Cloning, expression and regulation of l-acylglycerol-3-phosphate acyltransferase (AGAT)

Impaired uptake of nonesterified fatty acids (NEFA) is a component of insulinresistance. A driving force for NEFA uptake is the cytoplasmic NEFA level, which is controlled by triglycéride (TG) synthesis. We have cloned and characterized AGAT. which catalyzes the second step in TO synthesis. A full length c.DNA clone encoding a 283 amino-acid protein (31.7 kDa) and a cDNA clone encoding ihe ("-terminal 171 amino acids were isolated from a human fat cell cDNA library. The primary structure is homologous with yeast and C. elegans AGAT; AGAT has an \-termina! signal sequence, two transmembrane domains, an N glyrosylation site, multiple phosphorylation sites, and two prenyl attachment sites. A 2.4 kb transcript was widely expressed in human tissues with highest expression in skeletal muscle and heart. The AGAT gene is located in the human HLA class III region on chromosome 6. AGAT and a truncated form containing the ("-terminal 171 amino acids were expressed in an AGAT deficient E. Coli strain. Roth transformants contained AGAT activity indicating that the catalytic domain is in the G-terminal portion of the enzyme. AGAT expression during 3T3-I.1 adipocyte differentiation was examined. Northern blol analysis using the entire coding region as a probe showed: 1) high level expression of a 2.4 kb transcript in both 3T3-L1 fibroblasts in exponential growth and in 3T3-L1 adipocytes. 2) a prominent 3 kb message only detected in ft brohlasts. Western blot analysis using an antibody raised against the truncated form showed nearly equal expression in fibroblasts and adipocvtes.