ABSTRACT
Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid metabolism. The lipid-lowering effects of 10,12 CLA were unique, as other trans FA did not reduce TG content to the same extent. Using low levels of [(14)C]-CLA isomers, it was shown that both isomers were readily incorporated into acylglycerols and phospholipids, albeit at lower levels than [(14)C]-oleic or [(14)C]-linoleic acids. When using [(14)C]-acetic acid and [(14)C]-pyruvic acid as substrates, 30 µM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3-24 h. Treatment with 30 µM 10,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within 7-12 h, respectively. The mRNA levels of liver X receptor (LXR)α and sterol regulatory element binding protein (SREBP)-1c, transcription factors that regulate SCD-1, were decreased by 10,12 CLA within 5 h. These data suggest that the isomer-specific decrease in de novo lipid synthesis by 10,12 CLA is due, in part, to the rapid repression of lipogenic transcription factors that regulate MUFA synthesis, suggesting an anti-obesity mechanism unique to this trans FA.
Subject(s)
Adipocytes/drug effects , Adipocytes/metabolism , Linoleic Acids, Conjugated/pharmacology , Lipid Metabolism/drug effects , Adult , Cells, Cultured , Fatty Acids/metabolism , Fatty Acids, Monounsaturated/metabolism , Female , Humans , Isomerism , Liver X Receptors , Middle Aged , Orphan Nuclear Receptors/genetics , Orphan Nuclear Receptors/metabolism , Real-Time Polymerase Chain Reaction , Stearoyl-CoA Desaturase/genetics , Stearoyl-CoA Desaturase/metabolism , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/metabolism , Trans Fatty Acids/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Triglycerides/biosynthesis , Young AdultABSTRACT
Very-long-chain polyunsaturated fatty acids, such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have well-documented importance in human health and nutrition. Sustainable production in robust host organisms that do not synthesize them naturally requires the coordinated expression of several heterologous desaturases and elongases. In the present study we show production of EPA in Saccharomyces cerevisiae using glucose as the sole carbon source through expression of five heterologous fatty acid desaturases and an elongase. Novel Δ5-desaturases from the ciliate protozoan Paramecium tetraurelia and from the microalgae Ostreococcus tauri and Ostreococcus lucimarinus were identified via a BLAST search, and their substrate preferences and desaturation efficiencies were assayed in a yeast strain producing the ω6 and ω3 fatty acid substrates for Δ5-desaturation. The Δ5-desaturase from P. tetraurelia was up-to-2-fold more efficient than the microalgal desaturases and was also more efficient than Δ5-desaturases from Mortierella alpina and Leishmania major. In vivo investigation of acyl carrier substrate specificities showed that the Δ5-desaturases from P. tetraurelia, O. lucimarinus, O. tauri, and M. alpina are promiscuous toward the acyl carrier substrate but prefer phospholipid-bound substrates. In contrast, the Δ5-desaturase from L. major showed no activity on phospholipid-bound substrate and thus appears to be an exclusively acyl coenzyme A-dependent desaturase.
