We have shown that deficiency of neutral sphingomyelinase 2 (nSMase2), an enzyme generating the sphingolipid ceramide, improves memory in adult mice. Here, we performed sphingolipid and RNA-seq analyses on the cortex from 10-month-old nSMase2-deficient (fro/fro) and heterozygous (+ /fro) mice. fro/fro cortex showed reduced levels of ceramide, particularly in astrocytes. Differentially abundant transcripts included several functionally related groups, with decreases in mitochondrial oxidative phosphorylation and astrocyte activation transcripts, while axon guidance and synaptic transmission and plasticity transcripts were increased, indicating a role of nSMase2 in oxidative stress, astrocyte activation, and cognition. Experimentally induced oxidative stress decreased the level of glutathione (GSH), an endogenous inhibitor of nSMase2, and increased immunolabeling for ceramide in primary + /fro astrocytes, but not in fro/fro astrocytes. β-galactosidase activity was lower in 5-week-old fro/fro astrocytes, indicating delayed senescence due to nSMase2 deficiency. In fro/fro cortex, levels of the senescence markers C3b and p27 and the proinflammatory cytokines interleukin 1β, interleukin 6, and tumor necrosis factor α were reduced, concurrent with twofold decreased phosphorylation of their downstream target, protein kinase Stat3. RNA and protein levels of the ionotropic glutamate receptor subunit 2B (Grin2b/NR2B) were increased by twofold, which was previously shown to enhance cognition. This was consistent with threefold reduced levels of exosomes carrying miR-223-3p, a micro-RNA downregulating NR2B. In summary, our data show that nSMase2 deficiency prevents oxidative stress-induced elevation of ceramide and secretion of exosomes by astrocytes that suppress neuronal function, indicating a role of nSMase2 in the regulation of neuroinflammation and cognition., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Prostate apoptosis response-4 (Par-4) is a tumor suppressor that induces apoptosis in cancer cells. However, the physiological function of Par-4 remains unknown. Here we show that conventional Par-4 knockout (Par-4 -/- ) mice and adipocyte-specific Par-4 knockout (AKO) mice, but not hepatocyte-specific Par-4 knockout mice, are obese with standard chow diet. Par-4 -/- and AKO mice exhibit increased absorption and storage of fat in adipocytes. Mechanistically, Par-4 loss is associated with mdm2 downregulation and activation of p53. We identified complement factor c3 as a p53-regulated gene linked to fat storage in adipocytes. Par-4 re-expression in adipocytes or c3 deletion reversed the obese mouse phenotype. Moreover, obese human subjects showed lower expression of Par-4 relative to lean subjects, and in longitudinal studies, low baseline Par-4 levels denoted an increased risk of developing obesity later in life. These findings indicate that Par-4 suppresses p53 and its target c3 to regulate obesity., Competing Interests: Authors TS-B and BT are employed by Loxo Oncology. VR is owner of a start-up company Parcure, LLC, in Lexington, KY, USA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Araujo, Sledziona, Noothi, Burikhanov, Hebbar, Ganguly, Shrestha-Bhattarai, Zhu, Katz, Zhang, Taylor, Liu, Chen, Weiss, He, Wang, Morris, Cassis, Nikolova-Karakashian, Nagareddy, Melander, Evers, Kern and Rangnekar.)

We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hypertrophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two- to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16- and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphingosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth.

Ceramide and diacylglycerol (DAG) are bioactive lipids and mediate many cellular signaling pathways. Sphingomyelin synthase (SMS) is the single metabolic link between the two, while SMS2 is the only SMS form located at the plasma membrane. SMS2 functions were investigated in HepG2 cell lines stably expressing SMS2. SMS2 overexpression did not alter sphingomyelin (SM), phosphatidylcholine (PC), or ceramide levels. DAG content increased by approx. 40% and led to downregulation of DAG-dependent protein kinase C (PKC). SMS2 overexpression also induced senescence, characterized by positivity for β-galactosidase activity and heterochromatin foci. HepG2-SMS2 cells exhibited protruded mitochondria and suppressed mitochondrial respiration rates. ATP production and the abundance of Complex V were substantially lower in HepG2-SMS2 cells as compared to controls. SMS2 overexpression was associated with inflammasome activation based on increases in IL-1β and nlpr3 mRNA levels. HepG2-SMS2 cells exhibited lipid droplet accumulation, constitutive activation of AMPK based on elevated 172 Thr phosphorylation, increased AMPK abundance, and insensitivity to insulin suppression of AMPK. Thus, our results show that SMS2 regulates DAG homeostasis and signaling in hepatocytes and also provide proof of principle for the concept that offset in bioactive lipids' production at the plasma membrane can drive the senescence program in association with steatosis and, seemingly, by cell-autonomous mechanisms.

This chapter will discuss methods for analyses of the rates of sphingomyelin synthesis and turnover associated with lipid rafts or plasma membrane. These methods involve the use of fluorescently (NBD-C6-ceramide or NBD-C6-Sphingomyelin)) or radioactively labeled substrates ([ 3 H-methyl]-phosphatidylcholine, [ 3 H-acyl]-ceramide, [ 14 C-methyl]-sphingomyelin) to quantify in vitro the activity of the sphingomyelin synthase (SMS) (also known as phosphatidylcholine:ceramide phosphocholine transferase), acid sphingomyelinase (the endosomal/lysosomal (L-SMase) and the secretory (S-SMase) forms) and neutral sphingomyelinase-2 (nSMase-2). These methods allow to quantify changes in the activity of enzymes that affect the SM-to-ceramide ratio on the plasma membrane, and consequently, the lipid rafts biophysical properties, dynamics, and raft-associated receptor clustering and signaling events. Specific attention is paid to challenges caused by the fact that SMS and nSMase-2 are integral/membrane bound proteins and how to avoid the use of detergent that suppress their specific activities.

Background: Epidemiological studies suggest that higher fruits and vegetables (F&V) consumption correlates with reduced risk of hepatic steatosis, yet evidence for causality and the underlying mechanisms is lacking., Objectives: We aimed to determine the causal relation between F&V consumption and improved metabolic disorders in mice fed high-fat (HF) (Experiment-1) or normal-fat (Experiment-2) diets and its underlying mechanisms., Methods: Six-week-old male C57BL/6J mice were randomly grouped and fed diets supplemented at 0%-15% (wt:wt) with a freeze-dried powder composed of 24 commonly consumed F&V (human equivalent of 0-9 servings/d) for 20 wk. In Experiment-1, mice were fed an HF (45% kcal fat) diet with 0% (HF0), 5%, 10%, or 15% (HF15) F&V or a matched low-fat control diet (10% kcal fat). In Experiment-2, mice were fed an AIN-93 diet (basal) (B, 16% kcal fat) with 0% (B0), 5%, 10%, or 15% (B15) F&V supplementation. Body weight and composition, food intake, hepatic steatosis, inflammation, ceramide levels, sphingomyelinase activity, and gut microbiota were assessed., Results: In Experiment-1, mice fed the HF15 diet had lower weight gain (17.9%), hepatic steatosis (48.4%), adipose tissue inflammation, blood (24.6%) and liver (33.9%) ceramide concentrations, and sphingomyelinase activity (38.8%) than HF0 mice (P < 0.05 for all). In Experiment-2, mice fed the B15 diet had no significant changes in weight gain but showed less hepatic steatosis (28.5%), blood and adipose tissue inflammation, and lower blood (30.0%) ceramide concentrations than B0 mice (P < 0.05 for all). These F&V effects were associated with favorable microbiota changes., Conclusions: These findings represent the first evidence for a causal role of high F&V intake in mitigating hepatic steatosis in mice. These beneficial effects may be mediated through changes in ceramide and/or gut microbiota, and suggest that higher than currently recommended servings of F&V may be needed to achieve maximum health benefits., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Sphingolipids are class of metabolically distinct lipids that play structural and signaling functions in all organisms. Sphingolipid metabolism is deregulated during various diseases such as cancer, neurological and immune disorders, and metabolic syndrome. With the advancement of sphingo-lipidomics and sphingo-genomics, an understanding of the specific roles of ceramide, the quintessential bioactive sphingolipid, in fatty liver disease has taken shape. Two major pathways for ceramide generation, the de novo pathway and the sphingomyelinase pathway are activated in the course of both, the non-alcoholic and the alcoholic, forms of fatty liver disease. The mechanisms of activation of these two pathways are distinct and reflect the different disease etiology in each case; at the same time, common processes impacted by the resulting ceramide overproduction involve lipotoxocity, ER/mitochondrial stress, inflammation, and de-regulation of hepatic lipid metabolism. Studies in human patients and animal models have delineated specific enzymes and ceramide species that are involved at the different stages of the disease, and represent novel pharmaceutical targets for successful management of fatty liver disease., (Copyright © 2018. Published by Elsevier Ltd.)

The goals of this study were to determine if secretory sphingomyelinase (S-SMase) activity is elevated in patients with rheumatoid arthritis (RA) compared to control subjects and to examine the relationships of S-SMase activity with functional status, quality of life, and RA disease activity measurements. We collected data on 33 patients who were diagnosed with RA and 17 non-RA controls who were comparable in terms of age, sex, and race. Demographic, clinical data and self-reported measures of fatigue, pain, and physical function were obtained directly from patients and controls. RA patients also completed quantitative joint assessment using a 28-joint count and functional status and quality of life assessment using the Modified Health Assessment Questionnaire (MHAQ). Archived serum samples were used to analyze retrospectively serum S-SMase activity in patients and controls. The mean serum S-SMase activity was 1.4-fold higher in patients with RA (RA 2.8 ± 1.0 nmol/ml/h vs. controls 2.0 ± 0.8 nmol/ml/h; p = 0.014). Spearman's rho correlations between S-SMase activity and oxidant activity, markers of inflammation and endothelial activation with the exception of P-selectin (rho = 0.40, p = 0.034), measures of disease activity, functional status, and quality of life were not statistically significant in patients with RA. We confirmed that S-SMase activity is higher among RA patients compared to controls, as in other acute and chronic inflammatory diseases. Future studies can build on the present findings to understand more fully the biologic role(s) of S-SMase activity in RA.

Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient ( fro/fro ) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C 16 -ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Nonalcoholic fatty liver disease (NAFLD) is a group of liver disorders encompassing simple hepatic steatosis and its more aggressive forms of nonalcoholic steatohepatitis and cirrhosis. It is a rapidly growing health concern and the major cause for the increasing incidence of primary liver tumors. Unequivocal evidence shows that sphingolipid metabolism is altered in the course of the disease and these changes might contribute to NAFLD progression. Recent data provide solid support to the notion that deregulated ceramide and sphingosine-1-phosphate metabolism are present at all stages of NAFLD, i.e., steatosis, nonalcoholic steatohepatitis, advanced fibrosis, and hepatocellular carcinoma (HCC). Insulin sensitivity, de novo lipogenesis, and the resulting lipotoxicity, fibrosis, and angiogenesis are all seemingly regulated in a manner that involves either ceramide and/or sphingosine-1-phosphate. Sphingolipids might also participate in the onset of hepatocellular senescence. The latter has been shown to contribute to the advancement of cirrhosis to HCC in the classical cases of end-stage liver disease, i.e., viral- or alcohol-induced; however, emerging evidence suggests that senescence is also involved in the pathogenicity of NAFLD possibly via changes in ceramide metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The level of insulin-like growth factor-binding protein 1 (IGFBP1), a liver-produced serum protein that regulates insulin-like growth factor-I bioactivity, glucose homeostasis, and tissue regeneration, increases during inflammation. This manuscript describes a novel pathway for the regulation of hepatic IGFBP1 mRNA and protein levels by interleukin (IL)-1β. Experiments with the luciferase reporter system show that IL-1β stimulates transcriptional activity from the 1-kb promoter region of IGFBP1. Although IL-1β stimulation suppresses the insulin activation of protein kinase B, the major upstream regulator of IGFBP1 mRNA transcription, the induction of IGFBP1 by IL-1β did not require an intact insulin response element. Furthermore, neither overexpression nor silencing of FoxO-1 had any effect on the IL-1β-induced increase in IGFBP1 mRNA levels and promoter activity. However, inhibition of the ERK MAP kinases effectively prevented the IL-1β effects. Inhibition of neutral sphingomyelinase, a key player in the IL-1β signaling cascade that acts upstream of ERK, also suppressed the IL-1β effects, while increasing the ceramide, through the addition of C2-ceramide or via treatment with exogenous sphingomyelinase, was sufficient to induce IGFBP1 promoter-driven luciferase activity. Studies in primary rat hepatocytes where the levels of neutral sphingomyelinase were either elevated or suppressed using adenoviral constructs affirmed the key role of neutral sphingomyelinase and ceramide (exerted likely through ERK activation) in the IL-1β-induced IGFBP1 production. Finally, the IL-1β effects on IGFBP1 mRNA production and protein secretion could be abolished by the addition of insulin, either at very late time points or at very high doses., (Copyright © 2016 the American Physiological Society.)

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: Myocardial infarction was used 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. Diaphragm force was depressed and fatigue accelerated by CHF. 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 level 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., (© 2014 The Authors. European Journal of Heart Failure © 2014 European Society of Cardiology.)

Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resveratrol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demonstrated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apoptosis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading to increase of SM, the latter acting as a native membrane antioxidant. In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve the functional activity of the membrane lipids in the aged liver by influencing specific membrane parameters, associated with the aging process., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

Sphingomyelinase (SMase) hydrolyzes membrane sphingomyelin into ceramide, which increases oxidants in nonmuscle cells. Serum SMase activity is elevated in sepsis and heart failure, conditions where muscle oxidants are increased, maximal muscle force is diminished, and fatigue is accelerated. We tested the hypotheses that exogenous SMase and accumulation of ceramide in muscle increases oxidants in muscle cells, depresses specific force of unfatigued muscle, and accelerates the fatigue process. We also anticipated that the antioxidant N-acetylcysteine (NAC) would prevent SMase effects on muscle function. We studied the responses of C2C12 myotubes and mouse diaphragm to SMase treatment in vitro. We observed that SMase caused a 2.8-fold increase in total ceramide levels in myotubes. Exogenous ceramide and SMase elevated oxidant activity in C2C12 myotubes by 15-35% (P < 0.05) and in diaphragm muscle fiber bundles by 58-120% (P < 0.05). The SMase-induced increase in diaphragm oxidant activity was prevented by NAC. Exogenous ceramide depressed diaphragm force by 55% (P < 0.05), while SMase depressed maximal force by 30% (P < 0.05) and accelerated fatigue--effects opposed by treatment with NAC. In conclusion, our findings suggest that SMase stimulates a ceramide-oxidant signaling pathway that results in muscle weakness and fatigue.

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.

Aging is characterized by changes in the organism's immune functions and stress response, which in the elderly leads to increased incidence of complications and mortality following inflammatory stress. Alterations in the neuro-endocrine axes and overall decline in the immune system play an essential role in this process. Overwhelming evidence however suggests that many cellular cytokine signaling pathways are also affected, thus underscoring the idea that both, "cellular" and "systemic" changes contribute to aging. IL-1beta for example, induces more potent cellular responses in hepatocytes isolated from aged animals then in hepatocytes from young rats. This phenomenon is referred to as IL-1b hyperresponsiveness and is linked to abnormal regulation of various acute phase proteins during aging.Evidence has consistently indicated that activation of neutral sphingomyelinase and the resulting accumulation of ceramide mediate cellular responses to LPS, IL-1beta, and TNFalpha in young animals. More recent studies identified the cytokine-inducible neutral sphingomyelinase with nSMase2 (smpd3) that is localized in the plasma membrane and mediates cellular responses to IL-1beta and TNFalpha. Intriguingly, constitutive up-regulation of nSMase2 occurs in aging and it underlies the hepatic IL-1b hyperresponsiveness. The increased activity of nSMases2 in aging is caused by a substantial decline in hepatic GSH content linking thereby oxidative stress to the onset of pro-inflammatory state in liver. nSMase2 apparently follows a pattern of regulation consisting with "developmental-aging" continuum, since in animal models of delayed aging, like calorie-restricted animals, the aging-associated changes in NSMase activity and function are reversed.

Nitric oxide (NO)-induced oxidative stress contributes to a variety of diseases. Although numerous mechanisms have been described controlling the production of NO, the mechanisms to prevent NO-induced cytotoxicity after NO synthesis are largely unknown. Here we report that scavenger receptor BI (SR-BI) prevents NO-induced cytotoxicity. Using CHO cell lines expressing wild-type and single-site mutant SR-BI protein, we demonstrate that SR-BI prevents NO-induced cytotoxicity and that a highly conserved CXXS redox motif is required for the anti-NO cytotoxicity activity of SR-BI. Using genetically manipulated mice, we demonstrate that SR-BI-null mice have a 3- to 4-fold increase in tyrosine nitrated proteins in aorta and liver compared with wild-type littermates, indicating that expression of SR-BI prevents peroxynitrite formation in vivo. Using lipopolysacharide (LPS)-challenged mice as an in vivo model of NO-induced cytotoxicity, we found that a single dose of LPS (120,000 U/g IP) induced 90% fatality of SR-BI-null mice within 3 days, whereas all of the wild-type littermates survived (n=20), demonstrating that SR-BI is highly protective against NO cytotoxicity in vivo. Importantly, SR-BI prevents LPS-induced death without eliminating NO production, suggesting that SR-BI prevents NO-induced cytotoxicity post-NO synthesis. Our study describes a novel observation that may shed new light on the treatment of nitric oxidative stress-associated diseases, such as septic shock.

Spatially fractionated high dose (grid) radiation treatment (SFGRT) involves irradiation of bulky tumors with one high, grid-delivered, dose of 15 Gy followed by multiple consecutive doses of 2 Gy each. The goal of this study is to determine the effect of this treatment on serum ceramide content and to investigate possible involvement of ceramide in tumor regression after SFGRT. Serum ceramide and Secretory SMase (S-SMase) were quantified in 11 patients before and at 24, 48 and 72 h after the dose of 15 Gy. Furthermore, LDL particles were isolated from the serum and their apoptotic ability was tested in human endothelial cells by TUNEL assay. Sixty seven per cent (6/8) of the patients with partial (PR) or complete (CR) response showed statistically significant increase in serum ceramide levels. Of the nonresponders in the study, none showed an elevation in ceramide. S-SMase activity underwent similar changes. LDL particles from serum of patients collected 72 hours after SFGRT sensitized the endothelial cells to undergo apoptosis in response to 5 Gy radiation that by itself had only modest effect on cell death. Independent elevation of ceramide content of endothelial cells that were otherwise resistant to radiation-induced cell death also was sufficient to sensitize these cells to apoptosis. Serum S-SMase activity and ceramide content increase following SFGRT and correlate with the clinical response. Apparently, these changes are in the LDL-associated ceramide and may contribute to better tumor reduction after SFGRT, due to the ability of LDL-derived ceramide to sensitize endothelial cells for apoptosis.

Recent studies have indicated that ceramide generated in the liver is secreted into the bloodstream as component of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). This manuscript investigates the effect of host acute phase response to inflammation on lipoprotein ceramide levels. In humans, two different patterns of responses were found. One group of volunteers experienced transient increases in serum ceramide at 1.5h after LPS administration. Second group showed prolonged increases that reached up to 10-fold above the basal level and continued for up to 24h. Increases in ceramide were found only in VLDL and LDL particles. LPS administration induced similar increases in mice. These increases were accompanied by activation of secreted sphingomyelinase in serum and serine-palmitoyl transferase in liver. ASMase knockout mice retained LPS-induced increases in serum ceramide, thus suggesting that the elevation of VLDL and LDL ceramide content is attributed at least in part to activation of de novo synthesis of ceramide in the liver.

Ceramide is a bioactive molecule involved in cellular responses to stress and inflammation. The major pathway for ceramide accumulation is via agonist-induced activation of cellular sphingomyelinases. It has also been shown that the ceramide level in circulating low density lipoprotein (LDL) increases during systemic inflammation, hence it is of importance to understand whether LDL-derived ceramide also contributes to the cellular ceramide homeostasis and affects cell functions. This article provides evidence of uptake of ceramide-enriched LDL by human microvascular endothelial cells in a receptor-mediated fashion. This uptake can be competed by native LDL, indicating that the LDL-binding receptor may be involved. Following uptake, part of the LDL-derived ceramide is converted to sphingosine, but more importantly, part of it accumulates inside the cells (approximately 1.44 nmol/mg of cell protein). This accumulation of ceramide correlates with an increased incidence of apoptosis. The addition of tumor necrosis factor-alpha further enhances the accumulation of LDL-derived ceramide and the rate of apoptosis. In contrast, inhibitors of receptor-mediated endocytosis block both, the accumulation of LDL-derived ceramide and the concurrent increase in apoptosis. We also show that LDL-delivered ceramide is a more efficient inducer of apoptosis as compared with ethanol-delivered ceramide, the same apoptotic effect being achieved by substantially smaller increases in intracellular ceramide. Taken together, the presented data indicate that increases in lipoprotein ceramide concentration may result in changes in the bioactive properties of circulating lipoproteins such as the enhanced ability to induce apoptosis in the microvascular endothelium.

The purpose of this study was to further characterize Fas-mediated liver apoptosis. We investigated whether Fas-mediated apoptosis in the liver requires induction of apoptosis-related regulators and whether Kupffer cells play a role in this process. Mice were injected with GdCl(3) to deplete/suppress Kupffer cells, followed by treatment with an anti-Fas agonistic antibody, Jo2. Hepatic mRNA levels of several pro- and anti-apoptotic regulators were determined 0.5, 1.5 and 4.0 h after Jo2 injection. Liver histology, TUNEL response, the activity of caspases-3, -8, and -9, and reduced and oxidized glutathione, were also evaluated. Jo2 dramatically increased the number of apoptotic nuclei in the liver, up-regulated mRNA for Bcl-w, Bfl-1, and Bcl-X(L,) but did not affect pro-apoptotic regulator mRNA expression. Caspase-3, -8 and -9 activity increased at 1.5 h after Jo2-injection. GdCl(3) treatment was associated with an increase in the apoptotic effect of Jo2. No effect of Jo2 was recorded on redox state of the free cellular thiol system. These data suggest that: (1) the prompt apoptotic response to Fas-mediated signaling in the liver does not require induction of pro-apoptotic factors; (2) Kupffer cells may play a major role in the liver apoptotic response to Fas ligation by clearing apoptotic cells by phagocytosis; (3) oxidative stress does not seem to play an important role in Fas-mediated liver apoptosis.

Previously, we showed that macrophages (MØ) from old mice have significantly higher levels of lipopolysaccharide (LPS)-induced prostaglandin E(2) (PGE(2)) production than young mice, due to increased cyclooxygenase-2 (COX-2) mRNA levels. The aim of the current study was to determine the underlying mechanisms of age-associated increase in COX-2 gene expression. The results demonstrate that increased COX-2 mRNA expression in the old mice is due to a higher rate of transcription rather than increased stability of COX-2 mRNA. Furthermore, the results show that LPS-induced ceramide levels from the old mice are significantly higher than those of young mice, whereas there is no age-related difference in concentration of its down stream metabolite, sphingosine. The addition of ceramide in the presence or absence of LPS resulted in a significant increase in PGE(2) production in a dose- and time-dependent manner. Inhibition of ceramide conversion to sphingosine had no effect on this ceramide-induced effect. The ceramide-induced up-regulation in PGE(2) production was mediated through increase in COX activity and transcriptional up-regulation of COX-2 mRNA. Collectively, these data suggest that the age-associated increase in MØ COX-2 mRNA is due to transcriptional up-regulation. Furthermore, this increase in transcription is mediated by higher cellular ceramide concentration in old MØ compared with that of young MØ.

Recently it has been demonstrated that high density lipoprotein (HDL) binding to scavenger receptors, class B, type I (SR-BI) stimulates endothelial nitric-oxide synthase (eNOS) activity. In the present studies we used a Chinese hamster ovary cell system and a human microvascular endothelial cell line to confirm that HDL stimulates eNOS activity in a SR-BI-dependent manner. Importantly, we have extended these studies to examine the mechanism whereby HDL binding to SR-BI stimulates eNOS. eNOS can be stimulated by an increase in intracellular calcium, by phosphorylation by Akt kinase, or by an increase in intracellular ceramide. Calcium imagining studies and experiments with the calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester demonstrated that HDL binding to SR-BI does not induce an increase in intracellular calcium. Antibodies specific for activated Akt kinase demonstrated that HDL binding to SR-BI does not induce Akt kinase activation. However, HDL binding to SR-BI caused a reversible increase in intracellular ceramide levels from 97 +/- 14 pmol/mg of protein to 501 +/- 21 pmol/mg of protein. In addition, C(2)-ceramide stimulated eNOS to the same extent as HDL, whereas C(2)-dihydroceramide did not stimulate eNOS. We conclude that HDL binding to SR-BI stimulates eNOS by increasing intracellular ceramide levels and is independent of an increase in intracellular calcium or Akt kinase phosphorylation.

Ceramide, which is an integral component of the sphingomyelin signaling pathway, can attenuate voltage-gated Ca(2+) channel (VGCC) activity in a number of cell types. The aim of the present study was to determine whether ceramide can also modulate VGCC activity, and as a consequence nicotinic receptor-dependent Ca(2+) signaling and catecholamine secretion, in rat adrenal chromaffin cells. Short-term C(6)-ceramide (CER) treatment dose-dependently inhibited nicotine (NIC)-induced peak intracellular Ca(2+) transients. Sphingomyelinase elicited similar responses, whereas the inactive ceramide analog C(2)-dihydroceramide had no effect on NIC-induced Ca(2+) transients. CER suppressed KCl- and NIC-induced Ca(2+) transients to a similar extent, suggesting that the voltage-gated Ca(2+) channel was a primary site of inhibition. In direct support of this concept, whole-cell patch-clamp analysis demonstrated that CER and sphingomyelinase significantly reduced peak Ca(2+) currents. Pretreatment with staurosporine significantly attenuated CER-dependent inhibition of both NIC-induced Ca(2+) transients and peak Ca(2+) current, suggesting that the effects of CER are mediated at least in part by protein kinase C. Consistent with suppressed Ca(2+) signaling, CER also significantly inhibited NIC-induced catecholamine secretion measured at the single-cell level by carbon fiber amperometry. This effect of CER was also significantly attenuated by pretreatment with staurosporine These data demonstrate that the sphingomyelin signaling pathway can modulate nicotinic receptor-dependent Ca(2+) signaling and catecholamine secretion in rat chromaffin cells., (Copyright 2001 Wiley-Liss, Inc.)

Aging leads to a decreased ability of liver to metabolize drugs and increased expression and secretion of acute phase proteins, such as serum amyloid A (SAA), C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP). This phenomenon resembles some aspects of the acute phase response of host to inflammation; however, the molecular basis for the similarity is unclear. Ceramide and sphingosine are second messenger mediators of cellular responses to stress and inflammation. In liver, they play important role in mediating acute phase responses to IL1-beta. In this study, we use HPLC and thin layer chromatography to evaluate the effects of aging on steady-state levels of ceramide and sphingosine. We report that both lipids are elevated in liver of old (24 months) as compared to young (5 months) male Fisher 344 rats. To elucidate the mechanism(s) for ceramide elevation, we test the acidic (ASMase) and neutral sphingomyelinase (NSMase) in vitro using NBD-sphingomyelin as an exogenous substrate. SM synthase is also analyzed in vitro using NBD-ceramide and [3H]-dipalmitoylphosphatidylcholine (DPPC) as exogenous substrates. In accordance with the increases in the mass of ceramide, the activity of acid and neutral SMase is elevated in old animals. Michaelis-Menten analysis of NSMase implies that the apparent activation of this enzyme is caused by an increase in the Vmax of the enzyme. In contrast, SM synthase activity is lower in old animals as compared to young ones. These results show that aging is accompanied by an elevation in SM turnover and a decrease in its synthesis, resulting in accumulation of pro-inflammatory and growth inhibitory second messenger ceramide. Ceramidase, the only enzyme leading to sphingosine generation, is also measured in vitro using NBD-ceramide as a substrate and liver homogenate as an enzyme source. Its activity is higher in the old rats, as compared to young ones. The acid and neutral forms of the enzyme are affected the most, while the changes in the alkaline enzyme are not significant. The increases in the basal levels of ceramide and sphingosine in old animals may contribute to the onset of an inflammatory like state in liver during aging, exemplified by decreased P4502C11 mRNA expression and chronic induction of acute phase protein expression.

Stroke is a major cause of long-term disability, the severity of which is directly related to the numbers of neurons that succumb to the ischemic insult. The signaling cascades activated by cerebral ischemia that may either promote or protect against neuronal death are not well understood. One injury-responsive signaling pathway that has recently been characterized in studies of non-neural cells involves cleavage of membrane sphingomyelin by acidic and/or neutral sphingomyelinase (ASMase) resulting in generation of the second messenger ceramide. We now report that transient focal cerebral ischemia induces large increases in ASMase activity, ceramide levels, and production of inflammatory cytokines in wild-type mice, but not in mice lacking ASMase. The extent of brain tissue damage is decreased and behavioral outcome improved in mice lacking ASMase. Neurons lacking ASMase exhibit decreased vulnerability to excitotoxicity and hypoxia, which is associated with decreased levels of intracellular calcium and oxyradicals. Treatment of mice with a drug that inhibits ASMase activity and ceramide production reduces ischemic neuronal injury and improves behavioral outcome, suggesting that drugs that inhibit this signaling pathway may prove beneficial in stroke patients.

Background: The role of apoptosis in EtOH-induced liver injury has not been investigated much. Therefore, the question whether apoptosis is a contributory factor to alcoholic liver disease remains to be answered. The purpose of this study was to characterize the liver apoptotic response in a murine model of alcohol-enhanced lipopolysaccharide (LPS) hepatotoxicity., Methods: Mice were fed an alcohol-containing liquid diet for 49 days followed by an acute LPS challenge. The liver state was judged on the basis of histological appearance, plasma liver enzyme activity (alanine:2-oxoglutarate and aspartate:2-oxoglutarate aminotransferases, as markers of hepatocytolysis), and plasma hyaluronan levels (as a marker of the sinusoidal endothelial cell scavenging function). The liver apoptotic response was assessed by DNA fragmentation (TUNEL procedure), and caspases-3 and -8 activity. To determine if ceramide played a role in the liver apoptotic response, the activity of acidic sphingomyelinase and tissue content of ceramide were also quantified., Results: Alcohol exposure induced fat accumulation and sensitized the liver to LPS injurious effects. Plasma liver enzyme activity was elevated by alcohol and this effect was potentiated by LPS. Liver apoptosis was augmented by both alcohol and LPS treatment as reflected by high frequency of positive TUNEL staining nuclei and by an increased activity of caspase-3 and -8. Acidic sphingomyelinase activity was also increased and it was associated with an elevated tissue content of ceramide. In addition, LPS also increased plasma TNF-alpha levels. These changes were accompanied by elevated plasma hyaluronan, reflecting an impaired sinusoidal endothelial cell scavenging function., Conclusions: These results provide a more complete description of the liver apoptotic response to both alcohol and LPS and may constitute the basis for further mechanistic studies on a possible role apoptosis may play in alcoholic liver injury.

Sphingolipids modulate many aspects of cell function, including the expression of cytochrome P450, a superfamily of heme proteins that participate in the oxidation of a wide range of compounds of both endogenous (steroid hormones and other lipids) and exogenous (e.g. alcohol, drugs and environmental pollutants) origin. Cytochrome P450-2C11 (CYP 2C11) is down-regulated in response to interleukin-1beta (IL-1beta), and this response involves the hydrolysis of sphingomyelin to ceramide as well as ceramide to sphingosine, and phosphorylation of sphingosine to sphingosine 1-phosphate. Activation of ceramidase(s) are a key determinant of which bioactive sphingolipid metabolites are formed in response to IL-1beta. Ceramidase activation also appears to account for the loss of expression of CYP 2C11 when hepatocytes are placed in cell culture, and the restoration of expression when they are plated on Matrigel; hence, this pathway is influenced by, and may mediate, interactions between hepatocytes and the extracellular matrix. Recent studies using inhibitors of sphingolipid metabolism have discovered that sphingolipids are also required for the induction of CYP1A1 by 3-methylcholanthrene, however, in this case, the requirement is for de novo sphingolipid biosynthesis rather than the turnover of complex sphingolipids. These findings illustrate how changes in sphingolipid metabolism can influence the regulation of at least several isoforms of cytochrome P450.

1. The effect of a sunflower oil-enriched diet on plasma membrane-bound protein kinase C, protein kinase A, casein and tyrosine kinase activities was studied. 2. The diet induced an increase in the content of linoleic acid and a decrease in the content of palmitic acid. The anisotropy parameter (rs) of the fluorescence probe DPH and SDPH decreased strongly in the experimental group. 3. Protein kinase C was stimulated more than two times. Tyrosine kinase, protein kinase A and casein kinase activities were increased by 65, 57 and 40%, respectively. 4. We suggest that a more fluid lipid environment favours higher plasma membrane-bound protein kinase activities.

The effect of cholesterol-supplemented diet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcholine:ceramide-phosphocholine transferase, and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10, 20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcholine:ceramide-phosphocholine transferase, and r = 0.998 for protein kinase C and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide.