RESUMO
Free fatty acid receptor 4 (FFAR4)/GPR120 comprises a receptor for medium- and long-chain fatty acids. We previously identified phytosphingosine (PHS) as a novel ligand of FFAR4. Although many natural FFAR4 ligands have carboxyl groups, PHS does not, thus suggesting that binding to FFAR4 is driven by a completely different mechanism than other natural ligands such as α-linolenic acid (ALA). To test this hypothesis, we performed docking simulation analysis using a FFAR4 homology model based on a protein model derived from the crystal structure of activated turkey beta-1 adrenoceptor. The docking simulation revealed that the probable hydrogen bonds to FFAR4 differ between various ligands. In particular, binding was predicted between R264 of the FFAR4 and the oxygen of the carboxylate group in ALA, as well as between E249 of the FFAR4 and the oxygen of the hydroxy group at the C4-position in PHS. Alanine substitution at E249 (E249A) dramatically reduced PHS-induced FFAR4 activation but demonstrated a weaker effect on ALA-induced FFAR4 activation. Kinetic analysis and Km values clearly demonstrated that the E249A substitution resulted in reduced affinity for PHS but not for ALA. Additionally, we observed that sphingosine, lacking a hydroxyl group at C4-position, could not activate FFAR4. Our data show that E249 of the FFAR4 receptor is crucial for binding to the hydroxy group at the C4-position in PHS, and this is a completely different molecular mechanism of binding from ALA. Because GPR120 agonists have attracted attention as treatments for type 2 diabetes, our findings may provide new insights into their development.
Assuntos
Esfingosina/análogos & derivados , Esfingosina/metabolismo , Comunicação Celular , Ácidos Graxos , Células HEK293 , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular/métodos , Ligação Proteica , Receptores Acoplados a Proteínas G , Esfingosina/fisiologiaRESUMO
Post-fermented teas, produced by microbial fermentation, are attracting attention due to their health benefits that reduce the risk of hyperlipidemia and atherosclerosis. Although several novel polyphenols have been identified from post-fermented teas, their biological activities have not yet been fully elucidated. In this study, we found that teadenol A, a polyphenol recently isolated from Japanese post-fermented tea, acts as a novel ligand on a long-chain fatty acid receptor, GPR120. Teadenol A activated GPR120 was over-expressed in 293T cells, and this activation was inhibited by the GPR120 antagonist AH7614. Additionally, teadenol A induced Erk1/2 phosphorylation and increased the intracellular Ca2+ concentration in 293T cells, and these effects were completely dependent on GPR120 expression. Our results suggest that teadenol A binds and activates GPR120 directly. Furthermore, teadenol A enhanced the secretion of GLP-1 from intestinal endocrine STC-1 cells. GLP-1 suppresses appetite and increases insulin secretion, exhibiting anti-diabetic effects. GPR120/GLP-1 signaling is attracting attention as a potential target for pharmaceuticals against type 2 diabetes. Our results suggest that teadenol A is a key molecule in post-fermented tea responsible for beneficial effects on metabolic syndrome.
Assuntos
Secreções Corporais , Alimentos Fermentados , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Ligantes , Receptores Acoplados a Proteínas G/metabolismo , Chá , Diabetes Mellitus Tipo 2 , Ácidos Graxos , Fermentação , Alimentos Fermentados/microbiologia , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/genética , Transdução de SinaisRESUMO
Koji, which is manufactured by proliferating non-pathogenic fungus Aspergillus oryzae on steamed rice, is the base for Japanese traditional fermented foods. We have revealed that koji and related Japanese fermented foods and drinks such as amazake, shio-koji, unfiltered sake and miso contain abundant glycosylceramide. Here, we report that feeding of koji glycosylceramide to obese mice alters the cholesterol metabolism . Liver cholesterol was significantly decreased in obese mice fed with koji glycosylceramide. We hypothesized that their liver cholesterol was decreased because it was converted to bile acids. Consistent with the hypothesis, many bile acids were increased in the cecum and feces of obese mice fed with koji glycosylceramide. Expressions of CYP7A1 and ABCG8 involved in the metabolism of cholesterol were significantly increased in the liver of mice fed with koji glycosylceramide. Therefore, it was considered that koji glycosylceramide affects the cholesterol metabolism in obese mice.
Assuntos
Ceramidas/administração & dosagem , Colesterol/metabolismo , Alimentos Fermentados , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aspergillus oryzae/metabolismo , Ácidos e Sais Biliares/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Japão , Lipoproteínas/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos ObesosRESUMO
Previously, we proposed the following mechanism for konjac ceramide (kCer)-mediated neurite outgrowth inhibition: kCer binds to Nrp as a Sema3A agonist, resulting in Nrp1/PlexA complex formation and activation of the Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerization. The Sema3A/Nrp1 signaling pathway is known to be also expressed in normal human keratinocytes. To determine whether kCer can function in human keratinocytes as it does in neurites, that is, if it can bind to Nrp1 in place of Sema3A, we studied the effect of kCer on HaCaT cell migration activity. Using a trans-well chamber assay, we compared the effects of Sema3A and kCer on serum-derived cell migration activity. kCer showed Sema3A-like suppression of cell migration activity and induction of cellular Cofilin phosphorylation. In addition, kCer and Sema3A inhibited histamine (His)-enhanced migration of immature HaCaT cells. We have demonstrated that kCer does not interact with histaime receptors H1R or H4R directly, but we speculate that kCer may transduce a signal downstream of the His signaling pathway.
RESUMO
Ginkgolic acid obtained as a sphingomyelin synthase inhibitor from a plant extract library inspired the concept of sphingolipid mimics. Ginkgolic acid-derived N-acyl anilines and ginkgolic acid 2-phosphate (GA2P) respectively mimic ceramide and sphingosine 1-phosphate (S1P) in structure and function. The GA2P-induced phosphorylation of ERK and internalization of S1P receptor 1 (S1P1) indicated potent agonist activity. Docking studies revealed that GA2P adopts a similar binding conformation to the bound ligand ML5, which is a strong antagonist of S1P1.
Assuntos
Produtos Biológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Salicilatos/farmacologia , Esfingolipídeos/agonistas , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Animais , Produtos Biológicos/síntese química , Produtos Biológicos/química , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Receptores de Lisoesfingolipídeo/metabolismo , Salicilatos/síntese química , Salicilatos/química , Esfingolipídeos/metabolismo , Relação Estrutura-Atividade , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Sphingomyelin (SM) is required for cells to proliferate, but the reason is not fully understood. In order to asses this question, we employed a cell line, ZS, which lacks both SMS1 and SMS2, isolated from mouse embryonic fibroblasts in SMS1 and 2 double knockout mouse, and SMS1 or SMS2 re-expressing cells, ZS/SMS1 or ZS/SMS2, respectively. We investigated regulation of SM in activating the mammalian target of rapamycin (mTOR) signal induced by essential amino acids (EAA), using these cells. EAA-stimulated mTOR signal was more activated in ZS/SMS1 and ZS/SMS2 cells than in controls. Treatment with methyl-b-cyclodextrin dramatically inhibited the activation. Interestingly, we found that the expression of CD98, LAT-1 and ASCT-2, amino acid transporters concerned with mTOR activation, was down-regulated in ZS cells. Transporters localized in microdomains and formed a functional complex. Our results indicate that SM affect proliferation through the transport of amino acids via SM-enriched microdomains.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Esfingomielinas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismoRESUMO
GPR120 is a receptor for long chain fatty acids and is expressed in small intestinal endocrine cells, L cells and adipose tissue. Activation of GPR120 promotes the secretion of incretin GLP-1, which is known to have effects on anti-metabolic syndrome. As such, GPR120 is a potential target of pharmaceuticals for type II diabetes. In this study, we performed ligand-screening for GPR120 on glycero- and sphingo-type lipids and their derivatives using a Transforming Growth Factor α-shedding assay. We found that phytosphingosine (PHS) activates GPR120 in a manner comparable to the natural ligand α-linolenic acid (ALA) and superior to that of the synthetic ligand GW9508. The IC50 value of PHS was 33.4 µM, of ALA was 31.0 µM and of GW9508 was 41.7 µM. Additionally, PHS-induced activation of GPR120 was inhibited by the specific antagonist AH7614. Many of the natural or synthetic ligands found thus far are compounds with carboxyl groups. However, PHS does not possess a carboxyl group, suggesting that its manner of interaction with GPR120 may be significantly different from that of other ligands. Since PHS is rich in the plasma membrane of yeast, our results imply that PHS found in fermented food could have effects on anti-diabetes through activation of GPR120.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Esfingosina/análogos & derivados , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Conformação Molecular , Esfingosina/farmacologia , Relação Estrutura-AtividadeAssuntos
Membrana Celular/química , Membrana Celular/metabolismo , Esfingomielinas/metabolismo , Animais , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma. To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not. It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation.
Assuntos
Mediadores da Inflamação/imunologia , Inflamação/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Esfingomielinas/imunologia , Transferases (Outros Grupos de Fosfato Substituídos)/imunologia , Animais , Células Cultivadas , Fatores Imunológicos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Esfingomielinas/químicaRESUMO
Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus which infects target cells via the envelope protein JEV-E. However, its cellular targets are largely unknown. To investigate the role of sphingomyelin (SM) in JEV infection, we utilized SM-deficient immortalized mouse embryonic fibroblasts (tMEF) established from SM synthase 1 (SMS1)/SMS2 double knockout mice. SMS deficiency significantly reduced both intracellular and extracellular JEV levels at 48 h after infection. Furthermore, after 15 min treatment with JEV, the early steps of JEV infection such as attachment and cell entry were also diminished in SMS-deficient tMEFs. The inhibition of JEV attachment and infection were recovered by overexpression of SMS1 but not SMS2, suggesting SMS1 contributes to SM production for JEV attachment and infection. Finally, intraperitoneal injection of JEV into SMS1-deficient mice showed an obvious decrease of JEV infection and its associated pathologies, such as meningitis, lymphocyte infiltration, and elevation of interleukin 6, compared with wild type mice. These results suggest that SMS1-generated SM on the plasma membrane is related in JEV attachment and subsequent infection, and may be a target for inhibition of JEV infection.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Encéfalo/patologia , Encéfalo/virologia , Membrana Celular/virologia , Chlorocebus aethiops , Encefalite Japonesa/patologia , Encefalite Japonesa/virologia , Fibroblastos/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Células Vero , Ligação ViralRESUMO
[This corrects the article DOI: 10.1186/s40064-016-2950-6.].
RESUMO
BACKGROUND: The Japanese traditional cuisine, Washoku, considered to be responsible for increased longevity among the Japanese, comprises various foods fermented with the non-pathogenic fungus Aspergillus oryzae (koji). We have recently revealed that koji contains an abundant amount of glycosylceramide. Intestinal microbes have significant effect on health. However, the effects of koji glycosylceramide on intestinal microbes have not been studied. MATERIALS AND METHODS: Glycosylceramide was extracted and purified from koji. C57BL/6N mice were fed a diet containing 1 % purified koji glycosylceramide for 1 week. Nutritional parameters and faecal lipid constituents were analyzed. The intestinal microbial flora of mice on this diet was investigated. RESULTS: Ingested koji glycosylceramide was neither digested by intestinal enzymes nor was it detected in the faeces, suggesting that koji glycosylceramide was digested by the intestinal microbial flora. Intestinal microbial flora that digested koji glycosylceramide had an increased ratio of Blautia coccoides. Stimulation of B. coccoides growth by pure koji glycosylceramide was confirmed in vitro. CONCLUSIONS: Koji functions as a prebiotic for B. coccoides through glycosylceramide. Since there are many reports of the effects of B. coccoides on health, an increase in intestinal B. coccoides by koji glycosylceramide might be the connection between Japanese cuisine, intestinal microbial flora, and longevity.
RESUMO
In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.
Assuntos
Aspergillus/fisiologia , Grão Comestível/microbiologia , Aromatizantes/metabolismo , Glucosilceramidas/metabolismo , Membranas/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Grão Comestível/metabolismo , Etanol/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was â¼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.
Assuntos
HIV-1/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Transferases (Outros Grupos de Fosfato Substituídos)/fisiologia , Internalização do Vírus , Actinas/metabolismo , Animais , Membrana Celular/enzimologia , Membrana Celular/virologia , Ativação Enzimática , Quinase 2 de Adesão Focal/metabolismo , Expressão Gênica , Humanos , Células Jurkat , Camundongos Knockout , Multimerização Proteica , Transporte Proteico , Receptores de HIV/metabolismo , Ligação ViralRESUMO
Elevated levels of amyloid-ß peptide (Aß) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular Aß can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in Aß metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped Aß and with the associated Aß were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-ß precursor protein transgenic mice resulted in marked reductions in Aß levels, amyloid depositions, and Aß-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for Aß binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for Aß by carrying it on the exosome surface GSLs and suggest a role of exosomes in Aß clearance in the central nervous system. Improving Aß clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Exossomos/metabolismo , Glicoesfingolipídeos/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismoRESUMO
The public health burden of metabolic syndrome (MetS), a multiplex risk factor that arises from insulin resistance accompanying abnormal adipose conditions, and Alzheimer's disease (AD), the most common form of dementia, continues to expand. Current available therapies for these disorders are of limited effectiveness. Recent findings have indicated that alternations in sphingolipid metabolism contribute to the development of these pathologies. Sphingolipids are major constituents of the plasma membrane, where they are known to form several types of microdomains, and are potent regulators for a variety of physiological processes. Many groups, including ours, have demonstrated that membrane sphingolipids, especially ceramide and its metabolites such as ceramide 1-phosphate, have roles in arteriosclerosis, obesity, diabetes, and inflammation associated with MetS. Aberrant sphingolipid profiles have been observed in human AD brains, and accumulated evidence has demonstrated that changes in membrane properties induced by defective sphingolipid metabolism impair generation and degradation of amyloid-ß peptide (Aß), a pathogenic agent of AD. In this review, we summarize current knowledge and pathophysiological implications of the roles of SLs in MetS and AD, to provide insight into the SL metabolic pathways as potential targets for therapy of these diseases. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Ceramidas/metabolismo , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , HumanosRESUMO
Sphingolipids are major constituents of the plasma membrane, where they are known to form lipid microdomains with cholesterol. Lipid microdomains are thought to be important not only for cellular signal transduction but also for the absorption of extracellular lipids or nutrients. Inhibition of sphingolipid biosynthesis suggested an importance for sphingolipids in fatty acid uptake via lipid microdomains. Additionally, we recently reported that the function of lipid microdomains was dynamically regulated by the sphingomyelin synthase SMS2 on the plasma membrane and that SMS2-deficient mice exhibit resistance against high-fat diet-induced increases in body weight, glucose intolerance, and fatty liver. Now, biosynthesis or metabolism of sphingolipids is thought to be involved in obesity, diabetes, and cardiovascular diseases. In this review, I focus on the functions of sphingolipids in lipid microdomains and describe their contributions to obesity and diabetes.
Assuntos
Lipídeos/química , Microdomínios da Membrana/metabolismo , Animais , Humanos , Metabolismo dos Lipídeos/fisiologia , Microdomínios da Membrana/genéticaRESUMO
We have previously reported that phytoceramide and phytosphingosine (PHS) stimulated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in cells. PPARγ is a therapeutic target for type 2 diabetes. We found in this study that an oral administration of PHS improved diet-induced glucose intolerance in mice. Since PHS is highly expressed in yeast, PHS in fermented foods may improve diabetes.
Assuntos
Ceramidas/administração & dosagem , Diabetes Mellitus Tipo 2/dietoterapia , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Intestino Delgado/metabolismo , Fígado/metabolismo , Esfingosina/análogos & derivados , Adiponectina/genética , Adiponectina/metabolismo , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Expressão Gênica , Intolerância à Glucose/prevenção & controle , Teste de Tolerância a Glucose , Intestino Delgado/química , Fígado/química , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/genética , Oxirredutases/deficiência , Oxirredutases/genética , PPAR gama/genética , PPAR gama/metabolismo , Esfingosina/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Recent studies have revealed that sphingomyelin (SM) is involved in metabolic syndrome and is a new target of an anti-metabolic syndrome drug. Deficiencies in the enzyme SM synthase 1 (SMS1) result in severe abnormalities, whereas deficiencies in SMS2 do not. SMS1 and SMS2 synthesize SM under similar conditions, so their respective activities cannot be measured separately. We report here on a sensitive, high-throughput and reliable cell-based method to separately measure each SMS activity and to screen for SMS-specific inhibitors, using HPLC and fluorescent ceramide (Cer) analogs. We isolated SMS-null cells and stably transfected them with SMS1 or SMS2. Using these cells, individual SMS activities could be measured separately. Fluorescent Cer, SM, and glucosylceramide analogs could be separated within 4 min by HPLC using an NH(2) column. SMS activities of SMS1- or SMS2-expressing cells seeded in a single well of a 96-well plate could be measured using HPLC and fluorescent Cer analogs. This method clearly demonstrated that treatment of the cells with their respective siRNA or D609, an inhibitor of SMS, resulted in a significant decrease in each SMS activity. These results indicate that our newly developed method can be utilized for screening therapeutics against metabolic syndrome that target SMS2.