Tumor Suppressor Par-4 Regulates Complement Factor C3 and Obesity.

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.

Neutral Sphingomyelinase 2 Mediates Oxidative Stress Effects on Astrocyte Senescence and Synaptic Plasticity Transcripts.

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. ß-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 1ß, 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.

Skeletal Muscle Cell Growth Alters the Lipid Composition of Extracellular Vesicles.

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.

Onset of Senescence and Steatosis in Hepatocytes as a Consequence of a Shift in the Diacylglycerol/Ceramide Balance at the Plasma Membrane.

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 ß-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-1ß and nlpr3 mRNA levels. HepG2-SMS2 cells exhibited lipid droplet accumulation, constitutive activation of AMPK based on elevated 172Thr 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.

Methods to Characterize Synthesis and Degradation of Sphingomyelin at the Plasma Membrane and Its Impact on Lipid Raft Dynamics.

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 ([3H-methyl]-phosphatidylcholine, [3H-acyl]-ceramide, [14C-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.

A Novel Combination of Fruits and Vegetables Prevents Diet-Induced Hepatic Steatosis and Metabolic Dysfunction in Mice.

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.

Alcoholic and non-alcoholic fatty liver disease: Focus on ceramide.

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.

Sphingolipids at the Crossroads of NAFLD and Senescence.

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.

Increased liver tumor formation in neutral sphingomyelinase-2-deficient mice.

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 C16-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.

Secretory sphingomyelinase (S-SMase) activity is elevated in patients with rheumatoid arthritis.

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.

The expanding role of sphingolipids in lipid droplet biogenesis.

Sphingolipids are a diverse class of lipids that have regulatory, structural, and metabolic functions. Although chemically distinct from the neutral lipids and the glycerophospholipids, which are the main lipid components of the lipid droplets, sphingolipids have nonetheless been shown to influence lipid droplet formation. The goal of this article is to review the available information and provide a cohesive picture of the role sphingolipids play in lipid droplet biogenesis. The following topics are discussed: (i) the abundance of sphingolipids in lipid droplets and their functional significance; (ii) cross-talk between the synthetic pathways of sphingolipids, glycerophospholipids, and neutral lipids; (iii) the impact of bioactive sphingolipids on TAG synthesis and degradation; (iv) interactions between sphingolipids and other lipid droplet components, like cholesterol esters and proteins; (v) inhibition/genetic deletion of specific sphingolipid metabolic enzymes and the resulting effects on lipid droplet formation in mouse models. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

Outcomes of Spatially Fractionated Radiotherapy (GRID) for Bulky Soft Tissue Sarcomas in a Large Animal Model.

GRID directs alternating regions of high- and low-dose radiation at tumors. A large animal model mimicking the geometries of human treatments is needed to complement existing rodent systems (eg, microbeam) and clarify the physical and biological attributes of GRID. A pilot study was undertaken in pet dogs with spontaneous soft tissue sarcomas to characterize responses to GRID. Subjects were treated with either 20 Gy (3 dogs) or 25 Gy (3 dogs), delivered using 6 MV X-rays and a commercial GRID collimator. Acute toxicity and tumor responses were assessed 2, 4, and 6 weeks later. Acute Radiation Therapy Oncology Group grade I skin toxicity was observed in 3 of the 6 dogs; none experienced a measurable response, per Response Evaluation Criteria in Solid Tumors. Serum vascular endothelial growth factor, tumor necrosis factor α, and secretory sphingomyelinase were assayed at baseline, 1, 4, 24, and 48 hours after treatment. There was a trend toward platelet-corrected serum vascular endothelial growth factor concentration being lower 1 and 48 hours after GRID than at baseline. There was a significant decrease in secretory sphingomyelinase activity 48 hours after 25 Gy GRID ( P = .03). Serum tumor necrosis factor α was quantified measurable at baseline in 4 of the 6 dogs and decreased in each of those subjects at all post-GRID time points. The new information generated by this study includes the observation that high-dose, single fraction application of GRID does not induce measurable reduction in volume of canine soft tissue sarcomas. In contrast to previously published data, these data suggest that GRID may be associated with at least short-term reduction in serum concentration of vascular endothelial growth factor and serum activity of secretory sphingomyelinase. Because GRID can be applied safely, and these tumors can be subsequently surgically resected as part of routine veterinary care, pet dogs with sarcomas are an appealing model for studying the radiobiologic responses to spatially fractionated radiotherapy.

Novel Interconnections in Lipid Metabolism Revealed by Overexpression of Sphingomyelin Synthase-1.

This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids.

Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state.

Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys(617) to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state.

Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle.

AIMS: Sphingolipid 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. RESULTS: We 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. INNOVATION: These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production. CONCLUSION: Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity.

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

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.

Diaphragm dysfunction in heart failure is accompanied by increases in neutral sphingomyelinase activity and ceramide content.

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.

Resveratrol alters the lipid composition, metabolism and peroxide level in senescent rat hepatocytes.

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.

Interleukin 1ß regulation of FoxO1 protein content and localization: evidence for a novel ceramide-dependent mechanism.

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-1ß, a pro-inflammatory cytokine, has the opposite effects on FoxO1. IL-1ß stimulation of primary rat hepatocytes and HEK293 cells overexpressing the IL-1ß receptor (293-IL-1RI) results in increased nuclear and cytosolic FoxO1 protein but not mRNA levels. IL-1ß 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-1ß induction of FoxO1. The IL-1ß 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-1ß effects. We also show that transcription of insulin-like growth factor-binding protein-1 mRNA, which requires active FoxO1, is stimulated by IL-1ß and is suppressed by the inhibition of nSMase-2 and JNK. In conclusion, we propose that IL-1ß regulates FoxO1 activity through a novel nSMase-2-dependent pathway.

Characterization of secretory sphingomyelinase activity, lipoprotein sphingolipid content and LDL aggregation in ldlr-/- mice fed on a high-fat diet.

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.