RESUMO
Background: Many of the drugs used for obesity treatment have adverse effects on the central nervous system. Therefore, novel treatments, such as peripherally acting drugs, are needed. Monoacylglycerol acyltransferase 2 (MGAT2), highly expressed in the small intestine, catalyzes the first step of triacylglycerol re-synthesis. MGAT2 inhibition suppresses food intake in high-fat diet (HFD)-fed mice, but the mechanisms remain unclear. Here, the involvement of the vagus nerve in MGAT2 inhibition-induced feeding suppression was investigated. Methods: Fasted mice were administered an MGAT2 inhibitor. Food intake was measured after HFD re-feeding, and the effect of capsaicin pretreatment on changes in food intake was evaluated. The number of c-fos-positive cells in the nucleus tractus solitarius and levels of appetite regulators were determined after HFD re-feeding or lipid gavage. Results: The anorectic effect of the MGAT2 inhibitor was abolished when vagus nerve function was interrupted by capsaicin. MGAT2 inhibition increased the number of c-fos-positive cells in the nucleus tractus solitarius and elevated intestinal oleoylethanolamide, plasma peptide tyrosine-tyrosine and plasma glucagon-like peptide-1 levels. Conclusion: MGAT2 inhibition suppresses feeding behavior via peripheral vagus nerve signaling and may serve as a novel anti-obesity strategy with a low risk of unexpected central nervous system-related adverse effects.
RESUMO
Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) that may lead to liver cirrhosis or hepatocellular carcinoma. Here, we examined the diagnostic utility of tri-antennary tri-sialylated mono-fucosylated glycan of alpha-1 antitrypsin (AAT-A3F), a non-invasive glycobiomarker identified in a previous study of NASH diagnosis. This study included 131 biopsy-proven Japanese patients with NAFLD. We evaluated the utility of AAT-A3F in NASH diagnosis, and conducted genetic analysis to analyse the mechanism of AAT-A3F elevation in NASH. Serum AAT-A3F concentrations were significantly higher in NASH patients than in NAFL patients, and in patients with fibrosis, lobular inflammation, and ballooning. Hepatic FUT6 gene expression was significantly higher in NASH than in NAFL. IL-6 expression levels were significantly higher in NASH than in NAFL and showed a positive correlation with FUT6 expression levels. The serum-AAT-A3F levels strongly correlated with hepatic FUT6 expression levels. AAT-A3F levels increased with fibrosis, pathological inflammation, and ballooning in patients with NAFLD and may be useful for non-invasive diagnosis of NASH from the early stages of fibrosis.
Assuntos
Biomarcadores/sangue , Hepatopatia Gordurosa não Alcoólica/diagnóstico , alfa 1-Antitripsina/sangue , Adulto , Idoso , Área Sob a Curva , Feminino , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Glicosilação , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Polissacarídeos/química , Polissacarídeos/metabolismo , Curva ROC , alfa 1-Antitripsina/metabolismoRESUMO
Sialic acids form the terminal sugars in glycan chains on glycoproteins via α2,3, α2,6, or α2,8 linkages, and structural isomers of sialyl linkages play various functional roles in cell recognition and other physiological processes. We recently developed a novel procedure based on sialic acid linkage-specific alkylamidation via lactone ring opening (aminolysis-SALSA). Herein, we have investigated an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts. One limitation of SALSA using amine hydrochloride salts may be solved by adding only tert-butylamine (t-BA) as an acid scavenger, and comparative and quantitative glycomic analyses can be performed using iSALSA. We also developed quantitative glycomic analysis using dual isotope-labeled glycans by derivatizing with aminooxy-functionalized tryptophanylarginine methyl ester (aoWR) and iSALSA at the reducing and nonreducing end, respectively. Furthermore, we demonstrate that the amount of α2,3-linked sialoglycans in serum are altered during liver fibrosis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography MS (LC/MS) analyses. We revealed that the ratio of A33,6,6 to A3F3,6,6 was gradually decreased along with liver fibrosis progression. Therefore, these glycan alterations are potential diagnostic markers of nonalcoholic steatohepatitis (NASH) fibrosis progression.
Assuntos
Glicômica/métodos , Ácido N-Acetilneuramínico/química , Polissacarídeos/química , Aminas/química , Biomarcadores , Glicoproteínas/química , Humanos , Marcação por Isótopo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Serum N-glycans have been reported to be potential diagnostic and therapeutic biomarkers for many diseases and conditions, such as inflammation, fibrosis, and cancer progression. We previously described the focused protein glycomic analysis (FPG) from gel-separated serum proteins. With this methodology, we sought novel glycan biomarkers for nonalcoholic steatohepatitis (NASH) and successfully identified some N-glycans that were significantly elevated in NASH patients compared to nonalcoholic fatty liver patients. Among them, trisialylated monofucosylated triantennary glycan (A3F) of alpha-1 antitrypsin showed the most dynamic change. For rapid identification of N-glycans on the focused proteins, we constructed a simplified method called immunoprecipitation glycomics (IPG), where the target proteins were immunoprecipitated with affinity beads and subsequently subjected to glycomic analysis by MALDI-TOF MS. Focusing on alpha-1 antitrypsin and ceruloplasmin as the target proteins, we compared the values of N-glycans determined by FPG and IPG. The quantified values of each N-glycan by these two methods showed a statistically significant correlation, indicating that high throughput and quantitative N-glycomics of targeted proteins can be achieved by the simplified IPG method. Thus, an analytical strategy combining FPG and IPG can be adapted to general biomarker discovery and validation in appropriate disease areas.
Assuntos
Glicômica , Hepatopatia Gordurosa não Alcoólica/sangue , Polissacarídeos/sangue , alfa 1-Antitripsina/sangue , Biomarcadores/sangue , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/isolamento & purificação , Ceruloplasmina/metabolismo , Feminino , Glicosilação , Humanos , Imunoprecipitação/métodos , Fígado/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Polissacarídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , alfa 1-Antitripsina/genéticaRESUMO
Most advanced knee osteoarthritis (OA) patients experience chronic pain resistant to cyclooxygenase (COX) inhibitors. However, the cells and molecules involved in this advanced OA pain remain poorly understood. In this study, we developed a rat model of advanced knee OA by modification of the monoiodoacetate-induced OA pain model and examined involvement of synovial macrophages in advanced OA pain. Cyclooxygenase inhibitors, such as celecoxib and naproxen, and a steroid were ineffective, but an opioid and anti-nerve growth factor (NGF) antibody was effective for pain management in the advanced OA model. Similar to advanced OA patients, histological analysis indicated severe bone marrow damages, synovitis, and cartilage damage and an increase of macrophages with high expression of interleukin-1ß, NGF, nitric oxide synthase (NOS) 1, NOS2, and COX-2 in the knee joint of the advanced OA model. Intravenous injection of clodronate liposomes depleted synovial macrophages, which decreased the level of not only proinflammatory mediator interleukin-1ß but also NGF in the knee joint, leading to pain suppression in the advanced OA model. These data suggest the involvement of synovial macrophages in advanced knee OA pain resistant to COX inhibitors by increasing proinflammatory mediators, and that drugs targeting synovial macrophages might have potent analgesic effects.
Assuntos
Celecoxib/uso terapêutico , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Macrófagos/patologia , Osteoartrite do Joelho/complicações , Dor/etiologia , Líquido Sinovial/citologia , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Força da Mão/fisiologia , Ácido Iodoacético/toxicidade , Masculino , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Osteoartrite do Joelho/induzido quimicamente , Medição da Dor , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Líquido Sinovial/metabolismo , Suporte de Carga/fisiologiaRESUMO
Hepatocellular carcinoma (HCC) is the major subtype of primary liver cancer, and is typically diagnosed late in its course. Considering the limitations and the reluctance of patients to undergo a liver biopsy, a reliable, noninvasive diagnostic marker that predicts and assesses the treatment and prognosis of HCC is needed. With recent technological advances of mass spectrometry, glycomics is gathering momentum and holds substantial potential to discover new glycan markers in cancer research. Here, to discover specific glycan markers for the early diagnosis of HCC, we analyzed the glycan profiles of gel-separated serum proteins of progressive liver disease model mice. By focused protein glycomics of 12 gel-separated glycoproteins using sera from the mouse models, we revealed the entire profile of glycans in each major serum protein. We found that the levels of trisialylated triantennary glycans of haptoglobin and vitamin D-binding protein increased significantly as the disease progressed, while the alteration in these protein levels were modest. Furthermore, these glycan increases were not observed in age-matched control mice. In conclusion, our approach has identified specific glycan marker candidates for the early diagnosis of HCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Haptoglobinas/metabolismo , Neoplasias Hepáticas/metabolismo , Polissacarídeos/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Animais , Carcinoma Hepatocelular/sangue , Modelos Animais de Doenças , Progressão da Doença , Glicômica , Glicoproteínas/sangue , Glicosilação , Neoplasias Hepáticas/sangue , Camundongos , Isoformas de Proteínas/metabolismoRESUMO
Transient receptor potential vanilloid 4 (TRPV4) receptor modulates pain, and this has been noted in several animal models. However, the involvement of TRPV4 in osteoarthritic (OA) pain remains poorly understood. This study assessed the functional changes in TRPV4 and the expression of its endogenous ligand 5,6-epoxyeicosatrienoic acid (5,6-EET) in a rat monoiodoacetate (MIA)-induced OA pain model (MIA rats). Monoiodoacetate-treated rats showed reduced grip strength as compared to sham-treated rats, and this loss in function could be recovered by the intraarticular administration of a TRPV4 antagonist (HC067047 or GSK2193874). By contrast, the intraarticular administration of the TRPV4 agonist, GSK1016790A, increased the pain-related behaviors in MIA rats but not in sham rats. TRPV4 expression was not increased in knee joints of MIA rats; however, the levels of phosphorylated TRPV4 at Ser824 were increased in dorsal root ganglion neurons. In addition, 5,6-EET was increased in lavage fluids from the knee joints of MIA rats and in meniscectomy-induced OA pain model rats. 5,6-EET and its metabolite were also detected in synovial fluids from patients with OA. In conclusion, TRPV4 was sensitized in the knee joints of MIA rats through phosphorylation in dorsal root ganglion neurons, along with an increase in the levels of its endogenous ligand 5,6-EET. The analgesic effects of the TRPV4 antagonist in the OA pain model rats suggest that TRPV4 may be a potent target for OA pain relief.
Assuntos
Artrite Experimental/metabolismo , Osteoartrite/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Artrite Experimental/induzido quimicamente , Modelos Animais de Doenças , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Força da Mão , Ácido Iodoacético , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Osteoartrite/induzido quimicamente , Dor , Medição da Dor , Fosforilação , Pirróis/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
OBJECTIVE: 18F-fluoromisonidazole (FMISO), a well-known PET imaging probe for diagnosis of hypoxia, is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of the nitro group. Previously, we showed the majority of 18F-FMISO was incorporated into low-molecular-weight metabolites in hypoxic tumors, and the glutathione conjugate of reduced FMISO (amino-FMISO-GS) distributed in the tumor hypoxic regions as revealed by imaging mass spectrometry (IMS). The present study was conducted to clarify whether FMISO is metabolized to amino-FMISO-GS within tumor cells and how amino-FMISO-GS contributes to FMISO accumulation in hypoxic cells. We also evaluated the relationship between FMISO accumulation and the glutathione conjugation-related factors in the cells. METHODS: Tumor cells (FaDu, LOVO, and T24) were treated with 18F-FMISO and incubated under normoxic or hypoxic conditions for 4 h. The FMISO metabolites were analyzed with LC-ESI-MS. Several glutathione conjugation-related factors of tumor cells were evaluated in vitro. FaDu tumor-bearing mice were intravenously injected with 18F-FMISO and the tumors were excised at 4 h post-injection. Autoradiography, IMS and histologic studies were performed. RESULTS: Amino-FMISO-GS was the main contributor to FMISO incorporated in hypoxic FaDu cells in vitro and in vivo. Total FMISO uptake levels and amino-FMISO-GS levels were highest in FaDu, followed by LOVO, and then T24 (total uptake: 0.851 ± 0.009 (FaDu), 0.617 ± 0.021 (LOVO) and 0.167 ± 0.006 (T24) % dose/mg protein; amino-FMISO-GS: 0.502 ± 0.035 (FaDu), 0.158 ± 0.013 (LOVO), and 0.007 ± 0.001 (T24) % dose/mg protein). The glutathione level of FaDu was significantly higher than those of LOVO and T24. The enzyme activity of glutathione-S-transferase catalyzing the glutathione conjugation reaction in FaDu was similar levels to that in LOVO, and was higher than that in T24. Quantitative RT-PCR analysis revealed that the expression levels of efflux transporters of the glutathione conjugate (multidrug resistance-associated protein 1) were lowest in FaDu, followed by LOVO, and then T24. CONCLUSIONS: FMISO accumulates in hypoxic cells through reductive metabolism followed by glutathione conjugation. We illustrated the possibility that increased production and decreased excretion of amino-FMISO-GS contribute to FMISO accumulation in tumor cells under hypoxic conditions.
Assuntos
Glutationa/metabolismo , Misonidazol/análogos & derivados , Neoplasias Experimentais/metabolismo , Oxigênio/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Hipóxia Tumoral , Linhagem Celular Tumoral , Humanos , Taxa de Depuração Metabólica , Misonidazol/farmacocinética , Neoplasias Experimentais/diagnóstico por imagem , Ligação Proteica , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC50 of 0.95µM against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity.
Assuntos
Antivirais/química , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Serina Endopeptidases/metabolismo , Dengue/virologia , Vírus da Dengue/enzimologia , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologiaRESUMO
BACKGROUND: Olmsted syndrome (OS) is a congenital dermatosis characterized by palmoplantar keratoderma and periorificial keratotic plaque. TRPV3 (transient receptor potential vanilloid subtype 3) encodes a thermosensitive Ca2+ channel and is the causative gene of OS. However, the molecular mechanism that causes the pathological development of OS is unclear. OBJECTIVE: We aimed to investigate the molecular mechanisms underlying OS pathology from the perspective of lipid metabolism. METHODS: Comprehensive lipidomics and microarray analyses were conducted on tissue samples from a non-lesional skin area of OS model rats (Ht rats) and from wild type (WT) rats as the control. RESULTS: Infiltration of leukocytes such as eosinophils and neutrophils and an increase in the fibrotic region were detected in the unaffected skin area of Ht rats compared with the WT rats. Among about 600 lipid species examined, the levels of 15-lipoxygenase (LOX) metabolites, the precursors of anti-inflammatory and pro-resolving lipid mediators, and dihydroceramides decreased by ≥16-fold in Ht rats compared with WT rats. Consistent with the decreases in the 15-LOX metabolites, expression levels of the genes that encode the 15-LOXs, Alox15 and Alox15b, were largely reduced. Conversely, increased expression levels were detected of Il36b, Ccl20, Cxcl1, and Cxcl2, which encode cytokines/chemokines, and S100a8 and S100a9, which encode the Ca2+ binding proteins that are implicated in epidermal proliferation. CONCLUSION: The pro-inflammatory state in the unaffected skin of Ht rats caused by decreases in 15-LOX metabolites and increases in cytokines/chemokines may contribute to the pathogenesis of OS.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Citocinas/metabolismo , Epiderme/fisiologia , Ceratodermia Palmar e Plantar/metabolismo , Metabolismo dos Lipídeos/genética , Animais , Animais Geneticamente Modificados , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Células Epidérmicas , Epiderme/metabolismo , Epiderme/ultraestrutura , Ceratodermia Palmar e Plantar/genética , Leucócitos , Microscopia Eletrônica , Ratos , Síndrome , Canais de Cátion TRPV/genéticaRESUMO
Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-ß1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-ß signaling pathway. Our findings regarding the effects of AM251 on the TGF-ß signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.
Assuntos
Túbulos Renais/efeitos dos fármacos , Piperidinas/farmacologia , Pirazóis/farmacologia , Linhagem Celular Transformada , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Humanos , Túbulos Renais/citologiaRESUMO
Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS). Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH), implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH.
Assuntos
Glutationa/metabolismo , Espectrometria de Massas , Neoplasias Experimentais , Nitroimidazóis , Animais , Hipóxia Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Nitroimidazóis/farmacocinética , Nitroimidazóis/farmacologiaRESUMO
Model mice are frequently used in drug discovery research. Knowledge of similarities and differences between the mouse and human glycomes is critical when model mice are used for the discovery of glycan-related biomarkers and targets for therapeutic intervention. Since few comparative glycomic studies between human and mouse have been conducted, we performed a comprehensive comparison of the major classes of glycans in human and mouse sera using mass spectrometric and liquid chromatographic analyses. Up to 131 serum glycans, including N-glycans, free oligosaccharides (fOSs), glycosaminoglycans, O-glycans, and glycosphingolipid (GSL)-glycans, were quantified. In both serum samples, N-glycans were the most abundant in the total serum glycome, while fOSs were the least abundant. As expected, the diversity of sialic acid (i.e. Neu5Ac vs. Neu5Gc) was the major species difference between human and mouse in terms of N- and O-glycosylation, while GSL-glycomic profiles were completely different, even when the sialic acid diversity was taken into consideration. Furthermore, total serum glycomics of STAM mouse were unveiled as an initial step to identify novel biomarkers of liver diseases, with which we could identify several glycans with expression significantly increased or decreased expression.
Assuntos
Proteínas Sanguíneas/biossíntese , Glicômica/métodos , Proteoma/genética , Animais , Biomarcadores/sangue , Proteínas Sanguíneas/genética , Cromatografia Líquida , Glicosilação , Humanos , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sphingomyelin synthase 2 (SMS2) is a proposed potential therapeutic target for obesity and insulin resistance. However, the contributions of SMS2 to glucose metabolism in tissues and its possible therapeutic mechanisms remain unclear. Thus, to determine whole-body glucose utilization and the contributions of each insulin-targeted tissue to glucose uptake, we performed a glucose kinetics study, using the radiolabeled glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG), in wild-type (WT) and SMS2 knockout (KO) mice. Insulin signaling was enhanced in the liver, white adipose tissue and skeletal muscle of SMS2 KO mice compared with those of WT mice. In addition, compared with in WT mice, blood clearance of (18)F-FDG was accelerated in SMS2 KO mice when they were fed either a normal or a high fat diet. (18)F-FDG uptake was also increased in insulin-targeted tissues such as skeletal muscle in the SMS2 KO mice. Whereas skeletal muscle sphingolipid content was not clearly affected, plasma levels of very long-chain fatty acid (VLCFA)-containing ceramides were markedly increased in SMS2 KO mice, compared with in WT mice. We also generated liver-conditional SMS2 KO mice and performed glucose and insulin tolerance tests on mice with a high fat diet. However, no significant effect was observed. Thus, our study provided evidence that genetic inhibition of SMS2 elevated glucose clearance through activation of glucose uptake into insulin-targeted tissues such as skeletal muscle by a mechanism independent of hepatic SMS2. Our findings further indicate that this occurs, at least in part, via indirect mechanisms such as elevation of VLCFA-containing ceramides.
Assuntos
Tecido Adiposo Branco/enzimologia , Glucose/metabolismo , Resistência à Insulina , Fígado/enzimologia , Músculo Esquelético/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Gorduras na Dieta/farmacologia , Glucose/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18-C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18-C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.
Assuntos
Rim/metabolismo , Obesidade/metabolismo , Esfingolipídeos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Ceramidas/metabolismo , Dieta Hiperlipídica , Humanos , Rim/lesões , Rim/patologia , Fígado/lesões , Fígado/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Knockout , Obesidade/patologia , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
RATIONALE: Targeted oxidized fatty acid analysis has been widely used to understand the roles of fatty acids in the development of diseases. However, because of the extensive structural diversity of fatty acids, it is considered that unknown lipid metabolites will remain undetected. Here, to discover and identify unknown lipid metabolites in biological samples, a global analytical system and a method of synthesizing lipid standards were investigated. METHODS: Oxidized fatty acids in mouse lung tissues were extracted using mixed-mode spin columns. Separation was achieved via ultra-high-performance liquid chromatography, mass spectrometric (MS) analysis was conducted in full scan mode using a Q Exactive Plus instrument equipped with an electrospray ionization probe, and structure analysis was carried out by high-resolution data-dependent tandem mass spectrometry (dd-MS(2)). In addition, lipid standards, which are not commercially available, were synthesized by bioconversion using Bacillus circulans. RESULTS: Oxidized fatty acids in mouse lung tissues were analyzed by high-resolution accurate-mass analysis, and multiple unknown molecules were discovered and tentatively identified using high-resolution dd-MS(2). Among these molecules, 21-hydroxydocosahexaenoic acid (21-HDoHE) and 22-HDoHE, which are not commercially available, were synthesized by bioconversion. By comparing the exact masses, retention times, and characteristic fragment ions of the synthesized standards, 21-HDoHE and 22-HDoHE were definitively identified in the mouse lung tissue. CONCLUSIONS: Our strategy of global analysis and bioconversion can be used for the discovery and identification of unknown lipid molecules.
Assuntos
Bacillus/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/química , Animais , Ácidos Graxos/metabolismo , Pulmão/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , OxirreduçãoRESUMO
(18)F-fluoromisonidazole (FMISO) has been widely used as a hypoxia imaging probe for diagnostic positron emission tomography (PET). FMISO is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of its nitro group. However, its detailed accumulation mechanism remains unknown. Therefore, we investigated the chemical forms of FMISO and their distributions in tumours using imaging mass spectrometry (IMS), which visualises spatial distribution of chemical compositions based on molecular masses in tissue sections. Our radiochemical analysis revealed that most of the radioactivity in tumours existed as low-molecular-weight compounds with unknown chemical formulas, unlike observations made with conventional views, suggesting that the radioactivity distribution primarily reflected that of these unknown substances. The IMS analysis indicated that FMISO and its reductive metabolites were nonspecifically distributed in the tumour in patterns not corresponding to the radioactivity distribution. Our IMS search found an unknown low-molecular-weight metabolite whose distribution pattern corresponded to that of both the radioactivity and the hypoxia marker pimonidazole. This metabolite was identified as the glutathione conjugate of amino-FMISO. We showed that the glutathione conjugate of amino-FMISO is involved in FMISO accumulation in hypoxic tumour tissues, in addition to the conventional mechanism of FMISO covalent binding to macromolecules.
Assuntos
Diagnóstico por Imagem/métodos , Hipóxia/diagnóstico por imagem , Espectrometria de Massas/métodos , Metaboloma , Misonidazol/análogos & derivados , Sondas Moleculares/metabolismo , Animais , Masculino , Metabolômica , Camundongos Endogâmicos BALB C , Camundongos Nus , Misonidazol/metabolismo , Peso Molecular , Neoplasias/diagnóstico por imagem , Cintilografia , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 µl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1-21.4 µM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.
Assuntos
Glicosídeo Hidrolases/metabolismo , Glicoesfingolipídeos/sangue , Cromatografia em Camada Fina , Glicoesfingolipídeos/metabolismo , Humanos , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and regulatory synthases is not known. It has been reported that Cer synthase (CerS) has restricted substrate specificity, whereas SMS has no specificity for different lengths of acyl-chains. We hypothesized that the distribution of each SM molecular species was regulated by expression of the CerS family. Thus, we compared the distribution of SM species and CerS mRNA expression using molecular imaging. Spatial distribution of each SM molecular species was investigated using ultra-high-resolution imaging mass spectrometry (IMS). IMS revealed that distribution of SM molecular species varied according to the lengths of acyl-chains found in each brain section. Furthermore, a combination study using in situ hybridization and IMS revealed the spatial expression of CerS1 to be associated with the localization of SM (d18:1/18:0) in cell body-rich gray matter, and CerS2 to be associated with SM (d18:1/24:1) in myelin-rich white matter. Our study is the first comparison of spatial distribution between SM molecular species and CerS isoforms, and revealed their distinct association in the brain. These observations were demonstrated by suppression of CerS2 using siRNA in HepG2 cells; that is, siRNA for CerS2 specifically decreased C22 very long-chain fatty acid (VLCFA)- and C24 VLCFA-containing SMs. Thus, histological analyses of SM species by IMS could be a useful approach to consider their molecular function and regulative mechanism.
Assuntos
Encéfalo/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingomielinas/biossíntese , Esfingosina N-Aciltransferase/metabolismo , Animais , Química Encefálica/fisiologia , Células Hep G2 , Humanos , Masculino , CamundongosRESUMO
O-Linked glycosylation of serine/threonine residues is a posttranslational modification of proteins and is essential for protein recognition and lipid functions on cell surfaces and within cells. The characterization of differently structured O-linked glycans (O-glycans) is particularly challenging because there is no known endoglycosidase for such groups. Therefore, chemical digestion approaches have been widely used; however, it is sometimes difficult to suppress unwanted side reactions. Recently, we reported a novel O-glycomics procedure using ß-elimination in the presence of pyrazolone analogues (BEP). In the present study, we describe a microwave (MW)-assisted BEP procedure for rapid and quantitative O-glycomic analysis. Following optimization of the reaction conditions, the MW-assisted BEP reaction substantially improved the recovery of total O-glycans from model glycoproteins (PSM) and the reaction time was reduced from 16 to 2 h. Combined with sequential solid-phase extractions, this MW-assisted BEP procedure enabled O-glycomic analyses of various biological samples.