RESUMO
OBJECTIVE: Methylsulfonylmethane (MSM), which contains organic sulphur, has been used for a long time as a medicinal ingredient because of its benefits to human health. MSM is reported to be protective against certain skin disorders, but it is unknown whether it affects melanin synthesis. Therefore, in our current research, we examined the possibility of MSM controlling the production of melanin in Mel-Ab melanocytes. METHODS: In Mel-Ab cells, melanin contents and tyrosinase activities were assessed and quantified. The expression of microphthalmia-associated transcription factor (MITF) and tyrosinase was evaluated using western blot analysis, while MSM-induced signalling pathways were investigated. RESULTS: The MSM treatment significantly resulted in a dose-dependent increase in melanin production. Furthermore, MSM elevated melanin-related proteins, including MITF and tyrosinase. However, the rate-limiting enzyme of melanin production, tyrosinase, was not directly influenced by it. Therefore, we investigated potential melanogenesis-related signalling pathways that may have been triggered by MSM. Our findings showed that MSM did not influence the signalling pathways associated with glycogen synthase kinase 3ß, cAMP response-element binding protein, extracellular signal-regulated kinase, or p38 mitogen-activated protein kinase. However, MSM phosphorylated c-Jun N-terminal kinases/stress-activated protein kinase (JNK/SAPK), which is known to induce melanogenesis. SP600125, a specific JNK inhibitor, inhibited MSM-induced melanogenesis. CONCLUSION: Taken together, our study indicates that MSM induces melanin synthesis and may serve as a therapeutic option for hypopigmentary skin disorders such as vitiligo.
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BACKGROUND: Ceramide, a bioactive signaling sphingolipid, has long been implicated in cancer. Members of the ceramide synthase (CerS) family determine the acyl chain lengths of ceramides, with ceramide synthase 4 (CerS4) primarily generating C18-C20-ceramide. Although CerS4 is known to be overexpressed in breast cancer, its role in breast cancer pathogenesis is not well established. METHODS: To investigate the role of CerS4 in breast cancer, public datasets, including The Cancer Genome Atlas (TCGA) and two Gene Expression Omnibus (GEO) datasets (GSE115577 and GSE96058) were analyzed. Furthermore, MCF-7 cells stably overexpressing CerS4 (MCF-7/CerS4) as a model for luminal subtype A (LumA) breast cancer were produced, and doxorubicin (also known as Adriamycin [AD])-resistant MCF-7/ADR cells were generated after prolonged treatment of MCF-7 cells with doxorubicin. Kaplan-Meier survival analysis assessed the clinical significance of CERS4 expression, while Student's t-tests or Analysis of Variance (ANOVA) compared gene expression and cell viability in different MCF-7 cell lines. RESULTS: Analysis of the public datasets revealed elevated CERS4 expression in breast cancer, especially in the most common breast cancer subtype, LumA. Persistent CerS4 overexpression in MCF-7 cells activated multiple cancer-associated pathways, including pathways involving sterol regulatory element-binding protein, nuclear factor kappa B (NF-κB), Akt/mammalian target of rapamycin (mTOR), and ß-catenin. Furthermore, MCF-7/CerS4 cells acquired doxorubicin, paclitaxel, and tamoxifen resistance, with concomitant upregulation of ATP-binding cassette (ABC) transporter genes, such as ABCB1, ABCC1, ABCC2, ABCC4, and ABCG2. MCF-7/CerS4 cells were characterized by increased cell migration and epithelial-mesenchymal transition (EMT). Finally, CERS4 knockdown in doxorubicin-resistant MCF-7/ADR cells resulted in reduced activation of cancer-associated pathways (NF-κB, Akt/mTOR, ß-catenin, and EMT) and diminished chemoresistance, accompanied by ABCB1 and ABCC1 downregulation. CONCLUSIONS: Chronic CerS4 overexpression may exert oncogenic effects in breast cancer via alterations in signaling, EMT, and chemoresistance. Therefore, CerS4 may represent an attractive target for anticancer therapy, especially in LumA breast cancer.
Assuntos
Neoplasias da Mama , Esfingosina N-Aciltransferase , Feminino , Humanos , Transportadores de Cassetes de Ligação de ATP , beta Catenina/genética , beta Catenina/metabolismo , Neoplasias da Mama/patologia , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Esfingosina N-Aciltransferase/genética , Células MCF-7RESUMO
(1) Background: six mammalian ceramide synthases (CerS1-6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22-C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.
Assuntos
Asma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Esfingosina N-Aciltransferase/deficiência , Células Th17/imunologia , Células Th2/imunologia , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Camundongos , Camundongos Knockout , Ovalbumina/toxicidade , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Esfingosina N-Aciltransferase/imunologia , Células Th17/patologia , Células Th2/patologiaRESUMO
Sphingosine kinases (SK) catalyze the phosphorylation of sphingosine to generate sphingosine-1-phosphate. Two isoforms of SK (SK1 and SK2) exist in mammals. Previously, we showed the beneficial effects of SK2 inhibition, using ABC294640, in a psoriasis mouse model. However, ABC294640 also induces the degradation of SK1 and dihydroceramide desaturase 1 (DES1). Considering these additional effects of ABC294640, we re-examined the efficacy of SK2 inhibition in an IMQ-induced psoriasis mouse model using a novel SK2 inhibitor, HWG-35D, which exhibits nM potency and 100-fold selectivity for SK2 over SK1. Topical application of HWG-35D ameliorated IMQ-induced skin lesions and normalized the serum interleukin-17A levels elevated by IMQ. Application of HWG-35D also decreased skin mRNA levels of interleukin-17A, K6 and K16 genes induced by IMQ. Consistent with the previous data using ABC294640, HWG-35D also blocked T helper type 17 differentiation of naïve CD4+ T cells with concomitant reduction of SOCS1. Importantly, HWG-35D did not affect SK1 or DES1 expression levels. These results reaffirm an important role of SK2 in the T helper type 17 response and suggest that highly selective and potent SK2 inhibitors such as HWG-35D might be of therapeutic use for the treatment of psoriasis.
Assuntos
Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Psoríase/tratamento farmacológico , Psoríase/imunologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Humanos , Imiquimode/toxicidade , Técnicas In Vitro , Interleucina-17/sangue , Interleucina-17/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Psoríase/induzido quimicamente , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologiaRESUMO
Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Inibidores Enzimáticos/farmacologia , Lisofosfolipídeos/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Psoríase/tratamento farmacológico , Esfingosina/análogos & derivados , Administração Tópica , Animais , Diferenciação Celular/efeitos dos fármacos , Ceramidases/antagonistas & inibidores , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Imiquimode , Imunidade/efeitos dos fármacos , Inflamação/genética , Interleucina-17/sangue , Masculino , Camundongos , Psoríase/induzido quimicamente , Psoríase/patologia , Quinolonas/farmacologia , RNA Mensageiro/metabolismo , Esfingosina/biossíntese , Proteína 1 Supressora da Sinalização de Citocina , Células Th17RESUMO
Sortilin, a member of the vacuolar protein sorting 10 domain receptor family, traffics newly synthesized proteins from the trans-Golgi network to secretory pathways, endosomes, and cell surface. Sortilin-trafficked molecules, including IL-6 and acid sphingomyelinase (aSMase), mediate cholangiocyte proliferation and liver inflammation, hepatic stellate cell activation, hepatocyte apoptosis, and fibrosis. Based on these sortilin-regulated functions, we investigated its role in biliary damage leading to hepatocellular injury and fibrosis. Sortilin-/- mice displayed impaired inflammation and ductular reaction 3 days after bile duct ligation (BDL), as demonstrated by reduced cholangiocyte proliferation and activation and reduced serum IL-6. Interestingly, liver fibrosis was reduced in Sortilin-/- mice after both BDL and carbon tetrachloride treatment, in line with attenuated in vitro activation of Sortilin-/- hepatic stellate cells. Sortilin-/- hepatic aSMase activity was reduced in the BDL and carbon tetrachloride models and accompanied by reduced in vivo hepatocyte apoptosis. In addition, wild type (WT), but not Sortilin-/- hepatocytes, had increased aSMase-dependent susceptibility to bile acid-induced apoptosis in vitro. Mechanistically, short-term IL-6 neutralization in bile duct-ligated WT mice decreased hepatic inflammation and reactive cholangiocyte-derived cytokines and chemokines, without affecting fibrosis, whereas pharmacological inhibition of aSMase activity was not sufficient to attenuate hepatic fibrosis. Only combined IL-6 and aSMase inhibition significantly reduced fibrosis in bile duct-ligated WT mice. We conclude that sortilin regulates cholestatic liver damage and fibrosis via effects on both aSMase activity and serum IL-6.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Apoptose , Ductos Biliares/patologia , Colestase/complicações , Hepatócitos/patologia , Cirrose Hepática/patologia , Fígado/lesões , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proliferação de Células , Quimiocinas/metabolismo , Colestase/patologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Ligadura , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Camundongos Endogâmicos C57BL , Testes de Neutralização , Fenótipo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Alveolar echinococcosis (AE) caused by Echinococcus multilocularis metacestode is frequently associated with deleterious zoonotic helminthiasis. The growth patterns and morphological features of AE, such as invasion of the liver parenchyme and multiplication into multivesiculated masses, are similar to those of malignant tumors. AE has been increasingly detected in several regions of Europe, North America, Central Asia, and northwestern China. An isoform of E. multilocularis antigen B3 (EmAgB3) shows a specific immunoreactivity against patient sera of active-stage AE, suggesting that EmAgB3 might play important roles during adaptation of the parasite to hosts. However, expression patterns and biochemical properties of EmAgB3 remained elusive. The protein profile and nature of component proteins of E. multilocularis hydatid fluid (EmHF) have never been addressed. In this study, we conducted proteome analysis of EmHF of AE cysts harvested from immunocompetent mice. We observed the molecular and biochemical properties of EmAgB3, including differential transcription patterns of paralogous genes, macromolecular protein status by self-assembly, distinct oligomeric states according to individual anatomical compartments of the worm, and hydrophobic ligand-binding protein activity. We also demonstrated tissue expression patterns of EmAgB3 transcript and protein. EmAgB3 might participate in immune response and recruitment of essential host lipids at the host-parasite interface. Our results might contribute to an in depth understanding of the biophysical and biological features of EmAgB3, thus providing insights into the design of novel targets to control AE.
Assuntos
Equinococose Hepática/imunologia , Echinococcus multilocularis/imunologia , Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita , Lipoproteínas/imunologia , Proteoma/imunologia , Adaptação Fisiológica/genética , Adaptação Fisiológica/imunologia , Animais , Clonagem Molecular , Modelos Animais de Doenças , Equinococose , Equinococose Hepática/genética , Equinococose Hepática/parasitologia , Equinococose Hepática/patologia , Echinococcus multilocularis/crescimento & desenvolvimento , Echinococcus multilocularis/patogenicidade , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Homeostase/genética , Homeostase/imunologia , Humanos , Lipoproteínas/genética , Camundongos , Ácido Oleico/metabolismo , Proteoma/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long-chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very-long acyl chain ceramides with concomitant increase of long chain bases and C16-ceramides, were more susceptible to dextran sodium sulphate-induced colitis, and their survival rate was markedly decreased compared with that of wild-type littermates. Using mixed bone-marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule-A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco-2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2-knockdown via CRISPR-Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long-chain bases and C16-ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC-dextran levels, indicating that altered SLs including deficiency of very-long-chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.
Assuntos
Ceramidas/deficiência , Colite/metabolismo , Colo/metabolismo , Esfingosina N-Aciltransferase/genética , Animais , Sistemas CRISPR-Cas , Células CACO-2 , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/mortalidade , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Ácidos Graxos Monoinsaturados/farmacologia , Edição de Genes , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Permeabilidade , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Esfingosina N-Aciltransferase/deficiência , Análise de SobrevidaRESUMO
Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on ß-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus.
Assuntos
Transdiferenciação Celular , Técnicas de Reprogramação Celular , Células Secretoras de Insulina/citologia , Células-Tronco Mesenquimais/citologia , Tonsila Palatina/citologia , Tecido Adiposo/citologia , Animais , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Diabetes Mellitus Experimental/cirurgia , Humanos , Insulina/farmacologia , Células Secretoras de Insulina/transplante , Mercaptoetanol/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Tonsila Palatina/efeitos dos fármacos , Selênio/farmacologia , Sinaptotagminas/deficiência , Transferrina/farmacologiaRESUMO
Ceramide is located at a key hub in the sphingolipid metabolic pathway and also acts as an important cellular signaling molecule. Ceramide contains one acyl chain which is attached to a sphingoid long chain base via an amide bond, with the acyl chain varying in length and degree of saturation. The identification of a family of six mammalian ceramide synthases (CerS) that synthesize ceramide with distinct acyl chains, has led to significant advances in our understanding of ceramide biology, including further delineation of the role of ceramide in various pathophysiologies in both mice and humans. Since ceramides, and the complex sphingolipids generated from ceramide, are implicated in disease, the CerS might potentially be novel targets for therapeutic intervention in the diseases in which the ceramide acyl chain length is altered. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
Assuntos
Ceramidas/metabolismo , Doença , Terapia de Alvo Molecular , Oxirredutases/antagonistas & inibidores , Esfingolipídeos/metabolismo , Animais , Humanos , Camundongos , Oxirredutases/metabolismoRESUMO
Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in the liver but not in the adipose tissue or skeletal muscle of the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [VH]-palmitate was also abrogated in the hepa- tocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in the CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids.
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Ácidos Graxos/metabolismo , Fígado/metabolismo , Esfingolipídeos/química , Esfingolipídeos/metabolismo , Acilação , Animais , Antígenos CD36/metabolismo , Membrana Celular/metabolismo , Ceramidas/metabolismo , Dependovirus/metabolismo , Dieta Hiperlipídica , Proteínas de Transporte de Ácido Graxo/metabolismo , Absorção Intestinal , Camundongos , Oxirredução , Transporte Proteico , Esfingosina N-Aciltransferase/deficiência , Esfingosina N-Aciltransferase/metabolismo , Triglicerídeos/metabolismoRESUMO
BACKGROUND & AIMS: Sortilin traffics newly synthesized molecules from the trans-Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). METHODS: DIO in C57BL/6 (WT) and sortilin(-/-) mice was induced by high-fat diet feeding for 10 weeks. RESULTS: Sortilin(-/-) mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin(-/-) mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin(-/-) hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin(-/-) mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. CONCLUSIONS: Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.
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Proteínas Adaptadoras de Transporte Vesicular/genética , Fígado Gorduroso/genética , Hepatócitos/metabolismo , Resistência à Insulina/fisiologia , Obesidade/complicações , RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Western Blotting , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica , Hepatócitos/patologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Sphingolipids have emerged as an important lipid mediator in intracellular signalling and metabolism. Ceramide, which is central to sphingolipid metabolism, is generated either via a de novo pathway, by attaching fatty acyl CoA to a long-chain base, or via a salvage pathway, by degrading pre-existing sphingolipids. As a 'sphingolipid rheostat' has been proposed, the balance between ceramide and sphingosine-1-phosphate has been the object of considerable attention. Ceramide has recently been reported to have a different function depending on its acyl chain length: six ceramide synthases (CerS) determine the specific ceramide acyl chain length in mammals. All CerS-deficient mice generated to date show that sphingolipids with defined acyl chain lengths play distinct pathophysiological roles in disease models. This review describes recent advances in understanding the associations of CerS with various diseases and includes clinical case reports.
Assuntos
Esfingolipídeos/metabolismo , Animais , Ceramidas/metabolismo , Humanos , Camundongos , Transdução de Sinais/fisiologia , Esfingolipídeos/químicaRESUMO
Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as d-galactosamine, CCl4, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.
Assuntos
Ceramidas/biossíntese , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Hepatócitos/metabolismo , Microdomínios da Membrana/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Acetaminofen/efeitos adversos , Acetaminofen/farmacologia , Analgésicos não Narcóticos/efeitos adversos , Analgésicos não Narcóticos/farmacologia , Animais , Tetracloreto de Carbono/toxicidade , Intoxicação por Tetracloreto de Carbono/genética , Intoxicação por Tetracloreto de Carbono/metabolismo , Intoxicação por Tetracloreto de Carbono/patologia , Ceramidas/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Conexinas/genética , Galactosamina/toxicidade , Junções Comunicantes/genética , Junções Comunicantes/patologia , Glutationa/genética , Glutationa/metabolismo , Hepatócitos/patologia , Microdomínios da Membrana/genética , Microdomínios da Membrana/patologia , Camundongos , Camundongos Mutantes , Esfingosina N-Aciltransferase/genética , Proteína beta-1 de Junções ComunicantesRESUMO
UNLABELLED: Sphingolipids are important structural components of cell membranes and act as critical regulators of cell function by modulating intracellular signaling pathways. Specific sphingolipids, such as ceramide, glucosylceramide, and ganglioside GM3, have been implicated in various aspects of insulin resistance, because they have been shown to modify several steps in the insulin signaling pathway, such as phosphorylation of either protein kinase B (Akt) or of the insulin receptor. We now explore the role of the ceramide acyl chain length in insulin signaling by using a ceramide synthase 2 (CerS2) null mouse, which is unable to synthesize very long acyl chain (C22-C24) ceramides. CerS2 null mice exhibited glucose intolerance despite normal insulin secretion from the pancreas. Both insulin receptor and Akt phosphorylation were abrogated in liver, but not in adipose tissue or in skeletal muscle. The lack of insulin receptor phosphorylation in liver correlated with its inability to translocate into detergent-resistant membranes (DRMs). Moreover, DRMs in CerS2 null mice displayed properties significantly different from those in wild-type mice, suggesting that the altered sphingolipid acyl chain length directly affects insulin receptor translocation and subsequent signaling. CONCLUSION: We conclude that the sphingolipid acyl chain composition of liver regulates insulin signaling by modifying insulin receptor translocation into membrane microdomains.
Assuntos
Intolerância à Glucose/etiologia , Resistência à Insulina , Microdomínios da Membrana/efeitos dos fármacos , Esfingolipídeos/metabolismo , Animais , Glicemia/metabolismo , Membrana Celular/efeitos dos fármacos , Ceramidas/metabolismo , Intolerância à Glucose/sangue , Insulina/fisiologia , Fígado/metabolismo , Microdomínios da Membrana/fisiologia , Camundongos , Oxirredutases/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The use of edible insect protein in food products is contingent on their biological effects. Conventional protein extraction methods are not only time-consuming and costly but also energy-intensive. There is a need for alternative techniques that maintain the bioactivities of insect proteins and are environmentally sustainable. This study compares the health functionality of mealworm (Tenebrio molitor larvae) concentrates obtained by conventional methods-alkali and salt (MS) extraction-and nonconventional methods-enzyme (ME) and screw press (MP)-to enhance their applicability despite lower protein concentration. Overall, MP exhibited the highest essential amino acids content, whereas ME showed the highest in vitro digestibility, total phenolic contents, and antioxidant capacities among all the concentrates. ME also had a significant cell proliferative capacity at concentrations ≥500 µg/mL. MS significantly inhibited tumor necrosis factor-alpha and interleukin-1beta secretion in lipopolysaccharide-treated Hep3B cells compared to other samples. As for anti-hyperglycemia effects, treatment with MS and ME for 2 and 5 min significantly increased the p-Akt/Akt ratio (MS, 1.34- and 1.61-fold; ME, 2.26- and 2.70-fold, respectively). In conclusion, enzyme treatment enhanced nutritional value and antioxidant capacity, whereas salt treatment potentially contributed to anti-inflammatory and anti-hyperglycemia activities. Hybrid extraction techniques combining conventional and nonconventional methods are suggested based on target applications, considering health benefits, environmental impact, costs, and efficiencies. PRACTICAL APPLICATION: Four mealworm protein extraction methods (alkali/salt/enzyme/screw press) were compared for their nutritional and biological properties. Alkali extraction enhanced protein content, enzyme treatment improved nutritional value and antioxidant capacity, and salt-assisted extraction exhibited immunomodulatory effects in vitro. Notably, enzyme and salt treatments produced protein concentrates with significant antidiabetic and anti-hyperglycemic properties.
RESUMO
Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein and a member of LGI/epitempin family. We previously showed that LGI3 was highly expressed in brain and played regulatory roles in neuronal exocytosis and differentiation. Besides the nervous system, LGI3 was shown to be expressed in diverse tissues. In this study, we found that LGI3 and its receptor candidate ADAM23 were expressed in adipose tissues and 3T3-L1 cells. 3T3-L1 preadipocytes secreted a 60-kDa protein, a major secreted form of LGI3, which declined with adipocyte differentiation. LGI3 was also expressed in adipose tissue macrophages in the ob/ob mice and in macrophage cell line. The 60-kDa LGI3 protein was selectively increased in the ob/ob adipose tissues comparing with the lean mice. Pull-down experiments, coimmunoprecipitation and immunocytochemistry indicated that LGI3 associated with ADAM23 in adipose tissues and 3T3-L1 cells. Knockdown of LGI3 or ADAM23 by siRNA increased adipogenesis in 3T3-L1 cells. Treatment with LGI3 protein did not affect preadipocyte proliferation but attenuated adipogenesis and this effect was reversed by siRNA-mediated knockdown of ADAM23. Taken together, we propose that LGI3 may be a candidate adipokine that is perturbed in obesity and suppresses adipogenesis through its receptor, ADAM23.
Assuntos
Proteínas ADAM/metabolismo , Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células 3T3-L1 , Proteínas ADAM/genética , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Tecido Adiposo/citologia , Animais , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Expressão Gênica , Imuno-Histoquímica , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Our finding that human skin expresses leucine-rich glioma inactivated 3 (LGI3) raises the question of the function of this cytokine in keratinocytes. We have shown that LGI3 stimulates human HaCaT keratinocyte migration without affecting viability or proliferation. Western blot analysis showed that LGI3 induced focal adhesion kinase activation, Akt phosphorylation, and glycogen synthase kinase 3ß (GSK3ß) phosphorylation in these cells. Using the scratch wound assay and a modified Boyden chamber, we found that LY294002, a selective phosphatidylinositol 3-kinase inhibitor, and LiCl, a selective GSK3ß inhibitor, abolished LGI3-induced cell migration. We tested ß-catenin levels after LGI3 treatment because the Akt-GSK3ß pathway regulates ß-catenin accumulation, and ß-catenin promotes cell migration. LGI3 treatment increased ß-catenin protein and nuclear localization, whereas LY294002 prevented LGI3-induced focal adhesion kinase and Akt activation as well as ß-catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following ß-catenin accumulation through the Akt pathway.
Assuntos
Movimento Celular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Proteínas/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Queratinócitos/fisiologia , Proteínas do Tecido Nervoso , Proteína Oncogênica v-akt/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/citologia , Pele/metabolismo , beta Catenina/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Sarcopenia and obesity are emerging as major social problems. In this study, we examined whether Gryllus bimaculatus (GB), an edible insect, prevents dexamethasone-induced muscle atrophy (sarcopenia) or high-fat diet (HFD)-induced obesity in mice. We generated a standard chow diet (SCD) + GB (85% SCD and 15% GB powder) and HFD + GB (85% HFD and 15% GB powder). SCD + GB feeding increased gains in body weight and white adipose tissue (WAT). Despite no difference in weight change between HFD + GB- and HFD-fed mice, HFD + GB feeding aggravated insulin resistance compared with HFD feeding. SCD + GB or HFD + GB feeding did not change most gene expressions in the liver and WAT but did increase MyHC1 expression in the muscle, meaning that GB increased muscle generation. Therefore, we fed SCD + GB with dexamethasone, which induces muscle degeneration. As a result, muscle fiber size increased, as did grip strength compared with dexamethasone-injected mice. In addition, SCD + GB reduced the expression of muscle degradation factors, such as atrogin1 and muscle RING-finger protein 1 (MuRF1). Furthermore, SCD + GB feeding increased Akt, mTOR, and p70S6K phosphorylation and MyHC1 expression, meaning that it may have increased protein synthesis. In conclusion, GB has great potential for inhibiting dexamethasone-induced muscle mass loss by increasing muscle protein synthesis and inhibiting muscle protein degradation.
RESUMO
Macrophage inhibitory cytokine 1 (MIC-1), which is overproduced in various human cancers and associated with cachexia, acts on the hypothalamus to suppress appetite and reduce body weight. We investigated the mechanisms through which MIC-1 affects bile acid metabolism and gallstone formation, which are poorly understood. Over 6 weeks, male C57BL/6 mice fed either standard chow or a lithogenic diet were intraperitoneally injected with phosphate-buffered saline (PBS) or MIC-1 (200 µg/kg/week). Among lithogenic diet-fed mice, MIC-1 treatment resulted in increased gallstone formation compared with PBS treatment. Compared with PBS treatment, MIC-1 treatment decreased hepatic cholesterol and bile acid levels and reduced expression of HMG-CoA reductase (HMGCR), the master cholesterol metabolism regulator sterol regulatory element-binding protein 2, cholesterol 7α-hydroxylase (CYP7A1), mitochondrial sterol 27-hydroxylase, and oxysterol 7α-hydroxylase. Compared with PBS treatment, MIC-1 treatment had no effect on small heterodimer partner, farnesoid X receptor, or pregnane X receptor expression, and extracellular signal-related kinase and c-Jun N-terminal kinase phosphorylation decreased, suggesting that these factors do not contribute to the MIC-1-induced reduction in CYP7A1 expression. Compared with PBS treatment, MIC-1 treatment increased AMP-activated protein kinase (AMPK) phosphorylation. Treatment with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reduced CYP7A1 and HMGCR expression, whereas the AMPK inhibitor Compound C reversed MIC-1-induced reductions in CYP7A1 and HMGCR expression. Furthermore, in MIC-1-treated mice, total biliary cholesterol levels increased together with increased ATP-binding cassette subfamily G (ABCG)5 and ABCG8 expression. Compared with PBS treatment, MIC-1 treatment did not affect expression of liver X receptors α and ß, liver receptor homolog 1, hepatocyte nuclear factor 4α, or NR1I3 (also known as constitutive androstane receptor), which are upstream of ABCG5/8; however, MIC-1 treatment increased ABCG5/8 expression and promoter activities. Our study indicates that MIC-1 influences gallstone formation by increasing AMPK phosphorylation, reducing CYP7A1 and HMGCR expression, and increasing ABCG5 and ABCG8 expression.