大鼠腦組織及人類神經細胞株SH-SY5Y表現之1-acyl-sn-glycerol-3-phosphate acyltransferase的受質特性及年齡對其基因表現影響之探討

Abstract

1-acyl-sn-glycerol-3-phosphate acyltransferases (AGPATs), also known as lysophophatidic acid acyltransferase (LPAAT), are involved in the transfer of fatty acid, mostly unsaturated, to the sn-2 carbon during de novo synthesis of glycerolipids, such as phospholipids and triacylglycerol. At least nine isoforms of AGPATs have been identified based on activity and/or sequence similarity. All of them have two conserved motifs, NHX4D and EGTR. It is well known that the sn-2 fatty acid of phospholipids of the frontal cortex, hippocampus and retina of human and most animals are rich in docosahexainoic aicd (DHA, 22:6n-3). Accumulating evidences showed that DHA is important for the functions of neurons. Since AGPAT is responsible for the esterification of sn-2 position of lysophosphatidic acid(LPA), there may have a tissue specific AGPAT that responsible for the accumulation of DHA in brain. Among all the AGPATs, the AGPAT4 mRNA is mostly expressed in brain. We therefore speculate that AGPAT4 may involve in accumulation of DHA in the brain. To investigate the relationship of AGPAT4 on DHA accumulation in neurons, the expression levels of AGPAT4 mRNA and DHA content in rat brains were determined. Results showed that both AGPAT4 mRNA and DHA content in the brain are decreased with increasing of age. Moreover, the level of AGPAT4 mRNA of the DHA deficient rats was higher than that of the control rats. In order to investigate the function of AGPAT4, an expression plasmid containing rat AGPAT4 cDNA was constructed and transfected into SH-SY5Y cells. After transfection, the cells were given with a variety of fatty acids (FAs) for 24h and the FA composition in the cell was analyzed by gas chromatography (GC). Compared with control cells, SH-SY5Y cells transfected with AGPAT4 incorporated higher DHA and α-linolenic acid (ALA, 18:3n-3) but not eicosapentaenoic acid (EPA), arachidonic acid (AA), palmitoleic acid (16:1), and miristic acid (14:0) compared with that in cells transfected with vector. After incubating with 0-50μM of DHA for 24 hours, the incorporation of DHA in AGPAT4 transfected SH-SY5Y cells increased in a dose-dependent manner, and the variation between SH-SY5Y with or without AGPAT4 overexpression was gradually different. Moreover, we differentiated SH-SY5Y to a neuronal-like state using a combination of retinoic acid and mitotic inhibitors. Both of the expression level of Agpat4 and DHA incorporation increase during cell differentiation. Knockdown the AGPAT4 expression by specific siRNAs resulted in decreased DHA content of phospholipids in SH-SY5Y cell. Taken together, this study showed that AGPAT4 may play a important function in DHA accumulation in neurons and in the SH-SY5Y differentiation.