The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters

The final step of triacylglycerol biosynthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. The two known DGATs, DGAT1 and DGAT2, are encoded by unrelated genes. Although both DGAT1 and DGAT2 knockout mice have reduced tissue triacylglycerol contents, they have disparate phenotypes, prompting us to investigate whether the two enzymes have unrecognized functional differences. We now report that DGAT1 exhibits additional acyltransferase activities in vitro, including those of acyl CoA:monoacylglycerol acyltransferase (MGAT), wax monoester and wax diester synthases, and acyl CoA:retinol acyltransferase (ARAT), which catalyze the synthesis of diacylglycerols, wax esters, and retinyl esters, respectively. These activities were demonstrated in in vitro assays with membranes from insect cells or homogenates from COS7 cells overexpressing DGAT1. Wax synthase and ARAT activities were also demonstrated in intact COS7 cells expressing DGAT1. Additionally, cells and tissues from DGAT1-deficient mice exhibited reduced ARAT activity, and the mice had increased levels of unesterified retinol in their livers on a high-retinol diet. Our findings indicate that DGAT1 can utilize a variety of acyl acceptors as substrates in vitro and suggest that these activities may be relevant to the in vivo functions of DGAT1.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A: diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K(m) values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9+/-2.1 microM and 13.9+/-0.3 microM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits approximately 85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl-oleate formation without influencing the retinyl-palmitate formation. Using this inhibitor, we estimate that approximately 64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Synthesis of neutral ether lipid monoalkyl-diacylglycerol by lipid acyltransferases

In mammals, ether lipids exert a wide spectrum of signaling and structural functions, such as stimulation of immune responses, anti-tumor activities, and enhancement of sperm functions. Abnormal accumulation of monoalkyl-diacylglycerol (MADAG) was found in Wolman's disease, a human genetic disorder defined by a deficiency in lysosomal acid lipase. In the current study, we found that among the nine recombinant human lipid acyltransferases examined, acyl-CoA:diacylglycerol acyltransferase (DGAT)1, DGAT2, acyl-CoA:monoacylglycerol acyltransferase (MGAT)2, MGAT3, acyl-CoA:wax-alcohol acyltransferase 2/MFAT, and DGAT candidate 3 were able to use 1-monoalkylglycerol (1-MAkG) as an acyl acceptor for the synthesis of monoalkyl-monoacylglycerol (MAMAG). These enzymes demonstrated different enzymatic turnover rates and relative efficiencies for the first and second acylation steps leading to the synthesis of MAMAG and MADAG, respectively. They also exhibited different degrees of substrate preference when presented with 1-monooleoylglycerol versus 1-MAkG. In CHO-K1 cells, treatment with DGAT1 selective inhibitor, XP-620, completely blocked the synthesis of MADAG, indicating that DGAT1 is the predominant enzyme responsible for the intracellular synthesis of MADAG in this model system. The levels of MADAG in the adrenal gland of DGAT1 KO mice were reduced as compared with those of the WT mice, suggesting that DGAT1 is a major enzyme for the synthesis of MADAG in this tissue. Our findings indicate that several of these lipid acyltransferases may be able to synthesize neutral ether lipids in mammals.     

Properties of the mouse intestinal acyl-CoA:monoacylglycerol acyltransferase,MGAT2

Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays an important role in dietary fat absorption by catalyzing a rate-limiting step in the re-synthesis of diacylglycerols in enterocytes. The present study reports further characterization of MGAT2, a newly identified intestinal MGAT (Cao, J., Lockwood, J., Burn, P., and Shi, Y. (2003) J. Biol. Chem. 278, 13860-13866) for its substrate specificity, requirement for lipid cofactors, optimum pH and Mg2+, and other intrinsic properties. MGAT2 enzyme expressed in COS-7 cells displayed a broad range of substrate specificity toward fatty acyl-CoA derivatives and monoacylglycerols, among which the highest activities were observed with oleoyl-CoA and rac-1-monolauroylglycerol, respectively. MGAT2 appeared to acylate monoacylglycerols containing unsaturated fatty acyls in preference to saturated ones. Lipid cofactors that play roles in signal transduction were shown to modulate MGAT2 activities. In contrast to oleic acid and sphingosine that exhibited inhibitory effects, phosphatidylcholine, phosphatidylserine, and phosphatidic acid stimulated MGAT2 activities. Using recombinant murine MGAT2 expressed in Escherichia coli, we demonstrated conclusively that MGAT2 also possessed an intrinsic acyl-CoA:diacylglycerol acyltransferase (DGAT) activity, which could provide an alternative pathway for triacylglycerol synthesis in the absence of DGAT. In contrast to the inhibitory effect on MGAT2 activities, nonionic and zwitterionic detergents led to a striking activation of DGAT activity of the human DGAT1 expressed in mammalian cells, which further distinguished the behaviors of the two enzymes. The elucidation of properties of MGAT2 will facilitate future development of compounds that inhibit dietary fat absorption as a means to treat obesity.     

Retinol esterification in Sertoli cells by lecithin-retinol acyltransferase

Esterification of retinol occurs during the metabolism of vitamin A in the testis. An acyl-CoA:retinol acyltransferase (ARAT) activity has been described for microsomes isolated from testis homogenates. That activity was also observed here in microsomal preparations obtained from cultured Sertoli cells from 20-day-old (midpubertal) rats. ARAT catalyzed the synthesis of retinyl laurate when free retinol and lauroyl-CoA were provided as substrates. However, in the absence of exogenous acyl-CoA, retinol was esterified by a different activity in a manner similar to the lecithin:retinol acyltransferase (LRAT) activity described recently for liver and intestine. Microsomal preparations obtained from enriched Sertoli cell fractions from the adult rat testis had 75-fold higher levels of LRAT than the preparations from midpubertal animals, but ARAT activity was the same in both these preparations. LRAT utilized an endogenous acyl donor and either unbound retinol or retinol complexed with cellular retinol-binding protein (CRBP) to catalyze the synthesis of retinyl linoleate, retinyl oleate, retinyl palmitate, and retinyl stearate. The addition of exogenous dilaurylphosphatidylcholine (DLPC) resulted in the synthesis of retinyl laurate. The esterification from both exogenous DLPC and endogenous acyl donor was inhibited by 2 mM phenylmethanesulfonyl fluoride (PMSF). ARAT activity was not affected by similar concentrations of PMSF. Furthermore, retinol bound to CRBP, a protein known to be present in Sertoli cells, was not an effective substrate for testicular ARAT. When retinol uptake and metabolism were examined in cultured Sertoli cells from 20-day-old rats, the cells synthesized the same retinyl esters that were produced by microsomal LRAT in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family

Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. We used expression cloning to identify cDNAs encoding a wax synthase in the mouse preputial gland. The wax synthase gene is located on the X chromosome and encodes a member of the acyltransferase family of enzymes that synthesize neutral lipids. Expression of wax synthase in cultured cells led to the formation of wax monoesters from straight chain saturated, unsaturated, and polyunsaturated fatty alcohols and acids. Polyisoprenols also were incorporated into wax monoesters by the enzyme. The wax synthase had little or no ability to synthesize cholesteryl esters, diacylglycerols, or triacylglycerols, whereas other acyltransferases, including the acyl-CoA:monoacylglycerol acyltransferase 1 and 2 enzymes and the acyl-CoA:diacylglycerol acyltransferase 1 and 2 enzymes, exhibited modest wax monoester synthesis activities. Confocal light microscopy indicated that the wax synthase was localized in membranes of the endoplasmic reticulum. Wax synthase mRNA was abundant in tissues rich in sebaceous glands such as the preputial gland and eyelid and was present at lower levels in other tissues. Coexpression of cDNAs specifying fatty acyl-CoA reductase 1 and wax synthase led to the synthesis of wax monoesters. The data suggest that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily

The esterification of alcohols such as sterols, diacylglycerols, and monoacylglycerols with fatty acids represents the formation of both storage and cytoprotective molecules. Conversely, the overproduction of these molecules is associated with several disease pathologies, including atherosclerosis and obesity. The human acyl-CoA:diacylglycerol acyltransferase (DGAT) 2 gene superfamily comprises seven members, four of which have been previously implicated in the synthesis of di- or triacylglycerol. The remaining 3 members comprise an X-linked locus and have not been characterized. We describe here the expression of DGAT2 and the three X-linked genes in Saccharomyces cerevisiae strains virtually devoid of neutral lipids. All four gene products mediate the synthesis of triacylglycerol; however, two of the X-linked genes act as acyl-CoA wax alcohol acyltransferases (AWAT 1 and 2) that predominantly esterify long chain (wax) alcohols with acyl-CoA-derived fatty acids to produce wax esters. AWAT1 and AWAT2 have very distinct substrate preferences in terms of alcohol chain length and fatty acyl saturation. The enzymes are expressed in many human tissues but predominate in skin. In situ hybridizations demonstrate a differentiation-specific expression pattern within the human sebaceous gland for the two AWAT genes, consistent with a significant role in the composition of sebum.     

Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.     

Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT)

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary retinol primarily as free retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these (chylomicron) retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:retinol acyltransferase.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic properties of MGAT3, a putative triacylgycerol synthase

Acyl-coenzyme A:monoacylglycerol acyltransferase 3 (MGAT3) is a member of the MGAT family of enzymes that catalyze the synthesis of diacylglycerol (DAG) from monoacylglycerol (MAG), a committed step in dietary fat absorption. Although named after the initial identification of its MGAT activity, MGAT3 shares higher sequence homology with acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) than with other MGAT enzymes, suggesting that MGAT3 may also possess significant DGAT activity. This study compared the catalytic properties of MGAT3 with those of MGAT1 and MGAT2 enzymes using both MAG and DAG as substrates. Our results showed that in addition to the expected MGAT activity, the recombinant MGAT3 enzyme expressed in Sf-9 insect cells displayed a strong DGAT activity relative to that of MGAT1 and MGAT2 enzymes in the order MGAT3 > MGAT1 > MGAT2. In contrast, none of the three MGAT enzymes recognized biotinylated acyl-CoA or MAG as a substrate. Although MGAT3 possesses full DGAT activity, it differs from DGAT1 in catalytic properties and subcellular localization. The MGAT3 activity was sensitive to inhibition by the presence of 1% CHAPS, whereas DGAT1 activity was stimulated by the detergent. Consistent with high sequence homology with DGAT2, the MGAT3 enzyme demonstrated a similar subcellular distribution pattern to that of DGAT2, but not DGAT1, when expressed in COS-7 cells. Our data suggest that MGAT3 functions as a novel triacylglycerol (TAG) synthase that catalyzes efficiently the two consecutive acylation steps in TAG synthesis.     

Evidence for a lecithin-retinol acyltransferase activity in the rat small intestine

Cellular retinol-binding protein, type II (CRBP (II] is an abundant protein of the mature enterocytes of the small intestine. It has been shown to direct retinol to an acyl-CoA-independent esterifying activity that utilizes an endogenous acyl donor (Ong, D.E., Kakkad, B., and MacDonald, P.N. (1987) J. Biol. Chem. 262, 2729-2736). Here we report that this activity in intestinal microsomes will catalyze the transfer of acyl moieties from exogenous phosphatidylcholine (PC) to retinol-CRBP(II) to produce retinyl esters. The microsomal activity displayed positional selectivity as only the sn-1-acyl moiety of PC was transferred to retinol-CRBP(II). The retinyl ester synthase was selective for PC substrates as acyl transfer from phosphatidylethanolamine, phosphatidic acid, or free fatty acid to retinol-CRBP(II) was not observed. Some formation of retinyl esters was observed with exogenous acyl-CoA, but the amount produced was considerably lower than ester formation from exogenous PC and could be shown to be due to a different enzyme activity. Inhibitor studies clearly distinguished between the enzyme activities responsible for the acyl-CoA-dependent esterification and the phosphatidylcholine-dependent esterification of retinol. The results provide strong evidence that retinol-CRBP(II) esterification in the intestine proceeds via a phosphatidylcholine-dependent transacylase mechanism similar to that established for the esterification of cholesterol by lecithin-cholesterol acyltransferase.     

ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase

CoA-dependent transacylation activity in microsomes is known to catalyze the transfer of fatty acids between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acids. We previously found a novel acyl-CoA synthetic pathway, ATP-independent acyl-CoA synthesis from phospholipids. We proposed that: 1) the ATP-independent acyl-CoA synthesis is due to the reverse reaction of acyl-CoA:lysophospholipid acyltransferases and 2) the reverse and forward reactions of acyltransferases can combine to form a CoA-dependent transacylation system. To test these proposals, we examined whether or not recombinant mouse acyl-CoA:1-acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT) could catalyze ATP-independent acyl-CoA synthetic activity and CoA-dependent transacylation activity. ATP-independent acyl-CoA synthesis was indeed found in the membrane fraction from Escherichia coli cells expressing mouse LPAAT, whereas negligible activity was observed in mock-transfected cells. Phosphatidic acid (PA), but not free fatty acids, served as an acyl donor for the reaction, and LPA was formed from PA in a CoA-dependent manner during acyl-CoA synthesis. These results indicate that the ATP-independent acyl-CoA synthesis was due to the reverse reaction of LPAAT. In addition, bacterial membranes containing LPAAT catalyzed CoA-dependent acylation of LPA; PA but not free fatty acid served as an acyl donor. These results indicate that the CoA-dependent transacylation of LPA consists of 1) acyl-CoA synthesis from PA through the reverse action of LPAAT and 2) the transfer of the fatty acyl moiety of the newly formed acyl-CoA to LPA through the forward reaction of LPAAT.     

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.     

CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium

Washed, buffered microsomes from bovine retinal pigment epithelium catalyze retinyl ester synthesis from retinol in the absence of an exogenous acyl donor. A plot of retinyl ester synthesis versus time reaches a plateau at 123 +/- 26 nmol of retinyl ester mg-1 microsomal protein, providing a minimum value of the concentration of the endogenous acyl donor. Fatty acyl-CoA analysis by three different methods employing high performance liquid chromatography resulted in the detection of less than 1 nmol mg-1 protein of acyl-CoA, indicating that fatty acyl-CoA is not the endogenous acyl donor. Stimulation of the rate of retinyl ester synthesis by palmitoyl-CoA or ATP, CoA, and palmitate is observed following its addition at the beginning of the reaction or after the endogenous acyl source has been exhausted by 20 min of reaction with retinol. Palmitate from [14C]palmitoyl-CoA is incorporated into retinyl ester at a rate similar to that for the incorporation of [3H] retinol, demonstrating the presence of an apparent acyl-CoA:retinol acyl transferase activity. The acyl group from palmitoyl-CoA can be transferred initially to a component of the microsomes and subsequently to retinol. The product of retinyl ester synthesis from all-trans-retinol and palmitoyl-CoA is all-trans-retinyl palmitate, indicating that the stereochemical configuration is retained during esterification. The kinetic parameters for the esterification of 11-cis-retinol and all-trans-retinol are similar.     

Lecithin:retinol acyltransferase in retinal pigment epithelial microsomes

Microsomal preparations from retinal pigment epithelium carry out phosphatidylcholine synthesis upon incubation with 1-palmitoyllysophosphatidylcholine and fatty acyl-CoA. Phosphatidylcholine synthesized in situ in this manner is an acyl donor for retinyl ester synthesis, demonstrating the existence of lecithin:retinol acyltransferase. Although acyl transfer to retinol is from the 1-position of phosphatidylcholine, the fatty acid in the 2-position is important in substrate recognition. The finding of this novel enzyme activity in retinal pigment epithelial microsomes suggests that phosphatidylcholine is the endogenous acyl donor in CoA-independent retinol esterification observed in these preparations.     

A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1

Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.     

Acyl-CoA: retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activation during the lipocyte phenotype induction in hepatic stellate cells

We have examined retinol esterification in the established GRX cell line, representative of hepatic stellate cells, and in primary cultures of ex vivo purified murine hepatic stellate cells. The metabolism of [3H]retinol was compared in cells expressing the myofibroblast or the lipocyte phenotype, under the physiological retinol concentrations. Retinyl esters were the major metabolites, whose production was dependent upon both acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). Lipocytes had a significantly higher esterification capacity than myofibroblasts. In order to distinguish the intrinsic enzyme activity from modulation of retinol uptake and CRBP-retinol content of the cytosol in the studied cells, we monitored enzyme kinetics in the purified microsomal fraction. We found that both LRAT and ARAT activities were induced during the conversion of myofibroblasts to lipocytes. LRAT induction was dependent upon retinoic acid, while that of ARAT was dependent upon the overall induction of the fat storing phenotype. The fatty acid composition of retinyl-esters suggested a preferential inclusion of exogenous fatty acids into retinyl esters. We conclude that both LRAT and ARAT participate in retinol esterification in hepatic stellate cells: LRAT's activity correlates with the vitamin A status, while ARAT depends upon the availability of fatty acyl-CoA and the overall lipid metabolism in hepatic stellate cells.     

MGAT2, a monoacylglycerol acyltransferase expressed in the small intestine

Acyl CoA:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, a precursor of triacylglycerol. In the intestine, MGAT plays a major role in the absorption of dietary fat by catalyzing the resynthesis of triacylglycerol in enterocytes. This resynthesis is required for the assembly of lipoproteins that transport absorbed fat to other tissues. Despite intense efforts, a gene encoding an intestinal MGAT has not been found. Previously, we identified a gene encoding MGAT1, which in mice is expressed in the stomach, kidney, adipose tissue, and liver but not in the intestine. We now report the identification of homologous genes in humans and mice encoding MGAT2. Expression of the MGAT2 cDNA in either insect or mammalian cells markedly increased MGAT activity in cell membranes. MGAT activity was proportional to the level of MGAT2 protein expressed, and the amount of diacylglycerol produced depended on the concentration of MGAT substrates (fatty acyl CoA or monoacylglycerol). In humans, the MGAT2 gene is highly expressed in the small intestine, liver, stomach, kidney, colon, and white adipose tissue; in mice, it is expressed predominantly in the small intestine. The discovery of the MGAT2 gene will facilitate studies to determine the functional role of MGAT2 in fat absorption in the intestine and to determine whether blocking MGAT activity in enterocytes is a feasible approach to inhibit fat absorption and treat obesity.