New approaches to functional elucidation of unknown genes related to lipid metabolism : combination of mutational approaches using C. elegans and metabolite analyses using mass spectrometry

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Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The fatty acyl residues of individual phospholipids appear to be under strict metabolic regulation. In general, saturated fatty acids are esterified at the sn-1 position, whereas polyunsaturated fatty acids (PUFAs), such as arachidonic acid, are commonly found at the sn-2 position. The rapid turnover of the sn-2 acyl chains of glycerophospholipids was originally described by Lands, who proposed that acyl moieties of membrane phospholipids are rapidly metabolized by the action of phospholipase A_2s and subsequently by lysophospholipid acyltransferases (Lands' cycle); however the genes involved in this turnover have not been reported.In this project, we established a screening system to identify genes required for utilization of exogenous PUFAs in C. elegans and found that an uncharacterized gene, which we named mboa-7, is required for incorporation of PUFAs into phosphatidylinositol (P … More I). Incorporation of exogenous PUFA into PI of the living worms and lysoPI acyltransferase (LPIAT) activity in the microsomes were greatly reduced in mboa-7 mutants. Furthermore, the membrane fractions of transgenic worms expressing recombinant mboa-7 and its human homologue exhibited remarkably increased LPIAT activity. mboa-7 encodes a member of the membrane-bound O-acyltransferase family, suggesting that mboa-7 is LPIAT. We also identified an acyltransferase which catalyzes acyltransferase activity toward lysoPC, lysoPS, and lysoPE.In regards to phospholipase A_2s, we generated knockout mice of PAF-AH (II), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids and was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium and hepatocytes. Pafah2^<-/-> mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2% mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl_4) was injected into mice, Pafah2^<-/-> mice showed a delay in hepatic injury recovery. Moreover, following CCl_4 administration, liver levels of the esterified form of 8-iso-PGF_2a, a known in vitro substrate of PAF-AH (II), were higher in Pafah2^<-/-> mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-iso-PGF_2a and protects tissue from oxidative stress-induced injury. Less