BBA - Molecular and Cell Biology of Lipids (v.1631, #3)

This review tracks some of the changes in fatty acid chemistry that have occurred during the past 60 years. Once disparaged, this topic is now recognised as important in biochemistry and nutrition. Among the significant areas that are addressed are fatty acid oxidation and hydrogenation, fatty acid synthesis, and selected reactions of the carboxyl group and of unsaturated centres. Underlying many of the developments that have occurred have been important advances in lipid analysis and a clearer understanding of reaction mechanism and stereochemistry. Developments in the future will include greater use of enzymes in technological processes and will result from environmental pressures to conduct reactions under milder conditions, use less solvent, and produce less waste.
Keywords: Lipid oxidation; Fatty acid of novel structure; Fatty acid synthesis; Reaction of the carboxyl group; Reaction of the double bond; Neighbouring group participation;

Regulation of the scavenger receptor BI and the LDL receptor by activators of aldosterone production, angiotensin II and PMA, in the human NCI-H295R adrenocortical cell line by Antoine Pilon; Geneviève Martin; Stéphanie Bultel-Brienne; Didier Junquero; André Delhon; Jean-Charles Fruchart; Bart Staels; Véronique Clavey (218-228).
In human adrenal cells, cholesterol for steroidogenesis is derived from both high-density lipoproteins (HDL) via the Scavenger Receptor Class B Type I (SR-BI) and low-density lipoproteins (LDL) via the LDL receptor pathway. We have previously shown that, in the human adrenocortical carcinoma cell line, NCI-H295R, SR-BI and LDL receptor expression and steroidogenesis are coordinately regulated by activators of protein kinase A (PKA) leading to glucocorticoid synthesis. In the present study, we studied whether SR-BI and LDL receptor expression are regulated by activators of the protein kinase C (PKC) signaling pathway, such as angiotensin II, which stimulate mineralocorticoid synthesis. First, it is shown that, in NCI-H295R cells, aldosterone synthesis is stimulated by a phorbol ester (phorbol-12-myristate-13 acetate, PMA), a potent PKC activator. Northern blot analysis indicated that both angiotensin II and PMA stimulated SR-BI expression in a time-dependent manner. LDL receptor expression is slightly stimulated by PMA. The induction of SR-BI gene expression occurs at the transcriptional level, via an activation of the human SR-BI promoter, as shown by transient transfection experiments. Finally, SR-BI protein level was increased in angiotensin II- and PMA-stimulated cells, resulting in higher lipoprotein binding and specific cholesteryl ester (CE) uptake from HDL, as well from LDL after angiotensin II and PMA stimulation.
Keywords: Lipoprotein receptor; Steroidogenesis; Cholesterol metabolism; Adrenal cell; Angiotensin II;

Acetyl-CoA carboxylase and SREBP expression during peripheral nervous system myelination by Jérôme Salles; Françoise Sargueil; Anja Knoll-Gellida; Lee A. Witters; Claude Cassagne; Bertrand Garbay (229-238).
The expression of acetyl-CoA carboxylase (ACC) in mouse peripheral nervous system (PNS) was investigated. Both ACC 265 and ACC 280 isoforms were expressed in the sciatic nerve, although ACC 265 was predominant. ACC 265 transcripts originating from promoters P1 and P2 could be detected in the developing nerve, as well as the two splice products, which are characterized by the presence or the absence of a 24-base sequence before the codon serine-1200. The mRNA levels for ACC 265 parallel those of other lipogenic genes whose expression is linked to the myelination process. In addition, ACC 265 mRNA and protein levels in the nerves of the trembler mutant, which is a mouse model of PNS dysmyelination, represented around 30% of the normal values. The expression of the sterol regulatory element-binding proteins (SREBPs) was also studied. SREBP 1 mRNAs were expressed at a constant level during nerve development, and their quantities were normal in trembler. On the contrary, SREBP 2 mRNA quantities varied during the myelination period similarly to the lipogenic gene mRNAs, and the levels measured in trembler represented only 10% of the normal values. Taken together, these results suggest that the coordinate expression of several lipogenic genes, which occurs during PNS myelination, could possibly be regulated by SREBP 2.
Keywords: Acetyl-CoA carboxylase; Sterol regulatory element binding protein; Myelination; Gene expression;

The study of structural accessibility of free thiol groups in human low-density lipoproteins by Marina Kveder; Anita Kriško; Greta Pifat; Heinz-Jürgen Steinhoff (239-245).
The experimental evidence for the apolipoprotein B100 (apoB) domain structuring in low-density lipoprotein (LDL) was investigated focusing on the accessibility of free thiol groups. Three different spectroscopic methods were combined with the biochemical perturbations of LDL particle. The spectrophotometric method was adapted for LDL and the exposure of free thiols was analyzed in the native LDL and LDL exposed to sequential denaturation. The results indicate that 24-h denaturation does not expose all free thiols in LDL. Using thiol-specific spin labeling and electron paramagnetic resonance spectroscopy (EPR), different populations of labeled thiols were resolved. The comparison of the EPR spectra of native LDL and LDL with selectively blocked thiol groups revealed significant difference in the respective hyperfine splittings. The phenomenon can arise due to different polarity and/or mobility of the nitroxides in the microenvironments of spin label binding sites of these two LDL samples. The results indicate that nine thiol groups in apoB are distributed in different domains of LDL: two are more exposed, two are buried deeply in the lipid matrix of the particle and the rest are located in hydrophobic parts of this extremely complex protein–lipid assembly. These observations provide experimental support for the emerging theoretical models of apoB.
Keywords: LDL; Denaturation of apoB; EPR;

Activation of phospholipase D is not mediated by direct phosphorylation on tyrosine residues by Sanjoy Mehta; Jeff Maglio; Mike S. Kobayashi; Andrea M. Sipple; Joel Horwitz (246-254).
The activation of phospholipase D (PLD) in PC12/PC2 pheochromocytoma cells involves a tyrosine kinase. However, it is not clear whether this is due to direct phosphorylation of the enzyme or some other intermediary protein. In this manuscript, we examined this issue by two methods: (1) immunoprecipitation of phosphotyrosine containing proteins and assay of phospholipase D; (2) overexpression of HA–phospholipase D2 and susbsequent immunoprecipitation. The only agent that caused phosphorylation of phospholipase D on tyrosine residues was the phosphatase inhibitor, peroxyvanadate. Other agents that activate phospholipase D, including bradykinin, ionomycin, and phorbol dibutyrate did not cause phosphorylation of the enzyme. In addition, there was a lack of correlation between the peroxyvanadate-mediated phosphorylation and activation of phospholipase D, both in terms of time course and concentration dependence. These data demonstrate that phospholipase D is directly phosphorylated on tyrosine residues. However, phosphorylation of tyrosine residues does not correlate with activation of the enzyme.
Keywords: Phospholipase D; Tyrosine kinase; PC12 cell;

The influence of chylomicron remnants on lipid accumulation and synthesis and the activity and/or expression of mRNA for some of the key enzymes involved was investigated in the murine macrophage cell line J774. The effects of varying the polyunsaturated fatty acid (PUFA) composition and oxidation state of the remnants were also examined. Chylomicron remnants derived from corn oil (rich in n−6 PUFA) or fish oil (rich in n−3 PUFA) were prepared in vivo and oxidised by incubation with CuSO4. The native and oxidised remnants caused a marked rise in intracellular triacylglycerol levels, but the rise induced by corn oil remnants (four- to sixfold) was greater than that observed with fish oil remnants (<2-fold). Triacylglycerol synthesis, as measured by the incorporation of [3H]oleate and [3H]glycerol into cellular triacylglycerol, was increased by all four remnant types tested, and corn oil remnants had a significantly greater effect than fish oil remnants. Oxidation of the remnants did not affect the results obtained. Although the incorporation of [3H]oleate into cholesteryl ester by the cells was not significantly changed by any of the four types of remnants tested, the activity and expression of mRNA for acyl Co-enzyme A: cholesterol acyltransferase (ACAT) was increased by corn oil, but not by fish or oxidised corn, remnants. Neutral cholesteryl ester hydrolase (nCEH) activity, however, was also raised by corn oil remnants. These studies indicate that chylomicron remnants induce the accumulation of triacylglycerol in J774 macrophages, and that increased synthesis of triacylglycerol plays a major role in this process. Furthermore, they demonstrate that these effects are enhanced when the remnants are enriched in n−6 PUFA as compared with n−3 PUFA, but not after oxidation of the particles, suggesting that the fatty acid composition of chylomicron remnants may be more important than their oxidation state in their ability to induce foam cell formation.
Keywords: Chylomicron remnant; Triacylglycerol; Cholesterol esterification; Polyunsaturated fatty acid;

The effects of dietary conjugated linoleic acid (CLA) on the activity and mRNA levels of hepatic enzymes involved in fatty acid synthesis and oxidation were examined in mice. In the first experiment, male ICR and C57BL/6J mice were fed diets containing either a 1.5% fatty acid preparation rich in CLA or a preparation rich in linoleic acid. In the second experiment, male ICR mice were fed diets containing either 1.5% linoleic acid, palmitic acid or the CLA preparation. After 21 days, CLA relative to linoleic acid greatly decreased white adipose tissue mass but caused hepatomegaly accompanying an approximate 10-fold increase in the tissue triacylglycerol content irrespective of mouse strain. CLA compared to linoleic acid greatly increased the activity and mRNA levels of various lipogenic enzymes in both experiments. Moreover, CLA increased the mRNA expression of Δ6- and Δ5-desaturases, and sterol regulatory element binding protein-1 (SREBP-1). The mitochondrial and peroxisomal palmitoyl-CoA oxidation rate was about 2.5-fold higher in mice fed CLA than in those fed linoleic acid in both experiments. The increase was associated with the up-regulation of the activity and mRNA expression of various fatty acid oxidation enzymes. The palmitic acid diet compared to the linoleic acid diet was rather ineffective in modulating the hepatic lipid levels or activity and mRNA levels of enzymes in fatty acid metabolism. It is apparent that dietary CLA concomitantly increases the activity and mRNA levels of enzymes involved in fatty acid synthesis and oxidation, and desaturation of polyunsaturated fatty acid in the mouse liver. Both the activation of peroxisomal proliferator α and up-regulation of SREBP-1 may be responsible for this.
Keywords: Conjugated linoleic acid; Fatty acid synthesis; Fatty acid oxidation; Gene expression; Mouse;