Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

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Acyl-Lipid Metabolism

Triglycerides (or triacylglycerols) represent the major form of stored energy in eukaryotes Triglyceride synthesis has been assumed to occur primarily through acyl CoA:diacylglycerol transferase (Dgat), a microsomal enzyme that catalyses the final and only committed step in the glycerol phosphate pathway Therefore, Dgat has been considered necessary for adipose tissue formation and essential for survival Here we show that Dgat-deficient (Dgat-/-) mice are viable and can still synthesize triglycerides Moreover, these mice are lean and resistant to diet-induced obesity The obesity resistance involves increased energy expenditure and increased activity Dgat deficiency also alters triglyceride metabolism in other tissues, including the mammary gland, where lactation is defective in Dgat-/- females Our findings indicate that multiple mechanisms exist for triglyceride synthesis and suggest that the selective inhibition of Dgat-mediated triglyceride synthesis may be useful for treating obesity

Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat.

LXRα and -β are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXRα-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.

https://www.pnas.org/content/pnas/96/1/266.full.pdf

Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta.

We recently mapped a quantitative trait locus (QTL) with a major effect on milk composition--particularly fat content--to the centromeric end of bovine chromosome 14. We subsequently exploited linkage disequilibrium to refine the map position of this QTL to a 3-cM chromosome interval bounded by microsatellite markers BULGE13 and BULGE09. We herein report the positional candidate cloning of this QTL, involving (1) the construction of a BAC contig spanning the corresponding marker interval, (2) the demonstration that a very strong candidate gene, acylCoA:diacylglycerol acyltransferase (DGAT1), maps to that contig, and (3) the identification of a nonconservative K232A substitution in the DGAT1 gene with a major effect on milk fat content and other milk characteristics.

/pdf/positional-candidate-cloning-of-a-qtl-in-dairy-cattle-2k78nt1zoz.pdf

Positional candidate cloning of a QTL in dairy cattle: identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition.

Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis.

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 23120) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation

https://www.pnas.org/content/pnas/95/22/13018.full.pdf

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

https://www.jbc.org/content/273/41/26755.full.pdf

Carrie B. Welch

Papers

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

ACAT-2, A Second Mammalian Acyl-CoA:Cholesterol Acyltransferase ITS CLONING, EXPRESSION, AND CHARACTERIZATION