[Show abstract][Hide abstract] ABSTRACT: In recent years, obesity has been considered a pathological stage of early lifestyle-related diseases, and adipose tissue and adipocyte research has been active. Glycosphingolipids are involved in the pathogenesis of type 2 diabetes induced by insulin resistance, but the details of the glycosphingolipid molecular species composition of adipocytes have yet to be elucidated. We used 3T3-L1 adipocytes and the 1,2-dichloroethane-wash method to remove triacylglycerols, which are abundant in adipocytes, and analyzed the structures of glycosphingolipids, particularly neutral glycosphingolipids, using liquid chromatography–mass spectrometry.
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The online version of this article (doi:10.1007/s11745-015-4035-7) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Muscle atrophy, a reduction of muscle mass, strength, and volume, results from reduced muscle use and plays a key role in various muscular diseases. In the microgravity environment of space especially, muscle atrophy is induced by muscle inactivity. Exposure to microgravity induces muscle atrophy through several biological effects, including associations with reactive oxygen species (ROS). This study used 3D-clinostat to investigate muscle atrophy caused by oxidative stress in vitro, and sciatic denervation was used to investigate muscle atrophy in vivo. We assessed the effect of Oenothera odorata root extract (EVP) on muscle atrophy. EVP helped recover cell viability in C2C12 myoblasts exposed to microgravity for 24 h and delayed muscle atrophy in sciatic denervated mice. However, the expressions of HSP70, SOD1, and ceramide in microgravity-exposed C2C12 myoblasts and in sciatic denervated mice were either decreased or completely inhibited. These results suggested that EVP can be expected to have a positive effect on muscle atrophy by disuse and microgravity. In addition, EVP helped characterize the antioxidant function in muscle atrophy.
Full-text · Article · May 2015 · Evidence-based Complementary and Alternative Medicine
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the effect of plasma irradiation, which is used in plasma medicine on biological tissues (cells). We used phospholipids, the main components of the cell membrane, namely, phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). Reference samples of the three phospholipids were irradiated with an atmospheric-pressure argon (Ar) plasma jet for 5s. The phospholipids irradiated with a plasma jet were separated by thin-layer chromatography (TLC). The TLC results indicated that several degradation products were obtained for PC and PG but not for PE after plasma jet irradiation. One of the degradation products obtained from PC was identified as lysophosphatidylcholine (LPC) and one of the products obtained from PG was identified as lysophosphatidylglycerol (LPG) by liquid chromatography-mass spectrometry. Therefore, PC and PG are degraded into their respective lysophospholipids by plasma jet irradiation, which disrupts the cell membrane.
[Show abstract][Hide abstract] ABSTRACT: To elucidate function of glycolipid raft, analyses by live cell imaging and mass spectrometry were greatly paid attention. Especially, the former has been developed more and more as for methodology. This chapter shows the assay method by FRAP technique and nonlinear curve fitting to measure the fluidity of membrane molecules with confocal laser scanning microscope.
[Show abstract][Hide abstract] ABSTRACT: To clarify the mechanism underlying the inactivation of Escherichia coli (E. coli) cells subjected to atmosphericpressure argon (Ar) plasma jet irradiation, we focused on phospholipids, the main components of the cell wall and membrane of E. coli. Commercially available standard samples of phospholipids, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were irradiated with an atmospheric-pressure Ar plasma jet in air for 5 s. The phospholipids irradiated with the plasma jet were separated by thin-layer chromatography (TLC), and the results showed that PE was unchanged even after plasma jet irradiation, whereas PG was degraded into several substances. One of them was identified as lysophosphatidylglycerol (LPG), a lysophospholipid of PG, by liquid chromatography-mass spectrometry (LC-MS). From these results, we inferred that PG in the cell wall and membrane was degraded into LPG and a fatty acid by plasma jet irradiation, and that the cell wall and membrane were disrupted, inactivating E. coli. [DOI: 10.1380/ejssnt.2014.400]
No preview · Article · Aug 2014 · e-Journal of Surface Science and Nanotechnology
[Show abstract][Hide abstract] ABSTRACT: Extraction of thin-layer chromatography (TLC) spot with organic solvents is one of the convenient methods for glycolipid isolation from crude samples, but other background contaminants are also co-extracted during this process. Calcium sulfate (gypsum), starch and synthetic polymers are commonly used as binders in TLC plates, and the co-extracted synthetic polymers usually interfere with further structural analyses, especially in analysis by mass spectrometry (MS). Several column chromatographic processes are therefore used to purify these extracts. However, the purification processes are time consuming and suffer from sample loss. In our previous study, we established a method for detergent removal from glycolipids in a detergent-resistant membrane microdomain (DRM). The procedure is based on selective detergent extraction, in which the sample is dried in a glass tube, followed by washing with non-polar organic solvents, 1,2-dichloroethane (DCE). In this study, we have attempted to apply this method for the background removal from gangliosides and glycosylinositolphosphoceramides extracted from high performance TLC (HPTLC) spots. After DCE washing, these glycolipids were detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, and it was found that DCE washing could effectively remove HPTLC backgrounds.
No preview · Article · Dec 2013 · Analytical methods
[Show abstract][Hide abstract] ABSTRACT: MLS128 monoclonal antibody, which binds an epitope consisting of two or three consecutive Tn-antigens, inhibits colon cancer cell growth by binding to a 110 kDa glycoprotein (GP). Previous studies suggested a possible association of insulin-like growth factor-I receptor (IGF-IR) signaling in the inhibition of colon cancer cell growth by MLS128 (Morita et al. Biosci Trends. 3, 32-37, 2009; Zamri et al. ibid. 6, 303-312, 2012). The current study thus investigated the nature of 110 kDa GP and its possible association with IGF-IR. MLS128 treatment for 3 days caused down-regulation of IGF-IR and disappearance of 110 kDa GP in HT29 colon cancer cells. Immunoprecipitation/immunoblotting experiments did not reveal a direct association between the two molecules in HT29 cells. In LS180 and HT29 cells, however, 110 kDa GP and IGF-IR were found in microdomains. Treatment of these cells with MLS128 for 3 days caused a reduction in the IGF-IR and 110 kDa GP associated with microdomains. Two-dimensional gel electrophoresis/MLS128 immunoblotting of HT29 and LS180 cell lysates and immunoprecipitates revealed three spots, from which tryptic peptides were recovered for protein sequencing. Identification of 110 kDa GP was unsuccessful due to its heterogeneity and resistance to tryptic digestion. During this study, however, limited proteolysis of 110 kDa GP was observed in the microdomain-associated 110 kDa GP from HT29 and LS180 cells, suggesting that protease-susceptible sites or domains exist in the middle of 110 kDa GP. This information on limited proteolysis may provide a clue to identifying 110 kDa GP.
No preview · Article · Oct 2013 · Bioscience trends
[Show abstract][Hide abstract] ABSTRACT: Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4-methylumbelliferyl 5-acetyl neuraminic acid as a substrate. The main purpose of this study is to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP-40 or DOC/NP-40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same isoelectric point to that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris-buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane-rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2-transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.
Full-text · Article · Jun 2013 · Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: The Ludwig-Soret effect for aqueous solutions of α-cyclodextrin
(CD), β-CD, γ-CD, and glucose was studied by thermal
diffusion forced Rayleigh scattering (TDFRS) in the temperature range of
15-55°C. The Soret coefficient and the thermal diffusion coefficient
show the sign inversion behavior from negative to positive with
increasing temperature. The values of the temperature dependence of the
Soret coefficient for α-CD, β-CD, and γ-CD are
identical at all temperatures measured, although the Soret coefficient
of glucose is positive at all temperatures. The studies of dye
concentration dependence for CDs and glucose show an effect of the
inclusion ability of CDs on thermal diffusion behavior.
[Show abstract][Hide abstract] ABSTRACT: Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III(3)(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.
Full-text · Article · Aug 2012 · Glycoconjugate Journal
[Show abstract][Hide abstract] ABSTRACT: Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes, and the notion of the "raft/microdomain" gives specificity to this concept. Recently, fluorescence-based techniques such as fluorescence recovery after photobleaching (FRAP), single particle tracking (SPT), and fluorescence correlation spectroscopy (FCS) have shown promise for application to the dynamics of membrane molecules in microdomains. We previously revealed, by performing live-cell FRAP and SPT studies, a mechanism of insulin resistance in which dissociation of the insulin receptor (IR)-caveolin-1 (Cav1) complex was caused by an interaction between the IRβ subunit and the ganglioside GM3 cluster, a glycolipid-enriched membrane microdomain. We hoped to demonstrate that an alteration in the lipid component of microdomains affects lateral diffusion of membrane receptors. We therefore established an experimental system for monitoring the membrane organization of receptors by analyzing their lateral diffusion parameters in the plasma membranes of living cells using FRAP and SPT. In this study, measurement of the lateral diffusion of the IR was performed by fitting analysis to fluorescence recovery curves and trace analysis to individual fluorescent spots, which provided the diffusion constant. The results show how fast IR molecules diffuse before and after a change in membrane environment, such as stimulation by cholesterol depression or treatment with a glycosphingolipid (GSL) inhibitor. Using these techniques, we have established a method for determining the diffusion constant for the lateral movement of IR-EGFP, expressed in CHO-K1 cells. We will use these techniques for the lateral diffusion analysis of membrane receptors under other assay conditions, such as use of GSL-deficient cells or pathologic samples.
[Show abstract][Hide abstract] ABSTRACT: Although detergents are often essential in protocols, they are usually incompatible with further biochemical analysis. There are several methods for detergent removal, but the procedures are complicated or suffer from sample loss. Here, we describe a convenient and rapid method for detergent removal from sialic acid-containing glycosphingolipids (gangliosides) and neutral glycolipids in detergent-resistant membrane (DRM) microdomain. It is based on selective detergent extraction, in which the sample is dried on a glass tube, followed by washing with organic solvent. We investigated 18 organic solvents and used high performance thin-layer chromatography (HPTLC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) to confirm that dichloroethane (DCE) was the most suitable solvent and completely removed the nonionic detergent Triton X-100. Furthermore, DCE extraction effectively removed interference caused by other nonionic, zwitterionic, or ionic detergents in MALDI-QIT-TOF MS analysis.
No preview · Article · Jan 2012 · The Journal of Lipid Research
[Show abstract][Hide abstract] ABSTRACT: Previously, we demonstrated that an inhibitor of ganglioside biosynthesis, d-PDMP, could restore impaired insulin signaling in tumor necrosis factor α (TNFα)-treated adipocytes by blocking the increase of GM3 ganglioside. Here, we analyzed the interaction between insulin receptor (IR) and GM3 in the plasma membranes using immunoelectron microscopy. In normal adipocytes, most GM3 molecules localized at planar and non-caveolar regions. Approximately 19% of IR molecules were detected in caveolar regions. The relative ratio of IRs associated with caveolae in TNFα-treated adipocytes was decreased to one-fifth of that in normal adipocytes, but this decrease was restored by d-PDMP. Thus, we could obtain direct evidence that insulin resistance is a membrane microdomain disorder caused by aberrant expression of ganglioside.
[Show abstract][Hide abstract] ABSTRACT: The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function.
Full-text · Article · Jul 2009 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Caveolae are a subset of membrane microdomains (lipid raft) particularly abundant in adipocytes. Critical dependence of the
insulin metabolic signal transduction on caveolae/microdomains in adipocytes has been demonstrated. These microdomains can
be biochemically isolated with their detergent insolubility and were designated as detergent resistant microdomains (DRM).
Gangliosides are known as structurally and functionally important components in microdomains. We demonstrated that increased
GM3 expression was accompanied in the state of insulin resistance in mouse 3T3-L1 adipocytes induced by TNFα and in the adipose
tissues of obese/diabetic rodent models such as Zucker fa/fa rats and ob/ob mice (Tagami et al. 2002). We examined the effect of TNFα on the composition and function of DRM in adipocytes and demonstrated that increased GM3
levels result in the elimination of insulin receptor (IR) from the DRM while caveolin and flotillin remain in the DRM, leading
to the inhibition of insulin’s metabolic signaling (Kabayama et al. 2005). These findings are further supported by the report that mice lacking GM3 synthase exhibit enhanced insulin signaling (Yamashita et al. 2003). Thus, we present a new pathological feature of insulin resistance in adipocytes induced by TNFα.
[Show abstract][Hide abstract] ABSTRACT: Expression of gangliosides and alterations in their composition have been observed during cell proliferation and differentiation and in certain cell cycle phases, brain development and cancer malignancy. To investigate the characteristics of GM3 synthase, SAT-I mRNA and ganglioside GM3 expression levels in lung cancer, we examined the expression levels of SAT-I mRNA as well as GM3 in 40 tumor tissues surgically removed from non-small cell lung cancer patients. Adenocarcinoma tissues expressed SAT-I mRNA levels that were significantly higher than those of squamous and other carcinomas (P < 0.0001). Moreover, the SAT-I mRNA levels were high in the bronchioalveolar carcinoma subtype and low in the solid and mucin subtypes of adenocarcinomas (P = 0.049, 0.049 and 0.013, respectively). To clarify the relationship between SAT-I mRNA and epidermal growth factor receptor (EGFR)-tyrosine kinase (TK) inhibitor sensitivity, we carried out drug sensitivity tests for the EGFR-TK inhibitors gefitinib and AG1478 using eight adenocarcinoma cell lines expressing no EGFR mutations. The IC(50) values for gefitinib and AG1478 decreased dramatically with increasing SAT-I mRNA levels (R(2) = 0.81 and 0.59, respectively), representing a wide range of drug sensitivities among adenocarcinoma cell lines. To explore a possible mechanism of how GM3 could enhance the sensitivity to EGFR-TK inhibitors, the SAT-I gene was introduced stably into a GM3-negative clone of murine 3LL lung cancer cells to produce GM3-reconstituted clones. We found an increase in EGFR protein levels and gefitinib sensitivity in GM3-reconstituted cells, suggesting the involvement of GM3 in the turnover of EGFR protein. Therefore, it is highly expected that, by measuring the expression levels of SAT-I mRNA in lung biopsy samples from non-small cell lung cancer patients, enhanced pathological identification and individualized chemotherapeutic strategies can be established for the appropriate use of EGFR-TK inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNFalpha-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR-Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR beta-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR-Cav1 complex by the interactions of IR with GM3 in microdomains.
Full-text · Article · Sep 2007 · Proceedings of the National Academy of Sciences