The enzyme acyl coenzyme A:cholesterol acyltransferase 1 (ACAT1) mediates sterol esterification, a crucial component of intracellular lipid homeostasis. Two enzymes catalyze this activity in Saccharomyces cerevisiae (yeast), and several lines of evidence suggest multigene families may also exist in mammals. Using the human ACAT1 sequence to screen data bases of expressed sequence tags, we identified two novel and distinct partial human cDNAs. Full-length cDNA clones for these ACAT related gene products (ARGP) 1 and 2 were isolated from a hepatocyte (HepG2) cDNA library. ARGP1 was expressed in numerous human adult tissues and tissue culture cell lines, whereas expression of ARGP2 was more restricted. In vitro microsomal assays in a yeast strain deleted for both esterification genes and completely deficient in sterol esterification indicated that ARGP2 esterified cholesterol while ARGP1 did not. In contrast to ACAT1 and similar to liver esterification, the activity of ARGP2 was relatively resistant to a histidine active site modifier. ARGP2 is therefore a tissue-specific sterol esterification enzyme which we thus designated ACAT2. We speculate that ARGP1 participates in the coenzyme A-dependent acylation of substrate(s) other than cholesterol. Consistent with this hypothesis, ARGP1, unlike any other member of this multigene family, possesses a predicted diacylglycerol binding motif suggesting that it may perform the last acylation in triglyceride biosynthesis.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA: sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA: diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.A common element to diabetes, atherosclerosis and obesity, is the subcellular and extracellular accumulation of neutral lipids (1-3). The synthesis of these neutral lipids (triglyceride and steryl ester) results in both storage and detoxification of the alcohol and acyl substrates to these reactions. The reactions thus represent a pivotal component of lipid homeostasis in all eukaryotes and of the pathophysiology of some of the most prevalent human disease syndromes of the western world (reviewed in Refs. 4 -6).Significant progress has been made recently toward the complete identification of the enzymes that mediate the intracellular synthesis of cholesteryl esters and triglyceride (TG). 1 In mammals, these reactions are catalyzed, in part, by the three members of the O-acyltransferase gene family (5, 7-12). The ACAT1 and ACAT2 genes encode a ubiquitous and a tissuespecific acyl-CoA:cholesterol O-acyltransferase (ACAT), respectively (9, 10, 12), while the DGAT1 gene encodes an acylcoenzyme A (CoA) diacylglycerol O-acyltransferase (DGAT (9,11,13,14)). Although induced mutant mice for DGAT1 exhibit reduced body fat and are resistant to diet-induced obesity, these animals have normal serum TG levels, indicating that DGAT1-independent mechanism(s) for TG synthesis must also exist (15,16). Such a mechanism may be mediated, at least in part, by DGAT2, a human protein recently identified based on sequence similarity to two acyl-CoA:diacylglycerol acyltransferases purified from the oleaginous fungus Mortierella ramanniana (16 -18). The two M. ramanniana enzymes are 53% identical and show no se...

Triglyceride (TG), a water-insoluble energy-rich lipid, is secreted by the liver as part of very low density lipoproteins (VLDLs) to supply energy to extrahepatic tissues. Overproduction of VLDL is associated with increased risk of cardiovascular heart disease; this has renewed an interest in factors that affect hepatic TG production. The TG production rate is determined by measuring temporal increases in plasma TG under conditions in which TG hydrolysis by lipoprotein lipase (LPL) is inhibited. The nonionic detergent, Triton WR-1339 (Triton), has commonly been used to inhibit LPL for this purpose. Triton, in addition to inhibition of TG hydrolysis, has properties that have the potential to adversely influence lipoprotein metabolism. Another nonionic detergent, poloxamer 407 (P-407), also inhibits LPL. In these studies, we demonstrate that P-407 is comparable to Triton in the determination of TG production but without the unwanted side effects of Triton. Supplementary key words non-ionic detergents • hepatic lipids • lipoproteinsTriglyceride (TG) is an energy-rich compound, primarily stored in liver and adipose, and is mobilized in response to various metabolic signals. In plasma, TG, which is water insoluble, circulates as the neutral lipid core of lipoproteins, mainly chylomicrons, which carry dietary fat and are secreted by the small intestine, and very low density lipoproteins (VLDLs), which carry TG from the liver. Overproduction of VLDL has been associated with a number of disease states that result in an increased risk of cardiovascular heart disease; this has renewed an interest in factors that affect hepatic TG (lipoprotein) production (1).In the early 1950s, it was noted that intravenous injection of certain nonionic detergents resulted in milky serum that lasted up to 48 h (2). This was later shown to be due to the inhibition of TG hydrolysis by lipoprotein lipase (LPL) (3). Since then, lipolysis inhibition has been used to determine hepatic TG production rates, with Triton WR-1339 (also known as Tyloxapol) being widely used. Using this technique, the TG production rate is calculated from the increase in TG over time following detergent injection. Although this method is the basis for most studies on TG production in animals, there is considerable variation in its implementation. Variables include whether mice are fasted, fed chow or a fat-free diet, and what the plasma sampling period is (0 to 300 min) over which TG production rates are determined ( Table 1 ).In addition to inhibition of LPL, Triton has a number of other physiologic effects related to lipoprotein metabolism. Triton has been shown to cause dissociation of apolipoprotein A-I (apoA-I) and apoC-II from HDL (13). Triton is rapidly taken up by the liver, where it accumulates in the lysosomes, causes autophagic vacuole formation (14, 15), and is excreted in bile, possibly explaining a reduction in biliary phospholipid and cholesterol output (16). These hepatic and plasma effects of Triton may affect hepatic TG production, especial...

The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.Triglyceride (TG) 1 biosynthesis is a common method of energy storage and thus has an important role in energy balance. In humans, overaccumulation of TG, either as obesity or elevated serum triglyceride, has been shown to be an independent risk factor for a variety of diseases including diabetes (1) and atherosclerosis (2, 3). In the pathway described by Kennedy (4) for glyceride and glycerophosphatide synthesis, a branch point is reached at diacylglycerol (DG) that can serve as a precursor for several phospholipid species and as a substrate for acylCoA, diacylglycerol O-acyltransferase (DGAT) (EC 2.3.1.20), which catalyzes the terminal step in TG synthesis. Expression of a recently identified mammalian DGAT cDNA in insect and mammalian cells conferred elevated triglyceride synthesis but did not change incorporation of fatty acids into sterol ester (5-7). The DGAT gene belongs to the acyl-CoA cholesterol acyltransferase (ACAT) gene family that includes two mammalian ACATs (ACAT1, ACAT2) and two yeast ACAT-related enzymes (ARE1, ARE2) that catalyze intracellular sterol esterification (8). Whereas yeast can synthesize TG from oleoyl-CoA and DG, deletion mutants in ARE1 and ARE2 do not reduce [ 3 H]oleate incorporation into TG (9). Therefore, a conspicuous absence from the ACAT gene family is a yeast DGAT.Further examination of the Saccharomyces cerevisiae genome data base revealed two DGAT-like genes in addition to the ARE genes. We show here that these four genes do not have a major role in TG synthesis in yeast. By contrast, a yeast gene with sequence similarity to mammalian lecithin-cholesterol acyltransferase (LCAT) (EC 2.3.1.43) catalyzes the esterification of DG using phosphatidylcholine as the acyl donor. This novel enzymatic reaction mediates the majority of TG synthesis in the yeast cell during exponential growth. EXPERIMENTAL PROCEDURESGeneral-Molecular biology a...