Ceramide-mediated orchestration of oxidative stress response through filopodia-derived small extracellular vesicles.

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 HO 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.

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.

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.

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).

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.

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.

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 ([H-methyl]-phosphatidylcholine, [H-acyl]-ceramide, [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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

Interleukin 1β 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-1β, a pro-inflammatory cytokine, has the opposite effects on FoxO1.

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.

Protein phosphatase 2A and neutral sphingomyelinase 2 regulate IRAK-1 protein ubiquitination and degradation in response to interleukin-1beta.

The IL-1β signaling cascade is initiated by the phosphorylation of IL-1β receptor-associated kinase-1 (IRAK-1), followed by its ubiquitination and degradation. This paper investigates the regulation of IRAK-1 degradation in primary hepatocytes and in HEK cells overexpressing the IL-1β receptor. We provide evidence that protein phosphatase 2A (PP2A) is a negative regulator of the phosphorylation, Lys(48)-linked ubiquitination, and degradation of IRAK-1.

Sphingolipid metabolism, oxidant signaling, and contractile function of skeletal muscle.

Significance: Sphingolipids are a class of bioactive lipids that regulate diverse cell functions. Ceramide, sphingosine, and sphingosine-1-phosphate accumulate in tissues such as liver, brain, and lung under conditions of cellular stress, including oxidative stress. The activity of some sphingolipid metabolizing enzymes, chiefly the sphingomyelinases, is stimulated during inflammation and in response to oxidative stress.

Ceramide in stress response.

Evidence has consistently indicated that activation of sphingomyelinases and/or ceramide synthases and the resulting accumulation of ceramide mediate cellular responses to stressors such as lipopolysaccharide, interleukin 1beta, tumor necrosis factor alpha, serum deprivation, irradiation and various antitumor treatments. Recent studies had identified the genes encoding most of the enzymes responsible for the generation of ceramide and ongoing research is aimed at characterizing their individual functions in cellular response to stress. This chapter discusses the seminal and more recent discoveries in regards to the pathways responsible for the accumulation of ceramide during stress and the mechanisms by which ceramide affects cell functions.

The twists and turns of sphingolipid pathway in glucose regulation.

Palmitic acid is a saturated fat found in foods that lead to obesity, cardiovascular disease, and Type II diabetes. It is linked to the development of resistance to insulin stimulation in muscle, liver and other organs involved in glucose metabolism, which, in turn, underlines the onset of Type II diabetes. The cellular and molecular mechanisms of this insulin resistance are complex and not completely understood.