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Involvement of Sphingosine 1-Phosphate in Palmitate-Induced Non-Alcoholic Fatty Liver Disease
(2016)
Background/Aims: Ectopic lipid accumulation in hepatocytes has been identified as a risk factor for the progression of liver fibrosis and is strongly associated with obesity. In particular, the saturated fatty acid palmitate is involved in initiation of liver fibrosis via formation of secondary metabolites by hepatocytes that in turn activate hepatic stellate cells (HSCs) in a paracrine manner Methods: a-smooth muscle actin-expression (alpha-SMA) as a marker of liver fibrosis was investigated via western blot analysis and immunofluorescence microscopy in HSCs (LX-2). Sphingolipid metabolism and the generation of the bioactive secondary metabolite sphingosine I-phosphate (SIP) in response to palmitate were analyzed by LC-MS/MS in hepatocytes (HepG2). To identify the molecular mechanism involved in the progression of liver fibrosis real-time PCR analysis and pharmacological modulation of SIP receptors were performed. Results: Palmitate oversupply increased intra- and extracellular SIP-concentrations in hepatocytes. Conditioned medium from HepG2 cells initiated fibrosis by enhancing alpha-SMA-expression in LX-2 in a S1P-dependent manner In accordance, fibrotic response in the presence of SIP was also observed in HSCs. Pharmacological inhibition of SIP receptors demonstrated that S1P(3) is the crucial receptor subtype involved in this process. Conclusion: SIP is synthesized in hepatocytes in response to palmitate and released into the extracellular environment leading to an activation of HSCs via the S1P(3) receptor (C) 2016 The Author(s) Published by S. Karger AG, Basel
Dendritic cells (DCs) are the cutting edge in innate and adaptive immunity. The major functions of these antigen presenting cells are the capture, endosomal processing and presentation of antigens, providing them an exclusive ability to provoke adaptive immune responses and to induce and control tolerance. Immature DCs capture and process antigens, migrate towards secondary lymphoid organs where they present antigens to naive T cells in a well synchronized sequence of procedures referred to as maturation. Indeed, recent research indicated that sphingolipids are modulators of essential steps in DC homeostasis. It has been recognized that sphingolipids not only modulate the development of DC subtypes from precursor cells but also influence functional activities of DCs such as antigen capture, and cytokine profiling. Thus, it is not astonishing that sphingolipids and sphingolipid metabolism play a substantial role in inflammatory diseases that are modulated by DCs. Here we highlight the function of sphingosine 1-phosphate (S1P) on DC homeostasis and the role of SIP and SW metabolism in inflammatory diseases.
Controlled delivery of corticosteroids using nanoparticles to the skin and corneal epithelium may reduce their side effects and maximize treatment effectiveness. Dexamethasone-loaded ethyl cellulose, Eudragit® RS and ethyl cellulose/Eudragit® RS nanoparticles were prepared by the solvent evaporation method. Dexamethasone release from the polymeric nanoparticles was investigated in vitro using Franz diffusion cells. Drug penetration was also assessed ex vivo using excised human skin. Nanoparticle toxicity was determined by MTT and H2DCFDA assays. Eudragit® RS nanoparticles were smaller and positively charged but had a lower dexamethasone loading capacity (0.3–0.7%) than ethyl cellulose nanoparticles (1.4–2.2%). By blending the two polymers (1:1), small (105 nm), positively charged (+37 mV) nanoparticles with sufficient dexamethasone loading (1.3%) were obtained. Dexamethasone release and penetration significantly decreased with decreasing drug to polymer ratio and increased when Eudragit® RS was blended with ethyl cellulose. Ex vivo, drug release and penetration from the nanoparticles was slower than a conventional cream. The nanoparticles bear no toxicity potentials except ethyl cellulose nanoparticles had ROS generation potential at high concentration. In conclusion, the nanoparticles showed great potential to control the release and penetration of corticosteroids on the skin and mucus membrane and maximize treatment effectiveness.
The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor compound, has not yet been fully elucidated. Here, we show that human cancer cells have markedly lower levels of sphingomyelin (SM) than nontumor (MRC-5) cells. In this context, 2OHOA treatment strongly augments SM mass (4.6-fold), restoring the levels found in MRC-5 cells, while a loss of phosphatidylethanolamine and phosphatidylcholine is observed (57 and 30%, respectively). The increased SM mass was due to a rapid and highly specific activation of SM synthases (SMS). This effect appeared to be specific against cancer cells as it did not affect nontumor MRC-5 cells. Therefore, low SM levels are associated with the tumorigenic transformation that produces cancer cells. SM accumulation occurred at the plasma membrane and caused an increase in membrane global order and lipid raft packing in model membranes. These modifications would account for the observed alteration by 2OHOA in the localization of proteins involved in cell apoptosis (Fas receptor) or differentiation (Ras). Importantly, SMS inhibition by D609 diminished 2OHOA effect on cell cycle. Therefore, we propose that the regulation of SMS activity in tumor cells is a critical upstream event in 2OHOA antitumor mechanism, which also explains its specificity for cancer cells, its potency, and the lack of undesired side effects. Finally, the specific activation of SMS explains the ability of this compound to trigger cell cycle arrest, cell differentiation, and autophagy or apoptosis in cancer cells.
Studies over the past several years have demonstrated the important role of sphingolipids in cystic fibrosis (CF), chronic obstructive pulmonary disease and acute lung injury. Ceramide is increased in airway epithelial cells and alveolar macrophages of CF mice and humans, while sphingosine is dramatically decreased. This increase in ceramide results in chronic inflammation, increased death of epithelial cells, release of DNA into the bronchial lumen and thereby an impairment of mucociliary clearance; while the lack of sphingosine in airway epithelial cells causes high infection susceptibility in CF mice and possibly patients. The increase in ceramide mediates an ectopic expression of beta 1-integrins in the luminal membrane of CF epithelial cells, which results, via an unknown mechanism, in a down-regulation of acid ceramidase. It is predominantly this down-regulation of acid ceramidase that results in the imbalance of ceramide and sphingosine in CF cells. Correction of ceramide and sphingosine levels can be achieved by inhalation of functional acid sphingomyelinase inhibitors, recombinant acid ceramidase or by normalization of beta 1-integrin expression and subsequent re-expression of endogenous acid ceramidase. These treatments correct pulmonary inflammation and prevent or treat, respectively, acute and chronic pulmonary infections in CF mice with Staphylococcus aureus and mucoid or non-mucoid Pseudomonas aeruginosa. Inhalation of sphingosine corrects sphingosine levels only and seems to mainly act against the infection. Many antidepressants are functional inhibitors of the acid sphingomyelinase and were designed for systemic treatment of major depression. These drugs could be repurposed to treat CF by inhalation.
Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can’t achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients.
Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1(tmEx1) mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.