Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio i...

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Published in:Molecular and cellular biochemistry 2007-09, Vol.303 (1-2), p.121-130
Main Authors: de Aguirres, Aline B, Mello, Paola A, Andrade, Claudia M. B, Breier, Ana Carolina, Margis, Rogério, Guaragna, Regina M, Borojevic, Radovan, Guma, Fátima C. R, Trindade, Vera M. T
Format: Article
Language:English
Subjects:
Adipocytes
Adipocytes - cytology
Adipocytes - metabolism
Adipocytes - physiology
Animals
Biosynthesis
Biosynthetic Pathways
Cell Line
Ceramide
Fatty acids
Fibroblasts - cytology
Fibroblasts - metabolism
Fumonisin B1
Galactose
Ganglioside synthesis
Gangliosides
Gangliosides - metabolism
Gene expression
Genotype & phenotype
Glycosphingolipid salvage pathways
Golgi apparatus
Hepatic stellate cell
Hepatocytes - cytology
Hepatocytes - metabolism
Inhibitors
Liver
Mice
mRNA
Myoblasts - cytology
Myoblasts - physiology
Palmitoyltransferase
Phenotype
Phenotypes
Proteins
Serine - metabolism
Serine C-Palmitoyltransferase - metabolism
Serine palmitoyltransferase
Sphingolipids - chemistry
Sphingolipids - metabolism
Sphingomyelins - chemistry
Sphingomyelins - metabolism
β-chloroalanine
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Summary:GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM β-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 μM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-¹⁴C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain's composition.
ISSN:0300-8177
1573-4919
DOI:10.1007/s11010-007-9464-z