Advertisement
J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M500036-JLR200 on April 16, 2005

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M500036-JLR200v1
46/7/1502    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yen, C.-L. E.
Right arrow Articles by Farese, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yen, C.-L. E.
Right arrow Articles by Farese, R. V., Jr.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 46, 1502-1511, July 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

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

Chi-Liang Eric Yen1,*,{dagger}, Mara Monetti*,{dagger}, Betty J. Burri§ and Robert V. Farese, Jr.*,{dagger},**

* Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, CA 94158
{dagger} Cardiovascular Research Institute, University of California, San Francisco, CA 94158
** Department of Medicine, University of California, San Francisco, CA 94158
§ Western Human Nutrition Research Center, United States Department of Agriculture, Davis, CA 95616

Published, JLR Papers in Press, April 16, 2005. DOI 10.1194/jlr.M500036-JLR200

1 To whom correspondence should be addressed. e-mail: eyen{at}gladstone.ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

Abbreviations: ACAT, acyl CoA:cholesterol acyltransferase; ARAT, acyl CoA:retinol acyltransferase; CoA, coenzyme A; DGAT, acyl CoA:diacylglycerol acyltransferase; LRAT, lecithin:retinol acyltransferase; MEF, mouse embryonic fibroblast; MGAT, acyl CoA:monoacylglycerol acyltransferase; TLC, thin-layer chromatography

Supplementary key words acyl CoA:diacylglycerol acyltransferase • retinol (vitamin A) • wax ester • monoacylglycerol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Triacylglycerols (triglycerides) are the primary molecules of energy storage in eukaryotic organisms. In mammals, triacylglycerol biosynthesis is essential for normal physiology and is prominent in the adipose tissue, liver, small intestine, and mammary gland, which all store or secrete triacylglycerols. However, excessive storage of triacylglycerols in human adipose tissue results in obesity, and in nonadipose tissues, it is associated with tissue dysfunction, referred to as lipoxicity (1). Thus, an understanding of the molecular aspects of triacylglycerol biosynthesis is of great importance.

The final step in triacylglycerol biosynthesis, the joining of diacylglycerol and fatty acyl coenzyme A (CoA), is catalyzed by acyl CoA:diacylglycerol O-acyltransferase (DGAT). Two DGAT enzymes have been identified. DGAT1, a member of a gene family that includes acyl CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 (2–4), is a protein of ~500 amino acids in most species (5, 6). It is very hydrophobic, has multiple predicted membrane-spanning domains (5), and like ACAT1 (7), forms homodimers and homotetramers (8). DGAT1 is expressed in most tissues, with the highest expression levels in the small intestine, testis, adipose tissue, mammary gland, and skin (5). DGAT2, on the other hand, is a member of a larger gene family whose members include acyl CoA:monoacylglycerol acyltransferase (MGAT) 1, MGAT2, MGAT3, and wax synthases (9–16). DGAT2 enzymes of different species have ~400 amino acids, are less hydrophobic than DGAT1, and have only one or two predicted membrane-spanning domains (10). DGAT2 is expressed highly in tissues that figure prominently in triacylglycerol metabolism, including liver and adipose tissues (10). Despite their similar enzymatic properties in in vitro assays, DGAT1 and DGAT2 share little, if any, sequence similarity (5, 10).

The in vivo functions of DGAT1 and DGAT2 have been analyzed in gene-targeted mice deficient in either enzyme (17, 18). DGAT1-deficient (Dgat1–/–) mice are viable, have modest reductions in tissue triacylglycerol content, and have normal plasma triacylglycerol levels (17). These mice are protected from diet-induced and some genetic forms of obesity through an increase in energy expenditure (17, 19). They exhibit increased sensitivity to insulin and leptin and are protected from diet-induced insulin resistance (19, 20). These phenotypic effects appear to be due in part to altered endocrine function of the white adipose tissue (21). Unexpectedly, Dgat1–/– mice also have abnormalities in tissue-specific developmental processes. Their skin is characterized by atrophy of the sebaceous glands and hair loss, which is most prominent in postpubertal male mice. Their fur lacks wax diesters (22), which are the major component of skin surface lipids in mice. Female Dgat1–/– mice exhibit a lactation defect and do not secret milk due to impaired development of the mammary epithelium (17, 23). This aspect of the phenotype appears to reflect the absence of DGAT1 both in the mammary epithelium and in the surrounding stromal tissue (23).

The absence of DGAT2 has a more profound effect on triacylglycerol metabolism. DGAT2-deficient (Dgat2–/–) mice have intrauterine growth retardation and die within hours after birth (18). They have severely reduced triacylglycerols and lack substrates for energy metabolism. They also lack essential fatty acids and specific skin lipids that are crucial for the function of the epidermal water barrier (18). In yeast, the DGAT2 homolog Dga1p accounts for the majority of triacylglycerol synthesis (24), whereas the DGAT1 homologs Are1p and Are2p account for only a minor portion (25).

The disparate phenotypes of DGAT1- and DGAT2-deficient mice prompted us to investigate whether the two enzymes have previously unrecognized differences in their biochemical functions. In this study, we sought to determine whether DGAT1 and DGAT2 have other substrates and, more specifically, whether either DGAT can use additional fatty acyl acceptors as substrates.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression studies in insect cells
Mouse DGAT1 (accession number AF078752) and DGAT2 (accession number AF384160) were tagged with an N-terminal FLAG epitope (MGDYKDDDDG, epitope underlined) and expressed in Spodoptera frugiperda (Sf9) insect cells by baculovirus infections as described (5, 10–12). Wild-type baculovirus infections were used as a negative control. Expression of DGAT1 and DGAT2 proteins was demonstrated by immunoblotting of membrane proteins (5 µg) with an anti-FLAG M2 antibody (Sigma-Aldrich Co.; St. Louis, MO).

In vitro acyltransferase assays
For initial screening of acyl aceptors, acyltransferase activities were assayed with 25 µM acyl donor and 100 µM acyl acceptor as described (11). Nonpolar acyl acceptors (diacylglycerol, monoacylglycerol, 1-hexadecanol, 1,2-hexadecandiol, cholesterol, and retinol) were dissolved in acetone, which accounted for 5% of 200 µl reactions. Retinol was dissolved in dimethylsulfoxide in some experiments, and results were identical to those obtained with retinol dissolved in acetone. Reactions were started by adding membranes (from insect cells) or total protein homogenates (from mammalian cells or mouse tissues) and stopped after 10 min (increases in reaction products were linear for up to 30 min) by adding 4 ml of chloroform-methanol (2:1; v/v). The extracted lipids were dried, separated by thin-layer chromatography (TLC) with hexane-ethyl ether-acetic acid (80:20:1; v/v/v), visualized with iodine vapor, and identified by comparison with lipid standards. For experiments with radiolabeled substrates, TLC plates were scanned with an imaging scanner (Bioscan AR-2000; Washington, D.C.) or exposed to X-ray film, and bands were scraped to assess the incorporation of radioactivity into lipid products. All assays were performed in duplicate, and experiments were repeated two to three times.

MGAT (EC 2.3.1.22), DGAT (EC 2.3.1.20), acyl CoA:long-chain fatty alcohol O-acyltransferase (wax synthase; EC 2.3.1.75), and acyl CoA:retinol O-acyltransferase (ARAT; EC 2.3.1.76) activities were detected by incorporation of [14C]palmitoyl CoA (25 µM; specific activity, ~20,000 cpm/nmol; Amersham Biosciences, Piscataway, NJ) into diacylglycerol, triacylglycerol, wax monoester and wax diester, and retinyl ester in the presence of added sn-2 monooleoylglycerol, sn-1,2 dioleoylglycerol, 1-hexadecanol, 1,2-hexadecandiol, and all-trans retinol, respectively. Stereoisomers and derivatives of retinol (all-trans and 13-cis retinol, all-trans retinal, and all-trans retinoic acids) were from Sigma-Aldrich. The dependence of MGAT, wax synthase, and ARAT activities on acyl acceptors as substrates was determined with assays using various concentrations of acyl acceptor (sn-2 monooleoylglycerol, 1-hexadecanol, 1,2-hexadecandiol, and all-trans retinol, respectively) and 50 µM [14C]palmitoyl CoA (specific activity, ~20,000 cpm/nmol). The acyl acceptors were serially diluted beforehand to maintain the percentage of acetone in each reaction at 5%. Likewise, the dependence of MGAT, wax monoester synthase, and ARAT activity on acyl donor as substrates was determined with assays using various concentrations of palmitoyl CoA and the respective radiolabeled acyl acceptor (200 µM [3H]sn-2 monooleoylglycerol, specific activity ~3,000 cpm/nmol; 100 µM [14C]1-hexadecanol, specific activity ~6,000 cpm/nmol; 50 µM [3H]all-trans retinol, specific activity ~330,000 cpm/nmol; American Radiolabeled Chemicals, St. Louis, MO).

Expression studies in mammalian cells
The FLAG-tagged mouse DGAT1 cDNA was subcloned into a pcDNA3 expression vector and transfected into COS-7 cells with Fugene 6 (Roche Diagnostics; Chicago, IL). The human DGAT1 coding sequence (accession number NM_012079) was amplified from human liver cDNA (Clontech; Palo Alto, CA) by PCR (Takara Ex Taq; Panvera, Madison, WI) and was subcloned without the FLAG tag into pIRESneo2 vector (Clontech). ß-Galactosidase, FLAG-tagged mouse ACAT2 (accession number AF078751), DGAT2, MGAT1 (accession number AF078752), and human MGAT2, (accession number AY157608) were expressed as controls. Cells were harvested 48 h after transfection. Expression of FLAG-tagged proteins was analyzed by immunoblotting of homogenates (20 µg of protein) with the anti-FLAG antibody. The in vitro acyltransferase activities of total homogenates were assayed with labeled palmitoyl CoA as described above for proteins expressed in insect cells.

Wax and retinol esterification in cultured cells
The incorporation of [14C]1-hexadecanol into wax monoester and of [3H]retinol into retinyl esters in cultured cells was measured in COS-7 cells overexpressing DGAT1. COS-7 cells were plated in six-well dishes (105/well) for 24 h and transfected with vectors containing cDNAs for DGAT1 or control enzymes; 24 h later, cells were incubated in medium containing 5 µM [14C]1-hexadecanol (specific activity, ~5,500 cpm/nmol) or 2.5 µM [3H]retinol (specific activity, ~200,000 cpm/nmol) for 18 h. Neutral lipids were extracted, separated by TLC, visualized with a Bioscan imaging scanner, and scraped to assess the incorporation of radioactivity into wax and retinyl esters.

In vitro assay and labeling studies with mouse embryonic fibroblasts
Mouse embryonic fibroblasts (MEFs) were established from DGAT1-deficient embryos and their wild-type littermates as described (10). The in vitro acyltransferase activities of total homogenates from primary MEFs (passage 3) were assayed with labeled palmitoyl CoA as described above for insect proteins. For labeling of intact cells, primary MEFs were plated in triplicate (4 x 105/100 mm dish) and, 24 h later, incubated with medium containing 5 µM [3H]retinol (specific activity, ~70,000 cpm/nmol) for 18 h (similar results, with proportionally less incorporation of radioactivity, were observed after 6 h of incubation). Neutral lipids were analyzed as described above.

Measurements of retinol and retinyl esters
Retinoid levels were measured with reverse-phase HPLC methods. For MEFs, cellular retinol levels were measured in three samples of 3–10 x 106 cells from each cell line. Cells were cultured in media containing 10% fetal bovine serum that contained 0.5 µM retinol and 0.25 µM retinyl esters. For mouse studies, male wild-type and Dgat1–/– mice (19 weeks old; >96% C57BL/6J genetic background) were maintained on a regular chow diet (containing 10 kcal% fat, PicoLab 5053; Purina Mill, Brentwood, MO) and then either kept on chow or switched to a high-fat diet (containing 45 kcal% fat, TD88137; Harlan, Madison, WI) for 1 week before tissues were harvested. The chow diet contained ~10 IU/g of vitamin A (mainly retinyl acetate), and the high-fat diet contained ~20 IU/g of vitamin A (mainly retinyl palmitate).

Retinoid levels in MEFs and mouse livers were measured in the laboratory of B. J. Burri (USDA, Davis, CA) (26) and levels in diets and serum were measured by Craft Technologies (Wilson, NC) (27). In one experiment, 12-week-old male wild-type and Dgat1–/– mice were maintained on a high-vitamin A diet (containing 160 IU/g of retinyl palmitate and 10 kcal% fat, D99102101; Research Diets, New Brunswick, NJ) for 3 weeks, and tissue retinoid levels were measured by Craft Technologies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DGAT1 expressed in insect cells possesses multiple acyltransferase activities
To examine the acyltransferase activities of DGAT1 and DGAT2, FLAG-tagged versions of their murine cDNAs were expressed in insect cells. These proteins migrated on SDS-PAGE at the expected molecular masses, and the levels of expression were comparable, as demonstrated by immunoblotting with an anti-FLAG antibody (Fig. 1A). In control experiments, membranes expressing either DGAT incorporated more [14C]palmitoyl CoA into triacylglycerols than did membranes from cells infected with wild-type baculovirus (Fig. 1B), verifying that both expressed enzymes were active. As reported previously (11, 12), both DGAT1 and DGAT2 also possess in vitro MGAT activity, incorporating [14C]palmitoyl CoA into diacylglycerol and triacylglycerols in the presence of monoacylglycerol (Fig. 1B).



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 1. Expression of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) and DGAT2 in Sf9 insect cells. A: Immunoblots of insect cell membranes. Expression of DGAT1 and DGAT2 was demonstrated by immunoblotting with an anti-FLAG antibody. Membrane proteins (5 µg) from Sf9 cells infected with wild-type virus, FLAG-tagged DGAT1 (DGAT1), or FLAG-tagged-DGAT2 (DGAT2) recombinant baculoviruses were analyzed. B–D: Acyltransferase activities conferred by expression of DGAT1. Acyl CoA:monoacylglycerol acyltransferase (MGAT) and DGAT (B), wax monoester and diester synthase (C), and acyl CoA:retinol acyltransferase (ARAT) (D) activities were detected by incorporation of [14C]palmitoyl coenzyme A (CoA) into diacylglycerol (DAG), triacylglycerol (TAG), wax monoester (ME), wax diester (DE), and retinyl ester (RE) in the presence of 100 µM added sn-2 monooleoylglycerol (MAG), sn-1,2 dioleoylglycerol (DAG, top label), 1-hexadecanol (Mono-ol), 1,2-hexadecandiol (Di-ol), and retinol (all-trans and 13-cis retinol), respectively. FFA, free fatty acid. The lipid products were extracted, dried, separated by thin-layer chromatography, and detected by exposure to X-ray film as described. Arrows indicate the respective product(s) of each acyltransferase activity. In (C), the prominent acylation product when 1,2-hexadecandiol was provided is most likely the intermediate 2-hydroxylhecadecyl hexadecanoate (lower arrow). Chromatographs shown are representative of three independent experiments.

 
We next explored whether DGAT1 or DGAT2 could utilize other acyl acceptors as substrates in vitro. With [14C] palmitoyl CoA as the acyl donor, membranes expressing DGAT1, but not those expressing DGAT2 or control viral proteins, catalyzed the incorporation of the acyl group into wax monoesters, wax diesters, and retinyl esters in the presence of 1-hexadecanol, 1,2-hexadecandiol, and retinol, respectively (Fig. 1C, D). Both all-trans retinol and 13-cis retinol served as substrates for the DGAT1-mediated retinol acyltransferase activity, but all-trans retinal and retinoic acid did not, consistent with a requirement for a free hydroxyl group on the end of the retinoid side chain (Fig. 1D). Cholesterol, glycerol, glycerol-3-phosphate, and 1-acylglycerol-3-phosphate did not serve as substrates (not shown).

To examine whether the acyltransferase activities of DGAT1 depend on the presence of appropriate substrates, we first varied the acyl acceptor concentrations in assays containing 50 µM [14C]palmitoyl CoA. In general, enzymatic activity increased with the concentration of each acyl acceptor (Fig. 2A–D). Although these experiments were not designed to calculate apparent Km and Vmax, under the assay condition used, we found that the maximal enzymatic activity measured was highest with monoacylglycerol as the acceptor, a condition that reflects both MGAT and DGAT activities (Fig. 2A). The apparent maximal activities observed with other acceptors were ~10% of that for monoacylglycerol but were still considerable (between 0.5 and 1.5 nmol/min/mg protein) (Fig. 2A–D).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Dependency of acyltransferase activities in membrane expressing DGAT1 on acyl acceptor concentrations. A: Acylglycerol acyltransferase (both DGAT and MGAT) activities assessed by the amount of [14C]palmitoyl CoA incorporated into both diacylglycerol and triacylglycerol in the presence of different concentrations of sn-2 monooleoylglycerol. B: Wax monoester synthase activity assessed by the amount of [14C]palmitoyl CoA incorporated into wax monoester in the presence of different concentrations of 1-hexadecanol. C: Wax diester synthase activity assessed by the amount of [14C]palmitoyl CoA incorporated into wax diester in the presence of different concentrations of 1,2-hexadecandiol. D: ARAT activity assessed by the amount of [14C]palmitoyl CoA incorporated into retinyl ester in the presence of different concentrations of all-trans retinol. Values are the mean of duplicate measurements and are representative of two experiments.

 
We also examined these acyltransferase activities at different concentrations of palmitoyl CoA and a fixed concentration of radiolabeled acyl acceptor as tracers (200 µM [3H]sn-2-monooleoylglycerol for MGAT activity, 100 µM [14C]1-hexadecanol for wax synthase activity, and 50 µM [3H] all-trans retinol for ARAT activity). In each case, the acyltransferase activity increased with increasing acyl CoA concentrations (Fig. 3A–C). Again, the activity was highest with sn-2-monoacylglycerol as the acyl acceptor, and the levels of this MGAT activity did not reach plateau even when the concentration of palmitoyl CoA was as high as 200 µM (Fig. 3A–C). Because these assays utilized radiolabeled acyl acceptors as substrates, they provide strong evidence that DGAT1 possesses in vitro MGAT, wax synthase, and ARAT activities.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 3. Dependency of acyltransferase activities in membrane expressing DGAT1 on acyl donor concentrations. A: MGAT activity assessed by the amount of [3H]sn-2 monooleoylglycerol incorporated into both diacylglycerol and triacylglycerol in the presence of different concentrations of palmitoyl CoA. B: Wax monoester synthase activity assessed by the amount of [14C]1-hexadecanol incorporated into wax monoester in the presence of different concentrations of palmitoyl CoA. C: ARAT activity assessed by the amount of [3H]all-trans retinol incorporated into retinyl ester in the presence of different concentrations of palmitoyl CoA. Values are the mean of duplicate measurements and are representative of two experiments.

 
DGAT1 possesses wax synthase and ARAT activities in mammalian cells
To determine whether DGAT1 possesses wax synthase and ARAT activities in mammalian cells, we transiently overexpressed mouse DGAT1 and control enzymes in COS-7 cells and assessed their enzymatic activities in cellular homogenates. The expression of FLAG-tagged DGAT and control proteins was demonstrated by immunoblotting (Fig. 4A). In control assays with diacylglycerol and [14C]palmitoyl CoA as the tracer, homogenates of COS-7 cells overexpressing DGAT1 or DGAT2 incorporated more radioactivity into triacylglycerols than did controls overexpressing other enzymes, including ß-galactosidase (LacZ), ACAT2, MGAT1, and MGAT2 (Fig. 4B). Homogenates of cells overexpressing ACAT2 incorporated more [14C]palmitoyl CoA into cholesterol esters than did the other enzymes. With 1-hexadecanol as the acyl acceptor, only homogenates from cells expressing DGAT1 exhibited wax synthase activity (Fig. 4C). Similarly, with all-trans retinol as the acceptor, homogenates from cells expressing DGAT1 exhibited ARAT activity (Fig. 4D). In some experiments, homogenates from cells expressing MGAT1 also exhibited very low levels of ARAT activity.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 4. Expression of DGAT1 in mammalian cells confers wax synthase and ARAT activity in in vitro assays. A: Immunoblotting of FLAG-tagged proteins demonstrating expression of FLAG-tagged DGAT1 and control proteins. Cells were transfected with expression vectors containing cDNAs for ß-galactosidase (LacZ) or FLAG-tagged versions of acyl CoA:cholesterol acyltransferase 2 (ACAT2), DGAT1, DGAT2, MGAT1, or MGAT2. B–D: DGAT (B), wax synthase (C), and ARAT (D) activities in COS-7 cell homogenates conferred by expression of DGAT1. [14C]palmitoyl CoA was used for enzyme assays. Arrows indicate the incorporation of [14C]palmitoyl CoA into triacylglycerol (TAG), wax monoester (ME), and retinyl ester (RE). CE, cholesterol ester. FFA, free fatty acid. Chromatographs shown are representative of three independent experiments.

 
To determine whether DGAT1 exhibits wax synthase and ARAT activities in intact cells, we measured the accumulation of radiolabeled substrates ([14C]1-hexadecanol or [3H]all-trans retinol) in esterification products in COS-7 cells overexpressing mouse DGAT1 and control enzymes (Fig. 5A). (We did not examine MGAT activity in intact cells overexpressing DGAT1 because monoacylglycerol, the MGAT reaction substrate, can be hydrolyzed before uptake by cells, and because diacylglycerol, the MGAT reaction product, can be converted to other lipids and does not accumulate in significant amounts.) Wax ester synthesis was highest in cells overexpressing DGAT1 (~10-fold of LacZ controls; Fig. 5B). Cells expressing DGAT2, and to a lesser extent cells overexpressing MGAT1, also synthesized more wax esters than did control cells. Retinol esterification levels were highest in cells overexpressing DGAT1 (~5-fold of LacZ controls; Fig. 5C). Cells overexpressing MGAT1 also exhibited higher levels of retinol esterfication than did control cells.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. Expression of DGAT1 in mammalian cells increases esterification of fatty acyl alcohol and retinol in intact cells. A: Immunoblotting of FLAG-tagged proteins demonstrating expression of FLAG-tagged DGAT1 and control proteins. B and C: Increased incorporation of fatty acyl alcohol into wax esters and of retinol into retinyl esters in COS-7 cells overexpressing DGAT1. Cells were plated in six-well plates, transfected with DGAT1 or control proteins, and 24 h later, incubated in medium containing 5 µM [14C]1-hexadecanol (specific activity, ~5,500 cpm/nmol) or 2.5 µM [3H]retinol (specific activity, ~200,000 cpm/nmol) for 18 h. Neutral lipids were separated by TLC, visualized with a Bioscan imaging scanner, and scraped to assess the incorporation of radioactivity. Values represent results from six pooled six-well dishes of each group. Similar results were observed in three independent experiments.

 
In addition, we examined COS-7 cells expressing human DGAT1 without the FLAG tag epitope. Like mouse DGAT1, human DGAT1 exhibited wax synthase and ARAT activities both in in vitro assays and in intact cell esterification assays (not shown). These findings also showed that the presence of the FLAG tag at the N-terminus of DGAT1 did not affect these additional acyltransferase activities.

Cells and tissues lacking DGAT1 exhibit reduced ARAT activity
Because the genes encoding ARAT enzymes have not been identified, we further characterized the ARAT activity of DGAT1 by examining whether ARAT activity was diminished in mouse cells and tissues lacking DGAT1. We first examined whether primary MEFs from Dgat1–/– mice have decreased in vitro ARAT activity. In a control experiment with [14C]palmitoyl CoA as the acyl donor and diacylglycerol as the acceptor, homogenates from two independent lines of Dgat1–/– MEFs exhibited less triacylglycerol synthesis than did homogenates from wild-type MEFs, as expected (not shown). With [14C]palmitoyl CoA as the acyl donor and all-trans-retinol as the acyl acceptor, ARAT activity was detectable in homogenates of three different wild-type MEF cell lines (Fig. 6A). In contrast, ARAT activity was nearly absent in homogenates of two different lines of Dgat1–/– MEFs.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 6. Mouse embryonic fibroblasts (MEFs) lacking DGAT1 exhibit reduced ARAT activity. A: Reduced in vitro ARAT activities in MEF homogenates lacking DGAT1. Primary MEFs were established from DGAT1-deficient embryos and their wild-type littermates as described (10). Each number represents an independent line of MEF cells. [14C]palmitoyl CoA was used for enzyme assays. Values are mean ± SD of four measurements. B: Decreased incorporation of retinol into retinyl esters in MEFs lacking DGAT1. Cells were plated in triplicate and, 24 h later, incubated in medium containing 5 µM [3H]retinol (specific activity, ~70,000 cpm/nmol) for an additional 18 h. Values are mean ± SD of measurements from three plates of each clone and are representative of two repeated experiments.

 
We next examined retinol esterification in intact wild-type and Dgat1–/– MEFs by measuring the accumulation of radioactivity in retinyl esters after 18 h incubation with [3H]all-trans-retinol. Levels of radiolabeled retinyl esters in two different cell lines of Dgat1–/– MEFs were less than one-half of those in wild-type MEF lines (Fig. 6B). We excluded a dilution effect of the tracer as a cause of the reduced retinol esterification in Dgat1–/– MEFs by measuring the retinol content in wild-type and Dgat1–/– MEFs. We found that the retinol levels were similar in wild-type and Dgat1–/– MEFs [1.68 ± 1.25 vs. 1.45 ± 0.004 pmol/106 cell (mean ± SD)] and that the basal cellular retinol levels were 3 orders of magnitude lower than the levels of labeled retinol taken up by cells (0.74–3.06 pmol/106 cell vs. 2–3 nmol/106 cell). The residual retinol esterification in Dgat1–/– MEFs is presumably due to a nonARAT activity, possibly that of lecithin:retinol acyltransferase (LRAT), which uses lecithin (phosphatidylcholine) instead of fatty acyl CoA as the acyl donor.

We next examined in vitro ARAT activities in liver, testis, and kidney homogenates from wild-type and Dgat1–/– mice. In control assays, ACAT activities were similar in homogenates from wild-type and Dgat1–/– tissues (Fig. 7A). In Dgat1–/– tissue homogenates, DGAT activity was reduced by more than 80% (Fig. 7B) and ARAT activity was reduced by 75–90% (Fig. 7C).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7. Reduced in vitro ARAT activities in liver, testis, and kidney of DGAT1-deficient mice. Tissue homogenates were from wild-type and Dgat1–/– mice fed a regular chow. The activities of ACAT (A), DGAT (B), and ARAT (C) were assessed by the incorporation of [14C] palmitoyl CoA (25 µM) into cholesterol esters, retinyl ester, and triacylglycerol, respectively. Values are mean ± SD of measurements from four mice in each group. * P < 0.001 versus wild-type mice (two-way ANOVA followed by Tukey test for multiple comparisons).

 
Finally, we assessed whether retinol metabolism is perturbed in Dgat1–/– mice. Mice were fed diets containing different levels of retinol, and retinol and retinyl esters were measured in the liver, where the majority of retinol is stored. In mice fed standard chow (~10 IU/g retinol), the hepatic content of unesterified retinol was similar in wild-type and Dgat1–/– mice (Fig. 8A). However, in mice fed either a high-fat diet (~20 IU/g retinol) or a high-retinol diet (160 IU/g retinol), unesterified retinol was 2- to 3-fold more abundant in Dgat1–/– livers (Fig. 8A). Interestingly, the hepatic content of retinyl esters was not reduced in Dgat1–/– livers from mice fed any of the diets, and in fact, retinyl ester levels were significantly higher in livers of Dgat1–/– mice fed the high-fat diet (Fig. 8B).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 8. Retinol and retinyl ester content in the livers of DGAT1-deficient mice. Wild-type and Dgat1–/– mice were fed a regular chow (containing 10 IU/g retinol), a high-fat diet (HF, 20 IU/g retinol), or a high-vitamin A diet (HA, 160 IU/g retinol). Levels of retinol (A) and retinyl esters (B) were measured by HPLC methods. Values are mean ± SEM. (The number of mice is indicated in the bars.) * P < 0.05 versus wild-type mice (two-way ANOVA followed by Tukey test for multiple comparisons).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that mammalian DGAT1 possesses MGAT, wax synthase, and ARAT activities as well as DGAT activity in in vitro assays of membranes of insect cells or homogenates of mammalian cells overexpressing DGAT1. Wax synthase and ARAT activities catalyzed by DGAT1 were demonstrated in intact cells, and ARAT activity was reduced in DGAT1-deficient cells and tissues. These additional activities were not found for DGAT2, with the exception of increased wax synthesis in intact cells overexpressing DGAT2, which was reported previously (15). Thus, unlike DGAT2, which appears to function primarily as a DGAT, DGAT1 possesses multiple acyltransferase activities.

These results are consistent with the evolutionary history of the two enzymes. The DGAT2 homolog in Saccharomyces cerevisiae, Dga1p, plays a major role in triacylglycerol synthesis (24, 28), whereas the DGAT1 homologs, Are1p and Are2p, play minor roles (24). Instead, Are1p and Are2p are involved in sterol esterification (29). Because the relatives of DGAT1 in lower organisms were not primarily involved in esterifying diacylglycerol, DGAT1 must have acquired the ability to utilize diacylglycerol as a substrate in higher organisms during evolution. Therefore, it is plausible that the substrate recognition site of DGAT1 is less discriminating than the corresponding site of DGAT2. Moreover, DGAT1 may have also acquired the ability to utilize other fatty acyl acceptors, such as monoacylglycerol, long-chain alcohols, and retinol.

The demonstration that DGAT1 possesses MGAT activity, as noted briefly in reports of the cloning of MGAT genes (11, 12), suggests that DGAT1 can catalyze the sequential esterification of two fatty acyl moieties to convert monoacylglycerol to diacylglycerol and then to triacylglycerol. This ability is consistent with a role for DGAT1 in the reesterification of monoacylglycerol and diacylglycerol generated by triacylglycerol hydrolysis in cells. Whether this is a function of DGAT1 in intact cells requires further testing.

DGAT1 catalyzed the synthesis of both wax monoesters and wax diesters. The combination of wax monoester synthase and DGAT activities is similar to that of an unrelated wax synthase/DGAT found in bacteria (30). It is not known whether, like DGAT1, the bacterial bifunctional enzyme is active in forming wax diesters. The wax monoester synthase activity of DGAT1 agrees with a recent report showing that DGAT1 overexpression in intact cells increases wax synthesis (15). The wax diester synthase activity of DGAT1 may help to explain the deficiency of type II wax diesters in the fur lipids of Dgat1–/– mice (22). However, because wax diesters are present in the fur of leptin-deficient Dgat1–/– mice (22), other wax synthases must exist. Our results show that DGAT2 overexpression also increased wax monoester synthase activity in intact cells, a finding that was recently reported (15), although we did not detect either wax monoester or diester synthase activity in in vitro assays of DGAT2. Additional wax synthases (members of the DGAT2 gene family) that catalyze the synthesis of wax monoesters (15, 16), but not wax diesters, were recently identified (C-L. Yen et al., unpublished observations).

Although ARAT activity was reported decades ago (31–34), the ARAT genes have not been identified. The current findings suggest that DGAT1, which is expressed in tissues reported to have ARAT activity, contributes to ARAT activity in vivo. DGAT1 exhibited ARAT activity in in vitro assays and accounted for a large portion of ARAT activity in the liver, testis, and kidney of mice. DGAT1 deficiency also perturbed retinol metabolism in the livers of Dgat1–/– mice. The physiological significance of ARAT, however, is not clear. Retinyl esters can also be synthesized by LRAT (32, 35–38). LRAT has a significantly lower Km for retinol than does ARAT (~2 µM vs. ~15–30 µM), suggesting that LRAT plays the major role in retinol storage when cellular retinol concentrations are relatively low. Indeed, mice lacking LRAT have severe reductions in retinyl ester content in liver, blood, and eyes and exhibit abnormalities of the retinal pigmented epithelium (39). The preferred substrate for LRAT is believed to be retinol bound to cytosolic retinol binding protein, whereas ARAT prefers unbound retinol as a substrate. Therefore, ARAT may play a role in retinol metabolism in tissues with low levels of cytosolic retinol binding protein or when cellular retinol concentrations are relatively high (40), thereby protecting cells from toxicity resulting from excess unesterified retinol. Our data agree with this possible function for ARAT, because hepatic levels of unesterified retinol were increased in Dgat1–/– mice challenged with high-retinol diets. The high levels of unesterified retinol in the livers of Dgat1–/– mice also raise the possibility that alterations in retinoid metabolism may contribute to the phenotype of mice lacking DGAT1. Retinol is the precursor for retinoic acids (all-trans and its isomer 9-cis retinoic acids), which are ligands for the nuclear hormone receptors retinoic acid receptor and retinoid-X receptor (41–43), and retinoids affect sebaceous gland function (44, 45), hair growth (46), and proliferation of mammary gland epithelial cells (47, 48), all of which are part of phenotypic changes observed in Dgat1–/– mice (17, 22, 23).

Consistent with LRAT being the major enzyme responsible for maintaining retinyl ester levels in mouse liver (39), hepatic retinyl ester content was not reduced in Dgat1–/– livers. In fact, the hepatic retinyl ester content in Dgat1–/– mice on a high-fat diet was, paradoxically, increased. The explanation for this finding is unclear at present. It is possible that the increases in retinyl ester content resulted from increased LRAT activity or decreased hydrolysis of retinyl esters.

In summary, although both DGAT1 and DGAT2 share diacylglycerol as a common fatty acyl acceptor, the two enzymes differ in their ability to use other acyl acceptors. DGAT1, much more than DGAT2, can catalyze esterification reactions utilizing a variety of fatty acyl acceptors and is, therefore, a less-discriminating enzyme than DGAT2. Our findings indicate that DGAT1 is a multifunctional acyltransferase, and these additional activities of DGAT1 may be relevant to its in vivo functions.


   ACKNOWLEDGMENTS
 
The authors thank S. Cases for providing MEF cells, T. Neidlinger (USDA) for measuring retinoids, and G. Gildengorin (USDA) for statistical analysis of these measurements; S. Erickson, Y. Huang, R. Mahley, K. Weisgraber, and M. Zeiger for comments on the manuscript; S. Ordway and G. Howard for editorial assistance; and B. Taylor for manuscript preparation. This work was supported by National Institutes of Health Grant DK-56084 (R.V.F.), an American Heart Association fellowship (C-L.E.Y.), and the J. David Gladstone Institutes.

Manuscript received January 31, 2005 and in revised form April 7, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Unger, R. H., and L. Orci. 2002. Lipoapoptosis: its mechanism and its diseases. Biochim. Biophys. Acta. 1585: 202–212.[Medline]

  2. Chang, C. C. Y., H. Y. Huh, K. M. Cadigan, and T. Y. Chang. 1993. Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J. Biol. Chem. 268: 20747–20755.[Abstract/Free Full Text]

  3. Cases, S., S. Novak, Y-W. Zheng, H. M. Myers, S. R. Lear, E. Sande, C. B. Welch, A. J. Lusis, T. A. Spencer, B. R. Krause, et al. 1998. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization. J. Biol. Chem. 273: 26755–26764.[Abstract/Free Full Text]

  4. Oelkers, P., A. Behari, D. Cromley, J. T. Billheimer, and S. L. Sturley. 1998. Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes. J. Biol. Chem. 273: 26765–26771.[Abstract/Free Full Text]

  5. Cases, S., S. J. Smith, Y-W. Zheng, H. M. Myers, S. R. Lear, E. Sande, S. Novak, C. Collins, C. B. Welch, A. J. Lusis, S. K. Erickson, and R. V. Farese, Jr. 1998. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc. Natl. Acad. Sci. USA. 95: 13018–13023.[Abstract/Free Full Text]

  6. Buhman, K. K., H. C. Chen, and R. V. Farese, Jr. 2001. The enzymes of neutral lipid synthesis. J. Biol. Chem. 276: 40369–40372.[Free Full Text]

  7. Yu, C., J. Chen, S. Lin, J. Liu, C. C. Y. Chang, and T-Y. Chang. 1999. Human acyl-CoA:cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro. J. Biol. Chem. 274: 36139–36145.[Abstract/Free Full Text]

  8. Cheng, D., R. L. Meegalla, B. He, D. A. Cromley, J. T. Billheimer, and P. R. Young. 2001. Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein. Biochem. J. 359: 707–714.[CrossRef][Medline]

  9. Lardizabal, K. D., J. T. Mai, N. W. Wagner, A. Wyrick, T. Voelker, and D. J. Hawkins. 2001. DGAT2 is a new diacylglycerol acyltransferase gene family. Purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J. Biol. Chem. 276: 38862–38869.[Abstract/Free Full Text]

  10. Cases, S., S. J. Stone, P. Zhou, E. Yen, B. Tow, K. D. Lardizabal, T. Voelker, and R. V. Farese, Jr. 2001. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. J. Biol. Chem. 276: 38870–38876.[Abstract/Free Full Text]

  11. Yen, C-L. E., S. J. Stone, S. Cases, P. Zhou, and R. V. Farese, Jr. 2002. Identification of a gene encoding MGAT1, a monoacylglylcerol acyltransferase. Proc. Natl. Acad. Sci. USA. 99: 8512–8517.[Abstract/Free Full Text]

  12. Yen, C-L. E., and R. V. Farese, Jr. 2003. MGAT2, a monoacylglycerol acyltransferase expressed in the small intestine. J. Biol. Chem. 278: 18532–18537.[Abstract/Free Full Text]

  13. Cheng, D., T. C. Nelson, J. Chen, S. G. Walker, J. Wardwell-Swanson, R. Meegalla, R. Taub, J. T. Billheimer, M. Ramaker, and J. N. Feder. 2003. Identification of acyl coenzyme A:monoacylglycerol acyltransferase 3, an intestinal specific enzyme implicated in dietary fat absorption. J. Biol. Chem. 278: 13611–13614.[Abstract/Free Full Text]

  14. Cao, J., J. Lockwood, P. Burn, and Y. Shi. 2003. Cloning and functional characterization of a mouse intestinal acyl-CoA:monoacylglycerol acyltransferase, MGAT2. J. Biol. Chem. 278: 13860–13866.[Abstract/Free Full Text]

  15. Cheng, J. B., and D. W. Russell. 2004. Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family. J. Biol. Chem. 279: 37798–37807.[Abstract/Free Full Text]

  16. Turkish, A. R., A. L. Henneberry, D. Cromley, M. Padamsee, P. Oelkers, H. Bazzi, A. M. Christiano, J. T. Billheimer, and S. L. Sturley. 2005. Identification of two novel human Acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily. J. Biol. Chem. 280: 14755–14764.[Abstract/Free Full Text]

  17. Smith, S. J., S. Cases, D. R. Jensen, H. C. Chen, E. Sande, B. Tow, D. A. Sanan, J. Raber, R. H. Eckel, and R. V. Farese, Jr. 2000. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking DGAT. Nat. Genet. 25: 87–90.[CrossRef][Medline]

  18. Stone, S. J., H. Myers, B. E. Brown, S. M. Watkins, K. R. Feingold, P. M. Elias, and R. V. Farese, Jr. 2004. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice. J. Biol. Chem. 279: 11767–11776.[Abstract/Free Full Text]

  19. Chen, H. C., S. J. Smith, Z. Ladha, D. R. Jensen, L. D. Ferreira, L. K. Pulawa, J. G. McGuire, R. E. Pitas, R. H. Eckel, and R. V. Farese, Jr. 2002. Increased insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1. J. Clin. Invest. 109: 1049–1055.[CrossRef][Medline]

  20. Chen, H. C., Z. Ladha, and R. V. Farese, Jr. 2002. Deficiency of acyl coenzyme a:diacylglycerol acyltransferase 1 increases leptin sensitivity in murine obesity models. Endocrinology. 143: 2893–2898.[Abstract/Free Full Text]

  21. Chen, H. C., D. R. Jensen, H. M. Myers, R. H. Eckel, and R. V. Farese, Jr. 2003. Obesity resistance and enhanced glucose metabolismin in mice transplanted with white adipose tissue lacking acyl CoA:diacylglycerol acyltransferase 1. J. Clin. Invest. 111: 1715–1722.[CrossRef][Medline]

  22. Chen, H. C., S. J. Smith, B. Tow, P. M. Elias, and R. V. Farese, Jr. 2002. Leptin modulates the effects of acyl CoA:diacylglycerol acyltransferase deficiency on murine fur and sebaceous glands. J. Clin. Invest. 109: 175–181.[CrossRef][Medline]

  23. Cases, S., P. Zhou, J. Schillingford, B. Wiseman, J. Fish, C. S. Angle, L. Hennighausen, Z. Werb, and R. Farese, Jr. 2004. Development of the mammary gland requires DGAT1 expression in stromal and epithelial tissues. Development. 131: 3047–3055.[Abstract/Free Full Text]

  24. Oelkers, P., D. Cromley, M. Padamsee, J. T. Billheimer, and S. L. Sturley. 2002. The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J. Biol. Chem. 277: 8877–8881.[Abstract/Free Full Text]

  25. Oelkers, P., A. Tinkelenberg, N. Erdeniz, D. Cromley, J. T. Billheimer, and S. L. Sturley. 2000. A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J. Biol. Chem. 275: 15609–15612.[Abstract/Free Full Text]

  26. Burri, B. J., T. R. Neidlinger, A. O. Lo, C. Kwan, and M. R. Wong. 1997. Supercritical fluid extraction and reversed-phase liquid chromatography methods for vitamin A and beta-carotene heterogeneous distribution of vitamin A in the liver. J. Chromatogr. A. 762: 201–206.[CrossRef][Medline]

  27. Craft, N. E., T. B. Haitema, K. M. Garnett, K. A. Fitch, and C. K. Dorey. 2004. Carotenoid, tocopherol, and retinol concentrations in elderly human brain. J. Nutr. Health Aging. 8: 156–162.[Medline]

  28. Sorger, D., and G. Daum. 2002. Synthesis of triacylglycerols by the acyl-coenzyme A:diacyl-glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae. J. Bacteriol. 184: 519–524.[Abstract/Free Full Text]

  29. Yang, H., M. Bard, D. A. Bruner, A. Gleeson, R. J. Deckelbaum, G. Aljinovic, T. M. Pohl, R. Rothstein, and S. L. Sturley. 1996. Sterol esterification in yeast: a two-gene process. Science. 272: 1353–1356.[Abstract]

  30. Kalscheuer, R., and A. Steinbuchel. 2003. A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J. Biol. Chem. 278: 8075–8082.[Abstract/Free Full Text]

  31. Ross, A. C. 1982. Retinol esterification by rat liver microsomes. Evidence for a fatty acyl coenzyme A:retinol acyltransferase. J. Biol. Chem. 257: 2453–2459.[Abstract/Free Full Text]

  32. Saari, J. C., and D. L. Bredberg. 1988. CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium. J. Biol. Chem. 263: 8084–8090.[Abstract/Free Full Text]

  33. Helgerud, P., L. B. Petersen, and K. R. Norum. 1982. Acyl CoA:retinol acyltransferase in rat small intestine: its activity and some properties of the enzymic reaction. J. Lipid Res. 23: 609–618.[Abstract]

  34. Blomhoff, R., M. Rasmussen, A. Nilsson, K. R. Norum, T. Berg, W. S. Blaner, M. Kato, J. R. Mertz, D. S. Goodman, U. Eriksson, et al. 1985. Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells. J. Biol. Chem. 260: 13560–13565.[Abstract/Free Full Text]

  35. MacDonald, P. N., and D. E. Ong. 1988. A lecithin:retinol acyltransferase activity in human and rat liver. Biochem. Biophys. Res. Commun. 156: 157–163.[CrossRef][Medline]

  36. Yost, R. W., E. H. Harrison, and A. C. Ross. 1988. Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein. J. Biol. Chem. 263: 18693–18701.[Abstract/Free Full Text]

  37. Schmitt, M. C., and D. E. Ong. 1993. Expression of cellular retinol-binding protein and lecithin-retinol acyltransferase in developing rat testis. Biol. Reprod. 49: 972–979.[Abstract]

  38. Zolfaghari, R., and A. C. Ross. 2000. Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin A and retinoic acid. J. Lipid Res. 41: 2024–2034.[Abstract/Free Full Text]

  39. Batten, M. L., Y. Imanishi, T. Maeda, D. C. Tu, A. R. Moise, D. Bronson, D. Possin, R. N. Van Gelder, W. Baehr, and K. Palczewski. 2004. Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. J. Biol. Chem. 279: 10422–10432.[Abstract/Free Full Text]

  40. Randolph, R. K., K. E. Winkler, and A. C. Ross. 1991. Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes. Arch. Biochem. Biophys. 288: 500–508.[CrossRef][Medline]

  41. Petkovich, M., N. J. Brand, A. Krust, and P. Chambon. 1987. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature. 330: 444–450.[CrossRef][Medline]

  42. Giguere, V., E. S. Ong, P. Segui, and R. M. Evans. 1987. Identification of a receptor for the morphogen retinoic acid. Nature. 330: 624–629.[CrossRef][Medline]

  43. Heyman, R. A., D. J. Mangelsdorf, J. A. Dyck, R. B. Stein, G. Eichele, R. M. Evans, and C. Thaller. 1992. 9-Cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 68: 397–406.[CrossRef][Medline]

  44. Zouboulis, C. C., B. Korge, H. Akamatsu, L. Q. Xia, S. Schiller, H. Gollnick, and C. E. Orfanos. 1991. Effects of 13-cis-retinoic acid, all-trans-retinoic acid, and acitretin on the proliferation, lipid synthesis and keratin expression of cultured human sebocytes in vitro. J. Invest. Dermatol. 96: 792–797.[CrossRef][Medline]

  45. Strauss, J. S., M. E. Stewart, and D. T. Downing. 1987. The effect of 13-cis-retinoic acid on sebaceous glands. Arch. Dermatol. 123: 1538–1541.

  46. Bazzano, G., N. Terezakis, H. Attia, A. Bazzano, R. Dover, D. Fenton, N. Mandir, L. Celleno, M. Tamburro, and S. Jaconi. 1993. Effect of retinoids on follicular cells. J. Invest. Dermatol. 101 (Suppl. 1): 138–142.[CrossRef]

  47. Mehta, R. G., W. L. Cerny, and R. C. Moon. 1983. Retinoids inhibit prolactin-induced development of the mammary gland in vitro. Carcinogenesis. 4: 23–26.[Abstract/Free Full Text]

  48. Kistler, A. 1986. Structure-activity relationship of retinoids on lobuloalveolar differentiation of cultured mouse mammary glands. Carcinogenesis. 7: 1175–1182.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 FASEB J.Home page
K. Strom, T. E. Gundersen, O. Hansson, S. Lucas, C. Fernandez, R. Blomhoff, and C. Holm
Hormone-sensitive lipase (HSL) is also a retinyl ester hydrolase: evidence from mice lacking HSL
FASEB J, July 1, 2009; 23(7): 2307 - 2316.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. R. Turkish and S. L. Sturley
The genetics of neutral lipid biosynthesis: an evolutionary perspective
Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E19 - E27.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
Y. Shi and D. Cheng
Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism
Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E10 - E18.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Y. S. Shih, M. A. Kane, P. Zhou, C. L. E. Yen, R. S. Streeper, J. L. Napoli, and R. V. Farese Jr.
Retinol Esterification by DGAT1 Is Essential for Retinoid Homeostasis in Murine Skin
J. Biol. Chem., February 13, 2009; 284(7): 4292 - 4299.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
C.-L. E. Yen, S. J. Stone, S. Koliwad, C. Harris, and R. V. Farese Jr.
Thematic Review Series: Glycerolipids. DGAT enzymes and triacylglycerol biosynthesis
J. Lipid Res., November 1, 2008; 49(11): 2283 - 2301.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Cheng, J. Iqbal, J. Devenny, C.-H. Chu, L. Chen, J. Dong, R. Seethala, W. J. Keim, A. V. Azzara, R. M. Lawrence, et al.
Acylation of Acylglycerols by Acyl Coenzyme A:Diacylglycerol Acyltransferase 1 (DGAT1): FUNCTIONAL IMPORTANCE OF DGAT1 IN THE INTESTINAL FAT ABSORPTION
J. Biol. Chem., October 31, 2008; 283(44): 29802 - 29811.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Cao, D. Shan, T. Revett, D. Li, L. Wu, W. Liu, J. F. Tobin, and R. E. Gimeno
Molecular Identification of a Novel Mammalian Brain Isoform of Acyl-CoA:Lysophospholipid Acyltransferase with Prominent Ethanolamine Lysophospholipid Acylating Activity, LPEAT2
J. Biol. Chem., July 4, 2008; 283(27): 19049 - 19057.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Wongsiriroj, R. Piantedosi, K. Palczewski, I. J. Goldberg, T. P. Johnston, E. Li, and W. S. Blaner
The Molecular Basis of Retinoid Absorption: A Genetic Dissection
J. Biol. Chem., May 16, 2008; 283(20): 13510 - 13519.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Minahk, K.-W. Kim, R. Nelson, B. Trigatti, R. Lehner, and D. E. Vance
Conversion of Low Density Lipoprotein-associated Phosphatidylcholine to Triacylglycerol by Primary Hepatocytes
J. Biol. Chem., March 7, 2008; 283(10): 6449 - 6458.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
K. R. Smith and D. M. Thiboutot
Thematic review series: Skin Lipids. Sebaceous gland lipids: friend or foe?
J. Lipid Res., February 1, 2008; 49(2): 271 - 281.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
A. Ruiz, N. B. Ghyselinck, N. Mata, S. Nusinowitz, M. Lloyd, C. Dennefeld, P. Chambon, and D. Bok
Somatic Ablation of the Lrat Gene in the Mouse Retinal Pigment Epithelium Drastically Reduces Its Retinoid Storage
Invest. Ophthalmol. Vis. Sci., December 1, 2007; 48(12): 5377 - 5387.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. C. Levin, M. Monetti, M. J. Watt, M. P. Sajan, R. D. Stevens, J. R. Bain, C. B. Newgard, R. V. Farese Sr., and R. V. Farese Jr.
Increased lipid accumulation and insulin resistance in transgenic mice expressing DGAT2 in glycolytic (type II) muscle
Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1772 - E1781.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
M. Westerterp, J. F. P. Berbee, D. J. M. Delsing, M. C. Jong, M. J. J. Gijbels, V. E. H. Dahlmans, E. H. Offerman, J. A. Romijn, L. M. Havekes, and P. C. N. Rensen
Apolipoprotein C-I binds free fatty acids and reduces their intracellular esterification
J. Lipid Res., June 1, 2007; 48(6): 1353 - 1361.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
X.-H. Tang, M.-J. Suh, R. Li, and L. J. Gudas
Cell proliferation inhibition and alterations in retinol esterification induced by phytanic acid and docosahexaenoic acid
J. Lipid Res., January 1, 2007; 48(1): 165 - 176.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. J. Stone, M. C. Levin, and R. V. Farese Jr.
Membrane Topology and Identification of Key Functional Amino Acid Residues of Murine Acyl-CoA:Diacylglycerol Acyltransferase-2
J. Biol. Chem., December 29, 2006; 281(52): 40273 - 40282.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
S. Saha, B. Enugutti, S. Rajakumari, and R. Rajasekharan
Cytosolic Triacylglycerol Biosynthetic Pathway in Oilseeds. Molecular Cloning and Expression of Peanut Cytosolic Diacylglycerol Acyltransferase
Plant Physiology, August 1, 2006; 141(4): 1533 - 1543.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Golczak, Y. Imanishi, V. Kuksa, T. Maeda, R. Kubota, and K. Palczewski
Lecithin:Retinol Acyltransferase Is Responsible for Amidation of Retinylamine, a Potent Inhibitor of the Retinoid Cycle
J. Biol. Chem., December 23, 2005; 280(51): 42263 - 42273.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Liu and L. J. Gudas
Disruption of the Lecithin:Retinol Acyltransferase Gene Makes Mice More Susceptible to Vitamin A Deficiency
J. Biol. Chem., December 2, 2005; 280(48): 40226 - 40234.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
C.-L. E. Yen, C. H. Brown IV, M. Monetti, and R. V. Farese Jr.
A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters
J. Lipid Res., November 1, 2005; 46(11): 2388 - 2397.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. M. O'Byrne, N. Wongsiriroj, J. Libien, S. Vogel, I. J. Goldberg, W. Baehr, K. Palczewski, and W. S. Blaner
Retinoid Absorption and Storage Is Impaired in Mice Lacking Lecithin:Retinol Acyltransferase (LRAT)
J. Biol. Chem., October 21, 2005; 280(42): 35647 - 35657.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M500036-JLR200v1
46/7/1502    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yen, C.-L. E.
Right arrow Articles by Farese, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yen, C.-L. E.
Right arrow Articles by Farese, R. V., Jr.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Advertisement
spacer
Advertisement
Advertisement