An Animal Cell Mutant with a Deficiency in Acyl/Alkyl-dihydroxyacetone-phosphate Reductase Activity

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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...

Section: Discussionsupporting

confidence: 82%

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

Hajra¹,

Larkins²,

Das³

et al. 2000

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“…Homogenates from one of the isolates, FAA.K1B (lane 3), showed a labeling pattern that was similar to wild-type cells (lane 1). This mutant was further characterized and shown to have a defect in acyl/alkyl-DHAP reductase (the third step in ether lipid biosynthesis) as reported in the accompanying paper (29). We chose FAA.K1A (lane 2), an FAO Ϫ strain, for further analysis in this study.…”

Section: Tritium Suicide Selection Of Fao-deficient Mutants-ourmentioning

confidence: 99%

Isolation of Animal Cell Mutants Defective in Long-chain Fatty Aldehyde Dehydrogenase

James¹,

Zoeller

1997

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Using tritium suicide, we have isolated a variant of the Chinese hamster ovary cell line, CHO-K1, that is deficient in long-chain fatty alcohol:NAD ؉ oxidoreductase (FAO; EC 1.1.1.192). Specifically, it was the fatty aldehyde dehydrogenase component that was affected. The enzymatic deficiency found in this mutant strain, designated FAA.K1A, was similar to that displayed by fibroblasts from patients with Sjö gren-Larsson syndrome (SLS), an inheritable neurocutaneous disorder. Complementation analyses suggested that the deficiency in fatty alcohol oxidation in the FAA.K1A cells and the SLS fibroblasts is a result of lesions in homologous genes. The FAA.K1A cells were unable to convert long chain fatty aldehydes to the corresponding fatty acids. This resulted in a hypersensitivity of the FAA.K1A cells to the cytotoxic effects of long chain fatty aldehydes. The difference between the mutant and wild-type cells was most obvious when using fatty aldehydes between 14 and 20 carbons, with the greatest difference between wild-type and mutant cells found when using octadecanal. Fibroblasts from a patient with SLS also displayed the hypersensitivity phenotype when compared with FAldDH ؉ human fibroblasts. In both CHO and human FAldDH ؊ cell lines, addition of long chain fatty aldehydes to the medium caused a dramatic increase in aldehyde-modified phosphatidylethanolamine, presumably through Schiff's base addition to the primary amine of the ethanolamine head group. When 25 M hexadecanal was added to the growth medium, approximately 10% of the phosphatidylethanolamine was found in the fatty aldehyde-modified form in FAA.K1A, although this was not observed in wild-type cells. Modified phosphatidylethanolamine could be detected in FAldDH ؊ cells even when exogenous fatty aldehydes were not added to the medium. We propose a possible role for fatty aldehydes, or other aldehydic species, in mediating some of the symptoms associated with Sjö gren-Larsson syndrome.

“…For phospholipid composition, steady-state labeling with 32 P i was used (13). Cells were plated into sterile glass scintillation vials (2.5 ϫ 10 4 cells/vial) at 33°C and allowed to attach overnight.…”

Section: Materials-1-o-mentioning

confidence: 99%

Isolation of Novel Animal Cell Lines Defective in Glycerolipid Biosynthesis Reveals Mutations in Glucose-6-phosphate Isomerase

Haller

Smith

Liu

et al. 2010

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Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 3 Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.