Induction of the Peroxisomal Glycerolipid-synthesizing Enzymes during Differentiation of 3T3-L1 Adipocytes

Abstract

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using Preadipocyte cell lines (1) have been widely used to study the induction of a number of enzymes and receptors and also the mechanism of differentiation at the genetic level (2, 3). The fibroblast-like mouse 3T3-L1 cells differentiate in response to high concentrations of insulin and other agents to cells that synthesize large amounts of triglycerides and are morphologically similar to adipose cells (4). A similar cell line (3T3-F442A), on transplantation in mice has been reported to develop into adipose tissue (5). During differentiation, lipogenic enzymes of the triglyceride biosynthetic pathway, as well as the ancillary enzymes needed for the formation of substrates and cofactors are induced (6). As outlined in Fig. 1, enzymes starting from the cytosolic glycerophosphate dehydrogenase (GPD), 1 catalyzing the biosynthesis of sn-glycerol 3-phosphate (GP) followed by the glycerophosphate acyltransferase (GPAT), 1-acyl-GP acyltransferase, and diacylglycerol acyltransferase (DAGAT) are highly induced during differentiation (7-9). Coleman and Bell (10) reported that due to nonspecificity of microsomal GPAT, which catalyzes the acylation of both GP and DHAP, DHAPAT activity is also induced during differentiation of these cells. All animal cells, however, have been shown to contain a specific peroxisomal DHAPAT which does not contain GPAT activity (11,12). This DHAPAT, co-localized in peroxisomes with acyl/alkyl DHAP:NADPH reductase and alkyl-DHAP synthase, is generally believed to be involve...

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

“…Second, overexpression of peroxisomal DHAPAT activity in the NRel-4.15 transfectant did not result in increases in plasmalogen biosynthesis or increase their levels above normal. These results are in agreement with the observation that plasmalogen levels are not increased in differentiating 3T3-L1 cells, in which DHAPAT is induced several-fold (16).…”

“…The regulation of plasmalogen biosynthesis is likely to be complex and linked to the biosynthesis of nonether glycerolipids, because later steps in the pathway are also used in the synthesis of diacyl phospholipids and triglycerides (1). Recent evidence also suggests that dihydroxyacetonephosphate acyltransferase (DHAPAT), which catalyzes the first step in plasmalogen biosynthesis, may play a significant role in nonether glycerolipid biosynthesis as well; this activity is increased several-fold during the differentiation of 3T3-L1 cells to adipocytes (16). Therefore, the role of DHAPAT in the synthesis of this latter group of glycerolipids (nonether) remains to be determined.…”

CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes

“…Indeed, we found that many adipogenic markers were upregulated during adipogenesis, e.g. several transcription factors (C/EBP␣, PPAR␥, RXR␣, SREBP-1), enzymes involved in fatty acid and TG metabolism (DHAPAT,FABP4/aP2,FAS,GPAT,SCD1,SCD2), and adipokines (AGT, resistin) (MacDougald and Lane, 1995;Gregoire et al, 1998;Burton et al, 2004;Hajra et al, 2000;Song et al, 2002). We also observed the downregulation of anti-adipogenic markers, such as Pref-1, an autocrine and/or paracrine inhibitor of adipogenesis, and the transcription factors GATA-2 and GATA-3.…”

Thematic Review Series: Glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity

“…These studies found that the mitochondrial isoform accounts for ?50% of total GPAT activity in rat liver and is significantly decreased upon fasting (27,28). The microsomal isoform is the major isoform in brown and white adipose tissue, accounting for 80-90% of total GPAT activity (29,30), and is dramatically upregulated during adipocyte differentiation (16,31,32). Microsomal GPAT is also the major isoform in heart (?70% of total activity) (33,34), and in brain (35).…”

“…This pathway is thought to be of only minor quantitative importance for the formation of triglycerides and glycerophospholipids in most tissues, and functions primarily in the formation of ether phospholipids, as demonstrated by studies in tissue culture cells as well as by mutational analysis in mice and humans (11)(12)(13)(14)(15). However, it can contribute significantly to glycerolipid formation under certain circumstances (e.g., in differentiated 3T3-L1 adipocytes after an overnight culture in glucose-free medium) (16). It is also noteworthy that diacylglycerol can be produced via the monoacylglycerol pathway in a GPAT-independent manner.…”