RESUMEN
Niemann-Pick type C1 (NPC1) disease is a rare neurodegenerative cholesterol and sphingolipid storage disorder primarily due to mutations in the cholesterol-trafficking protein NPC1. In addition to catabolic-derived sphingolipids, NPC1 dysfunction also leads to an increase in de novo sphingolipid biosynthesis, yet little is known about the cellular mechanism involved. Although deletion of NPC1 or inhibition of the NPC1 sterol binding domain enhanced de novo sphingolipid biosynthesis, surprisingly levels of the ORMDLs, the regulatory subunits of serine palmitoyltransferase (SPT), the rate-limiting step in sphingolipid biosynthesis, were also greatly increased. Nevertheless, less ORMDL was bound in the SPT-ORMDL complex despite elevated ceramide levels. Instead, ORMDL colocalized with p62, the selective autophagy receptor, and accumulated in stalled autophagosomes due to defective autophagy in NPC1 disease cells. Restoration of autophagic flux with N-acetyl-L-leucine in NPC1 deleted cells decreased ORMDL accumulation in autophagosomes and reduced de novo sphingolipid biosynthesis and their accumulation. This study revealed a previously unknown link between de novo sphingolipid biosynthesis, ORMDL, and autophagic defects present in NCP1 disease. In addition, we provide further evidence and mechanistic insight for the beneficial role of N-acetyl-L-leucine treatment for NPC1 disease which is presently awaiting approval from the Food and Drug Administration and the European Medicines Agency.
Asunto(s)
Autofagia , Enfermedad de Niemann-Pick Tipo C , Esfingolípidos , Esfingolípidos/metabolismo , Esfingolípidos/biosíntesis , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Enfermedad de Niemann-Pick Tipo C/genética , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Animales , Proteína Niemann-Pick C1 , Serina C-Palmitoiltransferasa/metabolismo , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/antagonistas & inhibidoresRESUMEN
Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H2O2-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.
Asunto(s)
Apoptosis , Ceramidas , Cicloserina , Estrés Oxidativo , Esfingolípidos , Estrés Oxidativo/efectos de los fármacos , Cicloserina/farmacología , Animales , Ceramidas/metabolismo , Ceramidas/farmacología , Ratones , Esfingolípidos/metabolismo , Apoptosis/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Serina C-Palmitoiltransferasa/metabolismo , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Peróxido de Hidrógeno/toxicidad , Peróxido de Hidrógeno/farmacología , Línea Celular , Degeneración Retiniana/metabolismo , Degeneración Retiniana/prevención & control , Degeneración Retiniana/patología , Degeneración Retiniana/tratamiento farmacológico , Western Blotting , Inhibidores Enzimáticos/farmacología , Supervivencia Celular/efectos de los fármacosRESUMEN
Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects.
Asunto(s)
Membrana Celular/efectos de los fármacos , Ácidos Grasos Monoinsaturados/farmacología , Serina C-Palmitoiltransferasa/metabolismo , Esfingolípidos/biosíntesis , Animales , Células CHO , Membrana Celular/metabolismo , Cricetulus , Lipidómica , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/genéticaRESUMEN
Sphingolipids (SLs) are vital for cells as forming membrane and transducing signals. The first step for de novo biosynthesis of SLs is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT), which has been proven to be a promising drug target for treating various diseases. However, there are few SPT-specific inhibitors have been identified so far. Myriocin, a natural fungal product, is confirmed as the most potent inhibitor of SPT and has been widely used, but studies of its molecular mechanism are still underway. Besides, there is no intact co-crystal structure of SPT-binding myriocin until now. Aiming to uncover the interaction mechanism between SPT- and PLP-binding myriocin at the molecular level, a systematic computational strategy was performed in this present study. Firstly, covalent docking was implemented to preliminarily predict the binding pose SPT/PLP-myriocin aldimine and its structurally similar intermediate SPT/PLP-ß-ketoacid aldimine. Secondly, two binding complexes were treated as initial structures to perform molecular dynamics simulations and binding free energy calculations. The calculated docking scores and predicted binding energies were consistent with the reported bioactivities. Finally, the binding mechanism of myriocin binding with SPT was meticulously described, and the key residues making favorable contributions were highlighted. Taken together, the current study could provide some important information and valuable guidance for further rational screening, design, and modification of potent specific SPT inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Ácidos Grasos Monoinsaturados/química , Simulación del Acoplamiento Molecular , Sitios de Unión , Inhibidores Enzimáticos/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/metabolismo , TermodinámicaRESUMEN
Serine palmitoyltransferase (SPT) plays the key role on catalysing the formation of 3-ketodihydrosphingosine, which is the first step of the de novo biosynthesis of sphingolipids. SPT is linked to many diseases including fungal infection, making it a potential therapeutic target. Thus, a logical docking-based virtual screening method was used to screen selective SPT inhibitor against fungi, not human. We used myriocin-similarity database to identify compounds with good binding with fungal SPT and poor binding with homology human SPT model. Preliminary bio-assay led to the discovery of a promising inhibitor WXP-003, which displayed good inhibitory activity against diversity fungi strains with MIC ranging from 0.78 to 12.5 µg/mL. WXP-003 could significantly reduce sphingolipids content in fungi and no effect on mouse fibroblast cell line L929. Molecular dynamics simulation depicted that SPT/WXP-003 complex formed the favoured interactions. Taken together, discovery of WXP-003 provided valuable guide for the development of novel anti-fungal agents.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Animales , Antifúngicos/farmacología , Inhibidores Enzimáticos/química , Humanos , Ratones , Simulación del Acoplamiento MolecularRESUMEN
Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.
Asunto(s)
Neoplasias/metabolismo , Neoplasias/patología , Serina/deficiencia , Esfingolípidos/química , Esfingolípidos/metabolismo , Alanina/biosíntesis , Alanina/metabolismo , Alanina/farmacología , Animales , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Dieta , Femenino , Glicina/biosíntesis , Glicina/deficiencia , Glicina/metabolismo , Glicina/farmacología , Células HCT116 , Humanos , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Ratones , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Fosfoglicerato-Deshidrogenasa/metabolismo , Ácido Pirúvico/metabolismo , Serina/sangre , Serina/farmacología , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/metabolismo , Esferoides Celulares/patología , Esfingolípidos/biosíntesis , Estrés Fisiológico/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this study, we identify the natural product gambogic acid as well as structurally related synthetic xanthones as first-in-class covalent inhibitors of the de novo sphingolipid biosynthesis. We apply chemoproteomics to determine that gambogic acid binds to the regulatory small subunit B of the serine palmitoyltransferase complex (SPTSSB). We then test structurally related synthetic xanthones to identify 18 as an equally potent but more selective binder of SPTSSB and show that 18 reduces sphingolipid levels in situ and in vivo. Finally, using various biological methods, we demonstrate that 18 induces cellular responses characteristic for diminished sphingosine-1-phosphate (S1P) signaling. This study demonstrates that SPTSSB may become a viable therapeutic target in various diseases with pathological S1P signaling. Furthermore, we believe that our compound will become a valuable tool for studying the sphingolipid metabolism and serve as a blueprint for the development of a new generation of sphingolipid biosynthesis inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Xantonas/química , Xantonas/farmacología , Animales , Descubrimiento de Drogas , Células HEK293 , Humanos , Lisofosfolípidos/metabolismo , Células MCF-7 , Ratones , Ratones Endogámicos ICR , Proteómica , Serina C-Palmitoiltransferasa/metabolismo , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-ß mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-ß inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.
Asunto(s)
Células Epiteliales/virología , Proteínas de la Membrana/fisiología , Rhinovirus/fisiología , Replicación Viral , Células A549 , Asma/etiología , Bronquios/citología , Células Cultivadas , Cromosomas Humanos Par 17/genética , Estrés del Retículo Endoplásmico , Ácidos Grasos Monoinsaturados/farmacología , Predisposición Genética a la Enfermedad , Genotipo , Células HeLa , Humanos , Interferón beta/biosíntesis , Interferón beta/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Mucosa Nasal/citología , Infecciones por Picornaviridae/complicaciones , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes/metabolismo , Rhinovirus/genética , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/metabolismo , Esfingolípidos/metabolismo , Ácido Tauroquenodesoxicólico/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND/AIMS: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells. METHODS: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned. RESULTS: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin. CONCLUSION: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF.
Asunto(s)
Ácidos Grasos Monoinsaturados/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Esfingolípidos/metabolismo , Autofagia/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Línea Celular , Colesterol/análisis , Cromatografía Líquida de Alta Presión , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Espectrometría de Masas , PPAR gamma/genética , PPAR gamma/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/metabolismo , Esfingolípidos/análisis , Esfingomielinas/análisisRESUMEN
Curcumin, a hydrophobic polyphenol found in the rhizome of Curcuma longa, has been shown to reduce intracellular lipid accumulation in mouse models of lysosomal storage diseases such as Niemann-Pick type C. Exosomes are small extracellular vesicles secreted by cells in response to changes in intracellular ceramide composition. Curcumin can induce exosome/microvesicle release in cellular models of lipid deposition; however, the mechanism by which curcumin stimulates this release is unknown. In a model of lipid trafficking impairment in C6 glia cells, we show that curcumin stimulated ceramide synthesis by increasing the intracellular concentration of ceramide-dihydroceramide. Ceramide overload increased exosome/microvesicle secretion 10-fold, thereby reducing the concentration of lipids in the endolysosomal compartment. These effects were blocked by inhibitors of serine palmitoyltransferase (myriocin) and ceramide synthase (fumonisin B1). It is concluded that the decrease in intracellular lipid deposition induced by curcumin is mediated by increased ceramide synthesis and exosome/microvesicle release. This action may represent an additional health benefit of curcumin.
Asunto(s)
Micropartículas Derivadas de Células/efectos de los fármacos , Ceramidas/biosíntesis , Curcumina/farmacología , Exosomas/efectos de los fármacos , Neuroglía/efectos de los fármacos , Animales , Línea Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Exosomas/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Fumonisinas/farmacología , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteínas LDL/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuroglía/patología , Enfermedad de Niemann-Pick Tipo C/dietoterapia , Enfermedad de Niemann-Pick Tipo C/patología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Ratas , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/metabolismoRESUMEN
Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.
Asunto(s)
Proteínas de la Membrana/genética , Vaina de Mielina/genética , Esfingolípidos/genética , Animales , Ceramidas/genética , Células HeLa , Humanos , Metabolismo de los Lípidos/genética , Lipogénesis/genética , Ratones , Ratones Noqueados , Vaina de Mielina/metabolismo , Nervio Ciático/crecimiento & desarrollo , Nervio Ciático/metabolismo , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/genética , Transducción de Señal/genética , Esfingolípidos/biosíntesisRESUMEN
Ceramide and sphingosine display a unique profile during brain development, indicating their critical role in myelinogenesis. Employing advanced technology such as gas chromatography-mass spectrometry, high performance liquid chromatography, and immunocytochemistry, along with cell culture and molecular biology, we have found an accumulation of sphingosine in brain tissues of patients with multiple sclerosis (MS) and in the spinal cord of rats induced with experimental autoimmune encephalomyelitis. The elevated sphingosine leads to oligodendrocyte death and fosters demyelination. Ceramide elevation by serine palmitoyltransferse (SPT) activation was the primary source of the sphingosine elevation as myriocin, an inhibitor of SPT, prevented sphingosine elevation and protected oligodendrocytes. Supporting this view, fingolimod, a drug used for MS therapy, reduced ceramide generation, thus offering partial protection to oligodendrocytes. Sphingolipid synthesis and degradation in normal development is regulated by a series of microRNAs (miRNAs), and hence, accumulation of sphingosine in MS may be prevented by employing miRNA technology. This review will discuss the current knowledge of ceramide and sphingosine metabolism (synthesis and breakdown), and how their biosynthesis can be regulated by miRNA, which can be used as a therapeutic approach for MS.
Asunto(s)
Ceramidas/biosíntesis , MicroARNs/genética , Esclerosis Múltiple/genética , Serina C-Palmitoiltransferasa/metabolismo , Esfingosina/biosíntesis , Animales , Encéfalo/metabolismo , Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/uso terapéutico , Humanos , MicroARNs/antagonistas & inhibidores , Terapia Molecular Dirigida , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Vaina de Mielina/metabolismo , Ratas , Serina C-Palmitoiltransferasa/antagonistas & inhibidoresRESUMEN
Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4-/-). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4-/- mice. We noted similar results in mice infected with adeno-associated virus-expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA-mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4-ceramide-PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders.
Asunto(s)
Proteína 4 Similar a la Angiopoyetina/metabolismo , Ceramidas/metabolismo , Dexametasona/toxicidad , Hígado/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteína 4 Similar a la Angiopoyetina/deficiencia , Proteína 4 Similar a la Angiopoyetina/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Hígado Graso/etiología , Hígado Graso/metabolismo , Hipertrigliceridemia/etiología , Hipertrigliceridemia/metabolismo , Lipogénesis/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa C/antagonistas & inhibidores , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/metabolismo , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
Sulforaphane (SFA), a naturally active isothiocyanate compound from cruciferous vegetables used in clinical trials for cancer treatment, was found to possess potency to alleviate insulin resistance. But its underlying molecular mechanisms are still incompletely understood. In this study, we assessed whether SFA could improve insulin sensitivity and glucose homeostasis both in vitro and in vivo by regulating ceramide production. The effects of SFA on glucose metabolism and expression levels of key proteins in the hepatic insulin signaling pathway were evaluated in insulin-resistant human hepatic carcinoma HepG2 cells. The results showed that SFA dose-dependently increased glucose uptake and intracellular glycogen content by regulating the insulin receptor substrate 1 (IRS-1)/protein kinase B (Akt) signaling pathway in insulin-resistant HepG2 cells. SFA also reduced ceramide contents and downregulated transcription of ceramide-related genes. In addition, knockdown of serine palmitoyltransferase 3 (SPTLC3) in HepG2 cells prevented ceramide accumulation and alleviated insulin resistance. Moreover, SFA treatment improved glucose tolerance and insulin sensitivity, inhibited SPTLC3 expression and hepatic ceramide production and reduced hepatic triglyceride content in vivo. We conclude that SFA recovers glucose homeostasis and improves insulin sensitivity by blocking ceramide biosynthesis through modulating SPTLC3, indicating that SFA may be a potential candidate for prevention and amelioration of hepatic insulin resistance via a ceramide-dependent mechanism.
Asunto(s)
Ceramidas/biosíntesis , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Hepatocitos/efectos de los fármacos , Resistencia a la Insulina , Isotiocianatos/farmacología , Hígado/efectos de los fármacos , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Animales , Glucógeno/metabolismo , Células Hep G2 , Hepatocitos/enzimología , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ácido Palmítico/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/metabolismo , Transducción de Señal , Sulfóxidos , Triglicéridos/metabolismoRESUMEN
Alzheimer's disease (AD) is a slow-progressing disease of the brain characterized by symptoms such as impairment of memory and other cognitive functions. AD is associated with an inflammatory process that involves astrocytes and microglial cells, among other components. Astrocytes are the most abundant type of glial cells in the central nervous system (CNS). They are involved in inducing neuroinflammation. The present study uses astrocyte-neuron cocultures to investigate how ARN14494, a serine palmitoyltransferase (SPT) inhibitor, affects the CNS in terms of anti-inflammation and neuroprotection. SPT is the first rate-limiting enzyme in the de novo ceramide synthesis pathway. Consistent evidence suggests that ceramide is increased in AD brain patients. After ß-amyloid 1-42 injury in an in vitro model of AD, ARN14494 inhibits SPT activity and the synthesis of long-chain ceramides and dihydroceramides that are involved in AD progression. In mouse primary cortical astrocytes, ARN14494 prevents the synthesis of proinflammatory cytokines TNFα and IL1ß, growth factor TGFß1, and oxidative stress-related enzymes iNOS and COX2. ARN14494 also exerts neuroprotective properties in primary cortical neurons. ARN14494 decreases neuronal death and caspase-3 activation in neurons, when the neuroinflammation is attenuated in astrocytes. These findings suggest that ARN14494 protects neurons from ß-amyloid 1-42 induced neurotoxicity through a variety of mechanisms, including antioxidation, antiapoptosis, and anti-inflammation. SPT inhibition could therefore be a safe therapeutic strategy for ameliorating the pathology of Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Astrocitos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Muerte Celular/efectos de los fármacos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismoRESUMEN
Liver, as one of the most important organs involved in lipids and glucose metabolism, is perceived as a key tissue for pharmacotherapy of insulin resistance (IRes) and type 2 diabetes. Ceramides (Cer) are biologically active lipids, which accumulation is associated with the induction of muscle IRes. We sought to determine the role of intrahepatic bioactive lipids production on insulin action in liver of insulin-resistant rats and after myriocin administration. The experiments were conducted on male Wistar rats divided into three groups: Control, fed high-fat diet (HFD), and fed HFD and treated with myriocin (HFD/Myr). Before sacrifice, the animals were infused with a [U-13 C]palmitate to calculate lipid synthesis rate by means of tracer incorporation technique in particular lipid groups. Liver Cer, diacylglycerols (DAG), acyl-carnitine concentration, and isotopic enrichment were analyzed by LC/MS/MS. Proteins involved in lipid metabolism and insulin pathway were analyzed by western blot analysis. An OGTT and ITT was also performed. HFD-induced IRes and increased both the synthesis rate and the content of DAG and Cer, which was accompanied by inhibition of an insulin pathway. Interestingly, myriocin treatment reduced synthesis rate not only of Cer but also DAG and improved insulin sensitivity. We conclude that the insulin-sensitizing action of myriocin in the liver is a result of the lack of inhibitory effect of lipids on the insulin pathway, due to the reduction of their synthesis rate. This is the first study showing how the synthesis rate of individual lipid groups in liver changes after myriocin administration.
Asunto(s)
Glucemia/efectos de los fármacos , Ceramidas/metabolismo , Dieta Alta en Grasa , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Resistencia a la Insulina , Insulina/sangre , Hígado/efectos de los fármacos , Animales , Biomarcadores/sangre , Glucemia/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Humanos , Hígado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ratas Wistar , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/metabolismo , Transducción de SeñalRESUMEN
The majority of Merkel cell carcinoma, a highly aggressive neuroendocrine cancer of the skin, is associated with Merkel cell polyomavirus infection. Polyomavirus binding, internalization, and infection are mediated by glycosphingolipids. Besides receptor function, bioactive sphingolipids are increasingly recognized as potent regulators of several hallmarks of cancer. Merkel cell polyomavirus+ and Merkel cell polyomavirus- cells express serine palmitoyl transferase subunits and sphingosine kinase (SK) 1/2 mRNA. Induced expression of Merkel cell polyomavirus-large tumor antigen in human lung fibroblasts resulted in upregulation of SPTLC1-3 and SK 1/2 expression. Therefore, we exploited pharmacological inhibition of sphingolipid metabolism as an option to interfere with proliferation of Merkel cell polyomavirus+ Merkel cell carcinoma cell lines. We used myriocin (a serine palmitoyl transferase antagonist) and two SK inhibitors (SKI-II and ABC294640). In MKL-1 and WaGa cells myriocin decreased cellular ceramide, sphingomyelin, and sphingosine-1-phosphate content. SKI-II increased ceramide species but decreased sphingomyelin and sphingosine-1-phosphate concentrations. Aberrant sphingolipid homeostasis was associated with reduced cell viability, increased necrosis, procaspase-3 and PARP processing, caspase-3 activity, and decreased AKTS473 phosphorylation. Myriocin and SKI-II decreased tumor size and Ki-67 staining of xenografted MKL-1 and WaGa tumors on the chorioallantoic membrane. Our data suggest that pharmacological inhibition of sphingolipid synthesis could represent a potential therapeutic approach in Merkel cell carcinoma.
Asunto(s)
Carcinoma de Células de Merkel/tratamiento farmacológico , Ácidos Grasos Monoinsaturados/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Infecciones por Polyomavirus/tratamiento farmacológico , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Infecciones Tumorales por Virus/tratamiento farmacológico , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/patología , Recuento de Células , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Poliomavirus de Células de Merkel/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/patología , ARN Neoplásico/genética , Serina C-Palmitoiltransferasa/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/patologíaRESUMEN
We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Piperidinas/farmacología , Pirazoles/farmacología , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacocinética , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacocinética , Estereoisomerismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIM: This study assessed pulmonary outcomes generated by inhibiting key enzymes of sphingolipid metabolism pathways related to ceramide synthesis in a murine model of lung injury induced by lipopolysaccharide (LPS). METHODS: C57BL/6 male adult mice received LPS intratracheally and the expressions of acid sphingomyelinase (ASM), neutral sphingomyelinase (NSM), serine palmitoyl transferase (SPT) and dihydroceramide synthase (DS) were assessed at 2, 4, 6, 12 and 24â¯h after LPS instillation in lung homogenate (nâ¯=â¯30). The pharmacological inhibition of ASM, NSM, SPT and DS were assayed in other mice groups by three different doses of desipramine, GW4869, myriocin and fumonisin, respectively (nâ¯=â¯90). Their most effective doses were administered intraperitoneally 1 or 2â¯h before LPS to different animal groups (nâ¯=â¯120). Mice underwent determination of pulmonary mechanics, lung histopathological aspects and apoptosis. RESULTS: The expression levels of the enzymes reached their peak at 2-4 h after LPS administration. ASM inhibition attenuated alveolar collapse and GW4869 decreased lung elastance, proinflammatory cytokines' levels and was more effective to improve alveolar collapse than desipramine. On the other hand, SPT blockage aggravated lung lesion and no effects it was observed with fumonisin. Moreover, simultaneous administration of inhibitors (desipramine + GW4869, myriocin + fumonisin and all inhibitors together) resulted in no changes. CONCLUSION: Blockage of sphingomyelinases and the de novo pathways improved and aggravated lung injury, respectively, putatively suggesting specific targets to therapeutic strategies in LPS-induced lung injury.
Asunto(s)
Lipopolisacáridos/farmacología , Lesión Pulmonar/inducido químicamente , Esfingolípidos/metabolismo , Compuestos de Anilina/farmacología , Animales , Compuestos de Bencilideno/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/enzimología , Lesión Pulmonar/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/metabolismo , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Numerous studies have implicated dyslipidemia as a key factor in mediating insulin resistance. Ceramides have received special attention since their levels are inversely associated with normal insulin signaling and positively associated with factors that are involved in cardiometabolic disease. Despite the growing literature surrounding ceramide biology, there are limited data regarding the activity of ceramide synthesis and turnover in vivo. Herein, we demonstrate the ability to measure ceramide kinetics by coupling the administration of [2H]water with LC-MS/MS analyses. As a "proof-of-concept" we determined the effect of a diet-induced alteration on ceramide flux; studies also examined the effect of myriocin (a known inhibitor of serine palmitoyltransferase, the first step in sphingosine biosynthesis). Our data suggest that one can estimate ceramide synthesis and draw conclusions regarding the source of fatty acids; we discuss caveats in regards to method development in this area.