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Structural Characterization of Neutral Glycosphingolipids from 3T3-L1 Adipocytes.

Kojima H, Suzuki Y, Ito M, Kabayama K - Lipids (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Ritsumeikan University, Shiga, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

Thin layer chromatogram showing removal of TAG by DCE-wash. a Authentic GSL mixture with mouse epididymal fat was dried using a nitrogen stream, washed by pipetting with DCE, and separated into DCE-soluble (W) and residual (R) fractions. TAG were completely fractioned into the W fraction. TLC plate was developed by C/M/W, 60:40:10 (v/v/v) and visualized with cupric phosphate reagent. b Large amounts of isolated adipose tissue (mice epididymal fat) were washed with DCE as above. DCE washing also completely fractioned large amounts of TAG into the W fraction. GSL-like bands derived from epididymal fat appeared in the R fraction. c TAG-removal by DCE-wash of TNFα-treated 3T3-L1 adipocytes. Total lipid extract from adipocytes was washed with DCE, developed with C/M/0.2 % CaCl2, 60:40:9 (v/v/v) and visualized by orcinol–H2SO4 and primuline reagents. d Large amounts of TNFα-treated 3T3-L1 adipocyte were washed with DCE as above. Neutral GSL, including CMH and CDH, derived from TNFα-treated 3T3-L1 adipocyte appeared even by orcinol-H2SO4 staining
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Fig1: Thin layer chromatogram showing removal of TAG by DCE-wash. a Authentic GSL mixture with mouse epididymal fat was dried using a nitrogen stream, washed by pipetting with DCE, and separated into DCE-soluble (W) and residual (R) fractions. TAG were completely fractioned into the W fraction. TLC plate was developed by C/M/W, 60:40:10 (v/v/v) and visualized with cupric phosphate reagent. b Large amounts of isolated adipose tissue (mice epididymal fat) were washed with DCE as above. DCE washing also completely fractioned large amounts of TAG into the W fraction. GSL-like bands derived from epididymal fat appeared in the R fraction. c TAG-removal by DCE-wash of TNFα-treated 3T3-L1 adipocytes. Total lipid extract from adipocytes was washed with DCE, developed with C/M/0.2 % CaCl2, 60:40:9 (v/v/v) and visualized by orcinol–H2SO4 and primuline reagents. d Large amounts of TNFα-treated 3T3-L1 adipocyte were washed with DCE as above. Neutral GSL, including CMH and CDH, derived from TNFα-treated 3T3-L1 adipocyte appeared even by orcinol-H2SO4 staining

Mentions: Under the assumption that TAG were the dominant component of total lipids, we obtained mouse epididymal fat and applied the DCE-wash method to test the removal of TAG by our DCE-wash method. We observed that TAG were removed completely by this method (Fig. 1a, b). Next, we applied DCE-wash to 3T3-L1 adipocytes (Fig. 1c). TAG were efficiently removed and GSL-like bands appeared. However, phospholipid-like bands were also detected in abundance just above GM3. Alkaline hydrolysis was necessary to remove the phospholipids. After removing phospholipids via alkaline hydrolysis, groups of bands probably corresponding to CMH and CDH appeared in the residual fraction (Fig. 1d). However, large amounts of GSL were washed off in the DCE-soluble fraction, which may have been caused by large amounts of adipocyte-derived total lipids used for DCE-wash, containing high concentrations of TAG. A sufficient surface area inside the test tube for dried and solidified GSL adhesion is important in DCE-wash. GSL that did not adhere to the test tube wall appear to have been washed away with TAG. This can be prevented by increasing the surface area for solidification, an important step in DCE-wash. Although most GSL were found in the DCE soluble fraction, the 2 GM3 bands were not changed by DCE washing. This indicates that there was no selective loss of molecular species by DCE-wash. We performed molecular species analysis of GSL in the residual fraction using negative ion mode LC/MS/MS (Fig. 2; S1b, S1c, S2; Table 1). Here to define the structures of GSL, we used a device (Shimadzu LCMS-IT-TOF) capable of performing structural analysis by MSn. We set the molecular weight of ceramide in MS3 and checked the appearances of fatty acid and long-chain base fragments (P, R and V, U, T, S; Figs. S1, S2) to identify molecular species. In adipocytes, the major GSL was the GM3 ganglioside, followed by GD1a, CMH, and CDH (Fig. 2); molecular species with d18:1 long-chain base were the most abundant and saturated C16–C24 fatty acids predominated.Fig. 1


Structural Characterization of Neutral Glycosphingolipids from 3T3-L1 Adipocytes.

Kojima H, Suzuki Y, Ito M, Kabayama K - Lipids (2015)

Thin layer chromatogram showing removal of TAG by DCE-wash. a Authentic GSL mixture with mouse epididymal fat was dried using a nitrogen stream, washed by pipetting with DCE, and separated into DCE-soluble (W) and residual (R) fractions. TAG were completely fractioned into the W fraction. TLC plate was developed by C/M/W, 60:40:10 (v/v/v) and visualized with cupric phosphate reagent. b Large amounts of isolated adipose tissue (mice epididymal fat) were washed with DCE as above. DCE washing also completely fractioned large amounts of TAG into the W fraction. GSL-like bands derived from epididymal fat appeared in the R fraction. c TAG-removal by DCE-wash of TNFα-treated 3T3-L1 adipocytes. Total lipid extract from adipocytes was washed with DCE, developed with C/M/0.2 % CaCl2, 60:40:9 (v/v/v) and visualized by orcinol–H2SO4 and primuline reagents. d Large amounts of TNFα-treated 3T3-L1 adipocyte were washed with DCE as above. Neutral GSL, including CMH and CDH, derived from TNFα-treated 3T3-L1 adipocyte appeared even by orcinol-H2SO4 staining
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4541715&req=5

Fig1: Thin layer chromatogram showing removal of TAG by DCE-wash. a Authentic GSL mixture with mouse epididymal fat was dried using a nitrogen stream, washed by pipetting with DCE, and separated into DCE-soluble (W) and residual (R) fractions. TAG were completely fractioned into the W fraction. TLC plate was developed by C/M/W, 60:40:10 (v/v/v) and visualized with cupric phosphate reagent. b Large amounts of isolated adipose tissue (mice epididymal fat) were washed with DCE as above. DCE washing also completely fractioned large amounts of TAG into the W fraction. GSL-like bands derived from epididymal fat appeared in the R fraction. c TAG-removal by DCE-wash of TNFα-treated 3T3-L1 adipocytes. Total lipid extract from adipocytes was washed with DCE, developed with C/M/0.2 % CaCl2, 60:40:9 (v/v/v) and visualized by orcinol–H2SO4 and primuline reagents. d Large amounts of TNFα-treated 3T3-L1 adipocyte were washed with DCE as above. Neutral GSL, including CMH and CDH, derived from TNFα-treated 3T3-L1 adipocyte appeared even by orcinol-H2SO4 staining
Mentions: Under the assumption that TAG were the dominant component of total lipids, we obtained mouse epididymal fat and applied the DCE-wash method to test the removal of TAG by our DCE-wash method. We observed that TAG were removed completely by this method (Fig. 1a, b). Next, we applied DCE-wash to 3T3-L1 adipocytes (Fig. 1c). TAG were efficiently removed and GSL-like bands appeared. However, phospholipid-like bands were also detected in abundance just above GM3. Alkaline hydrolysis was necessary to remove the phospholipids. After removing phospholipids via alkaline hydrolysis, groups of bands probably corresponding to CMH and CDH appeared in the residual fraction (Fig. 1d). However, large amounts of GSL were washed off in the DCE-soluble fraction, which may have been caused by large amounts of adipocyte-derived total lipids used for DCE-wash, containing high concentrations of TAG. A sufficient surface area inside the test tube for dried and solidified GSL adhesion is important in DCE-wash. GSL that did not adhere to the test tube wall appear to have been washed away with TAG. This can be prevented by increasing the surface area for solidification, an important step in DCE-wash. Although most GSL were found in the DCE soluble fraction, the 2 GM3 bands were not changed by DCE washing. This indicates that there was no selective loss of molecular species by DCE-wash. We performed molecular species analysis of GSL in the residual fraction using negative ion mode LC/MS/MS (Fig. 2; S1b, S1c, S2; Table 1). Here to define the structures of GSL, we used a device (Shimadzu LCMS-IT-TOF) capable of performing structural analysis by MSn. We set the molecular weight of ceramide in MS3 and checked the appearances of fatty acid and long-chain base fragments (P, R and V, U, T, S; Figs. S1, S2) to identify molecular species. In adipocytes, the major GSL was the GM3 ganglioside, followed by GD1a, CMH, and CDH (Fig. 2); molecular species with d18:1 long-chain base were the most abundant and saturated C16–C24 fatty acids predominated.Fig. 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Ritsumeikan University, Shiga, Japan.

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.

No MeSH data available.


Related in: MedlinePlus