Paper 6

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 16, pp. 11082–11089, April 21, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

A Regulatory Role for 1-Acylglycerol-3-phosphateO-acyltransferase 2 in Adipocyte Differentiation*
Received for publication, August 31, 2005, and in revised form, January 30, 2006 Published, JBC Papers in Press,February 22, 2006, DOI 10.1074/jbc.M509612200

Sarah E. Gale‡§, Andrey Frolov‡§, Xianlin Han§, Perry E. Bickel§¶1, Li Cao , Anne Bowcock , Jean E. Schaffer‡§**, and Daniel S. Ory‡§¶2 From the ‡Center for Cardiovascular Research and Departments of §Internal Medicine, Genetics, **Molecular Biology and Pharmacology, and ¶Cell Biology and Physiology, School of Medicine, Washington University, St.Louis, Missouri 63110-1010
Mutations in the 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) gene have been identified in individuals affected with congenital generalized lipodystrophy (CGL). AGPAT2 catalyzes acylation of lysophosphatidic acid to phosphatidic acid, a precursor for both triacylglycerol (TAG) and phospholipid synthesis. Recent studies suggest that reduced AGPAT2 enzymaticactivity may underlie the CGL clinical phenotype. To gain insight into how altered AGPAT2 activity causes lipodystrophy, we examined the effect of knockdown of AGPAT2 expression in preadipocytes on TAG synthesis and storage, and on adipocyte differentiation. We show that AGPAT2 mRNA expression is induced 30-fold during adipocyte differentiation and that AGPAT2 enzymatic activity is required for TAGmass accumulation in mature adipocytes. We demonstrate that small interference RNA-mediated knockdown of AGPAT2 expression prevents appropriate early induction of C/EBP and PPAR , key transcriptional activators of the adipogenic program, and delays expression of multiple adipocyte-related genes. The unexpected finding, that levels of several phospholipid species, including phosphatidic acid (PA),are elevated in TAG-depleted adipocytes with AGPAT2 knockdown, suggests that impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synthesis. These findings underscore the importance of an AGPAT2-mediated metabolic pathway in adipocyte differentiation. referred to as lysophosphatidic acid acyltransferase or LPAAT- )encodes an enzyme that catalyzes the acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA), which serves as a precursor for triacylglycerol (TAG) and phospholipid synthesis (4, 5). AGPAT2 is one of five murine lysophosphatidic acid acyltransferase isoforms that have been identified (AGPAT1–5), each of which contains a highly conserved catalytic (NHX4D) and substrate binding motif(EGTR) (6). Although there are differences in the tissue distribution of the AGPAT isoforms, the physiological significance of this diversity is not understood. AGPAT1 and AGPAT3 are ubiquitously expressed, whereas AGPAT2, AGPAT4, and AGPAT5 are expressed in a tissue-specific manner (6). In human tissues AGPAT2 is highly expressed in liver, pancreas, skeletal muscle, and small intestine (7) and is themost highly expressed AGPAT isoform in adipose tissue (3). Structure-function studies of AGPAT2 mutations identified in CGL patients demonstrated reduced conversion of LPA to PA, suggesting that reduced AGPAT2 enzymatic activity may underlie the CGL clinical phenotype (8). Decreased availability of PA for TAG biosynthesis could cause lipodystrophy by resulting in reduced triglyceride synthesisand storage in adipocytes (9). On the other hand, individuals affected with CGL have reduced serum adipokines, such as leptin and adiponectin, suggesting that AGPAT2 function is critical for adipocyte growth and differentiation (9). It is possible that altered AGPAT2 activity causes lipodystrophy by affecting formation of intermediates in the TAG and phospholipid biosynthetic pathways, such as PA...