Cloning and Functional Characterization of a Mouse Intestinal
Acyl-CoA:Monoacylglycerol Acyltransferase, MGAT2*
Jingsong
Cao,
John
Lockwood,
Paul
Burn, and
Yuguang
Shi
From the Endocrine Research, Lilly Research Laboratories, Eli Lilly
and Company, Indianapolis, Indiana 46285
Received for publication, January 7, 2003, and in revised form, February 4, 2003
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ABSTRACT |
Acyl-CoA:monoacylglycerol transferase (MGAT)
plays a predominant role in dietary fat absorption in the small
intestine, where it catalyzes the first step of triacylglycerol
resynthesis in enterocytes for chylomicron formation and secretion.
Although the mouse small intestine exhibits the highest MGAT enzyme
activity among all of the tissues studied, the gene encoding the enzyme has not been identified so far. In the present studies, we report the
identification and characterization of a mouse intestinal MGAT, MGAT2.
Transient expression of MGAT2 in AV-12, COS-7, and Caco-2 cells led to
a more than 70-, 30-, and 35-fold increase in the synthesis of
diacylglycerol, respectively. MGAT2 expressed in mammalian cells can
catalyze the acylation of rac-1-, sn-2-, and
sn-3-monoacylglycerols, and the enzyme prefers
monoacylglycerols containing unsaturated fatty acyls as substrates.
MGAT2 also demonstrates weak DGAT activity, which can be distinguished
from its MGAT activity by detergent treatment that abolishes DGAT but
not MGAT activity. We also analyzed the biochemical features of MGAT2
and demonstrated homogenate protein-, time-, and substrate
concentration-dependent MGAT enzyme activity in transiently
transfected COS-7 cells. Northern blot analysis indicates that the
mouse MGAT2 is most abundantly expressed in the small intestine,
suggesting that MGAT2 may play an important role in dietary fat absorption.
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INTRODUCTION |
When total energy input is in excess of energy output, most excess
calories are stored as triacylglycerol and deposited into adipose
tissue. The excess storage of triacylglycerol in fat tissues causes
obesity. There are two major sources of triacylglycerol syntheses.
One is the endogenous pathway where triacylglycerol is
resynthesized from circulating free fatty acids, and the other is from
dietary fat absorption. The latter becomes a major contributing factor
for the ongoing epidemic of obesity in people who consume western diets
with high fat content. In mammals, the intestine plays a predominant
role in fat absorption (1, 2). Neutral fat or triacylglycerol is
emulsified to fine lipid droplets and further enzymatically digested to
monoacylglycerol and fatty acids, mainly by pancreatic lipase and its
cofactor. Facilitated by bile salts, the produced monoacylglycerol and
fatty acids are up-taken by enterocytes (3), where they are used to
resynthesize triacylglycerol allowing for lipids to be transported into
the circulation system (1).
Diacylglycerol is a precursor for phospholipids and
triacylglycerol synthesis, which play important roles in cellular
signal transduction (4), intestinal fat absorption (1, 5, 6), energy
storage in muscle and adipose tissues (7), and lactation (5, 8).
Diacylglycerol is synthesized by two pathways, the glycerol phosphate
pathway and the monoacylglycerol pathway (5, 6). The glycerol phosphate
pathway is present in almost all tissues (6). During fat absorption by
intestine, the monoacylglycerol pathway is the major route of
diacylglycerol resynthesis because of the high influx of
monoacylglycerol from breakdown of dietary triacylglycerol (9). In the
intestinal enterocytes, the resynthesis of triacylglycerol is
predominantly catalyzed by two types of enzymes:
acyl-CoA:monoacylglycerol acyltransferase
(MGAT)1 and
acyl-CoA:diacylglycerol acyltransferase (DGAT). Sequentially, MGAT
catalyzes the joining of monoacylglycerol and fatty acyl-CoAs to form
diacylglycerol, whereas DGAT catalyzes the final reaction in which
fatty acyl-CoAs are covalently joined to diacylglycerol, producing
triacylglycerol (10). It was reported that MGAT, DGAT, and acyl-CoA
synthase form a triacylglycerol synthase complex, which is distributed
within intestinal microsomes (11-13).
Two mammalian DGAT enzymes and one MGAT enzyme have been identified so
far (14-16). The identification and characterization of these gene
products and thereafter the generation of knockout mice lacking these
enzymes have greatly extended our understanding of lipid metabolism and
their relevance with human diseases such as obesity (8, 17-19). These
works also provide valuable molecular probes to further explore
additional enzymes involved in lipid metabolism. A gene encoding MGAT1
was recently identified in mice where expression of MGAT1 was detected
in the stomach, kidney, white and brown adipose tissue, and liver (16).
However, expression of MGAT1 was not detected in small intestine, where
high levels of MGAT activity had been observed (16, 20), thereby
implying other potential candidates for intestinal MGAT enzymes. In the present study, we identified and characterized a mouse intestinal MGAT
gene (MGAT2) that is most abundantly expressed in small
intestine. The murine MGAT2 demonstrates a strong MGAT activity and a
weak DGAT activity, with the latter being sensitive to inactivation with detergent.
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EXPERIMENTAL PROCEDURES |
Cloning of Full-length MGAT2 cDNA--
A cDNA clone was
identified in NCBI databases based on sequence homology to the known
mouse MGAT1. According to flanking cDNA ends, primer pairs were
designed to amplify the full-length coding region of MGAT2 from
Marathon-Ready cDNA prepared from mouse small intestine (BD
Biosciences Clontech, Palo Alto, CA). Amplification was performed by PCR using Pfu DNA polymerase (Stratagene,
La Jolla, CA), resulting in a ~1.1-kb cDNA product. The PCR
product was cloned into the pPCR-script Amp SK(+) vector (Stratagene) and sequenced.
Expression of MGAT2 in Mammalian Cells--
A mammalian
expression plasmid coding full-length mouse MGAT2 was engineered by
unidirectional subcloning of the 1.1-kb cDNA fragment from the
pPCR-script Amp SK(+) vector described above into the NotI
and HindIII sites of pcDNA3.1/Hygro(
) mammalian expression vector (Invitrogen) according to the manufacturer's instruction. Transient transfection was performed on AV-12, COS-7, or
Caco-2 cell lines using the mouse MGAT2 expression plasmids. Cells were
maintained under the conditions recommended by the ATCC (Manassas, VA).
A day before transfection, 2-4 million cells were subcultured into
20 × 150-mm plates, resulting in ~70% confluence. Cells were
transfected with 15 µg of DNA with the Fugene 6 mixture (Roche
Molecular Biochemicals) according to the manufacturer's instructions.
Forty-eight hours after transfection, cells were harvested in ice-cold
phosphate-buffered saline, pelleted by centrifugation, lysed, and
assayed immediately or frozen in liquid N2 for later use.
In Vitro Assays for MGAT and DGAT Activity--
Cell pellets
were homogenized in 20 mM NaCl with three short 10-s pulses
from a Brinkmann Instruments Polytron homogenizer. The resultant
homogenates were used to assess the activity of MGAT and DGAT in
transfected mammalian cells. The protein concentration in homogenates
was determined by a BCA protein assay kit (Pierce) according to the
manufacturer's instructions. MGAT and DGAT activity was determined at
room temperature in a final volume of 200 µl as previously described
(14, 16). Briefly, MGAT activity was determined by measuring the
incorporation of [14C]oleoyl moiety into
dioleoylglycerol with [14C]oleoyl-CoA (American
Radiolabeled Chemical Inc., St. Louis, MO) and acyl acceptors
(rac-1-oleoylglycerol, sn-2-oleoylglycerol, rac-1-stearoylglycerol, and
sn-3-stearoylglycerol). The incorporation of the
[14C]oleoyl moiety into trioleoylglycerol with
[14C]oleoyl-CoA and sn-1,2-dioleoylglycerol
was measured to obtain DGAT activity. The acyl acceptors were
introduced into the reaction mixture by liposomes. Briefly, acyl
acceptors and phosphatidylcholine (molar ratio 1:5) were dissolved in
chloroform and dried by vacuum. Liposomes were formed by adding water
and shaking for 1 min. The reaction mixture contained 100 mM Tris/HCl, pH 7.4, 5 mM MgCl2, 1 mg/ml bovine serum albumin free fatty acids (Sigma), 200 mM sucrose, 25 µM [14C]oleoyl-CoA (50 mCi/mmol; American Radiolabeled Chemicals), 200 µM acyl
acceptors in liposomes, and various amounts of cellular homogenate.
Unless indicated elsewhere, these experimental conditions were used
throughout the study. The reaction was initiated by adding protein
homogenate and terminated by adding 4 ml of chloroform/methanol (2:1,
v/v) after predetermined incubation periods. Lipids were extracted with
chloroform/methanol (2:1, v/v) by vortexing for 30 s. After
centrifugation to remove debris, aliquots of the organic phase
containing lipids were dried under a N2 stream and
separated by the Linear-K Preadsorbent TLC plate (Waterman Inc.,
Clifton, NJ) with hexane/ethyl ether/acetic acid (80:20:1, v/v/v). The separation was always performed under the conditions where
sn-1,2(2,3)- and sn-1,3-diacylglycerols were
clearly resolved. Individual lipid moieties were identified by
standards with exposure to I2 vapor. The TLC plates were
exposed to x-ray film or a phosphor screen to assess the incorporation
of 14C-labeled acyl moieties into respective lipid
products. Phosphorimaging signals were visualized using a Storm 860 PhosphorImager (Amersham Biosciences) and quantitated using the
ImageQuant software. The labeled products in TLC plates were also
scraped into scintillation vials to quantitate the incorporation of
radioactivity into lipid products by a Beckman LS 6500 scintillation
system (Fullerton, CA). The isomers of monoacylglycerols
(rac-1-oleoylglycerol, sn-2-oleoylglycerol, rac-1-stearoylglycerol, and
sn-3-stearoylglycerol) were from Sigma. Diacylglycerols
(sn-1,2-dioleoylglycerol,
rac-1,2-dioleoylglycerol, sn-1,3-
dioleoylglycerol, rac-1,2-distearoylglycerol, and
sn-1,3-distearoylglycerol) and triacylglycerols (glycerol
trioleate and glycerol tristearate) were purchased from Doosan Serdary
Research Laboratories (Toronto, Canada).
Northern Blot Analysis--
To analyze the tissue distribution
pattern of mouse MGAT2 mRNA, multiple tissue poly(A)+
RNA Northern blots from OriGene Technologies Inc. (Rockville, MD) or
BioChain Institute Inc. (Hayward, CA) were hybridized with [
-32P]dCTP (3000 Ci/mmol; ICN Radiochemicals) labeled
probes prepared from full-length cDNA of mouse MGAT2 using a random
primer DNA labeling system (Invitrogen). Hybridization was carried out
in ULTRAhyb (Ambion, Austin, TX) at 50 °C overnight, followed by three washes at 55 °C in 2× SSC buffer containing 0.1% SDS and 1 mM EDTA. Blots were stripped with boiling 1% SDS to remove
radiolabeled probe and reprobed with
-actin cDNA as an internal
control. The blots were exposed to Bio-Max MR film or a PhosphorImager
screen to visualize the signals, which were quantitated using
ImageQuant (Amersham Biosciences).
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RESULTS |
Identification of the Mouse MGAT2 Gene--
Yen et al.
(16) recently reported the cloning and characterization of the first
mammalian MGAT, MGAT1. Surprisingly, the mouse
MGAT1 gene is not expressed in the small intestine, a tissue that exhibits the highest MGAT enzyme activity (16); hence, the gene
responsible for the intestinal MGAT activity remains to be identified.
Using the mouse MGAT1 cDNA as a query sequence for blast
analysis of the public genomic and expressed sequence tag data bases,
we identified and cloned a MGAT candidate gene, MGAT2, by
PCR amplification. The mouse MGAT2 gene predicts a 334-amino acid protein of 38.6 kDa and possesses at least one transmembrane region, as suggested by a 40-amino acid signal peptide at the N
terminus (MVEFAPLLVPWERRLQTFAVLQWVFSFLALAQLCIVIFVG), and one potential
tyrosine phosphorylation site. There is no obvious N-linked glycosylation site within the protein predicted by the sequence analysis. Mouse MGAT2 is localized at chromosome 7 at 7E1.
Sequence comparison indicates that the mouse MGAT2 protein shares
extensive homology (52.5% identity) with MGAT1, which spans the entire
molecule (Fig. 1). The mouse MGAT2 also
shares close similarity in hydrophobicity profile with MGAT1 (Fig.
2). Although the MGAT2 shares significant sequence homology (47.5%) with the mouse DGAT2 as well, the homology does not cover the entire molecule (data not shown). Likewise, the
MGAT2 protein also differs from the mouse DGAT2 in hydrophobicity profile (Fig. 2). These features predict the mouse MGAT2 as a novel
member of the MGAT family of enzymes.

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Fig. 1.
Sequence comparison between mouse MGAT2 and
MGAT1. Amino acid residues identical for both enzymes are
indicated with vertical lines; residues with
strong conservation are indicated with a colon; weakly
conserved residues are indicated with a period.
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Fig. 2.
Comparison of hydrophobicity profiles among
the mouse MGAT2, MGAT1, and DGAT2 proteins. The hydrophobicity
profiles are assessed by Kyte-Doolittle analysis.
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Tissue Distribution of Mouse MGAT2 mRNA--
The tissue
distribution of mouse MGAT2 mRNA was analyzed by
Northern blot analysis (Fig. 3). A 1.8-kb
MGAT2 mRNA transcript was detected in small intestine,
kidney, adipose, and stomach (Fig. 3, A and B).
Very low expression levels of MGAT2 were also detected in
liver, skeletal muscle, and spleen. When normalized with the mRNA
level of
-actin, the expression level was the highest in intestine,
followed by kidney, adipose, stomach, liver, skeletal muscle, and
spleen (Fig. 3C). In contrast, no observable level of
expression was detected in murine brain, heart, lung, skin, testis, and
thymus (Fig. 3A).

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Fig. 3.
Tissue distribution of mouse MGAT2 mRNA
as examined by Northern blot analysis. Mouse multiple tissue blots
from Origene (A) and Biochain (B) containing 2 µg of poly(A)+ RNA were hybridized with radiolabeled
probes prepared from full-length cDNA of mouse MGAT2 as described
under "Experimental Procedures." The hybridized blots were stripped
of residual radioactivity and reprobed with -actin cDNA as an
internal control. The blots were exposed to a PhosphorImager Screen for
appropriate times to visualize the signals. The detectable signals of
MGAT2 were quantitated by ImageQuant software and normalized by
-actin (C).
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Functional Analysis of MGAT2 Activity in Mammalian Cells--
To
determine whether the protein encoded by MGAT2 cDNA
possesses MGAT, DGAT, or both activities, MGAT2 was transiently
expressed in AV-12, COS-7, and Caco-2 cells followed by in
vitro enzyme assay analyses. Since MGAT2 shares the closest
sequence homology with MGAT1, we first examined MGAT activity in cells
transfected with MGAT2 cDNA. In all three cell lines tested, MGAT
activity in protein homogenates from MGAT2-transfected cells
was significantly higher than that observed in untransfected and
mock-transfected (with empty vector) cells (Fig.
4A) as assessed by a
significant increase of radioactive 1,2(2,3)-dioleoylglycerol product
when 2-monooleoylglycerol was used as acyl acceptor, thereby
establishing the function of cloned MGAT2 as a synthetic enzyme of
diacylglycerol. Radioactivity incorporated into
1,2(2,3)-diacylglycerol by homogenate prepared from
MGAT2-transfected AV-12, COS-7, and Caco-2 cells was
70-, 30-, or 35-fold higher, respectively, compared with that from the
untransfected or mock-transfected cells (Fig. 4B). Since MGAT2 also shares sequence homology with the mouse DGAT2, we next analyzed DGAT activity using the same cell homogenates for the MGAT
enzyme assays. As indicated in Fig. 4, C and D,
the incorporation of 14C-acyl into triacylglycerol by
homogenates from MGAT2-transfected AV-12, COS-7, and Caco-2 cells was
7-, 3-, and 1.5-fold higher than that observed in control or
mock-transfected cells, respectively, indicating that MGAT2 also
possesses weak DGAT activity. However, the detected DGAT activity is
10-fold lower when compared with the MGAT activity.

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Fig. 4.
Enzymatic activity analysis of MGAT2
expressed in mammalian cells. AV-12, COS-7, and Caco-2 cells were
untransfected, or transfected with blank vector (mock-transfected) or
expression vector containing mouse MGAT2 cDNA (mMGAT2).
Forty-eight hours after transfection, cell homogenates were prepared,
followed by MGAT and DGAT activity assays. A, MGAT activity
assayed by TLC. MGAT activity was measured by detecting the
incorporation of the [14C]oleoyl moiety of oleoyl-CoA
(oleoyl-1-14C) into 1,2(2,3)-dioleoylglycerol
(1,2(2,3)-DAG) with
2-monooleoylglycerol (2-MAG). Note that the major product is
1,2(2,3)-diacylglycerol, whereas detectable 1,3-diacylglycerol
(1,3-DAG) was also formed, possibly using endogenous or
spontaneously isomerized 1-monoacylglycerol (or 3-monoacylglycerol) as
acyl acceptor, by the enzyme from MGAT2-transfected cells.
B, quantitative analysis of MGAT activity as determined by
the TLC system in A. C, DGAT activity assayed by
TLC. DGAT activity was measured by detecting the incorporation of
[14C]oleoyl moiety of oleoyl-CoA
(oleoyl-1-14C) into trioleoylglycerol (TAG)
with sn-1,2-dioleoylglycerol. D, quantitative
analysis of DGAT activity as determined by TLC system in C.
The free fatty acids (FA) came from the hydrolysis of the
labeled oleoyl-CoA. The same results were obtained in three separate
experiments.
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To investigate substrate stereospecificity, we examined whether MGAT2
can catalyze the acylation of the other stereoisomers of
monoacylglycerol. The experiments were conducted under conditions where
a 50 µM concentration of each acyl acceptor
(rac-1-oleoylglycerol, sn-2-oleoylglycerol,
sn-3-stearoylglycerol, or rac-1-stearoylglycerol) was incubated with 25 µM [14C]oleoyl-CoA
for 20 min. Cell homogenate was prepared from mock-transfected (as
negative control), mouse MGAT2 (mMGAT2)-, or mouse
MGAT1 (mMGAT1)-transfected COS-7 cells. As shown in Fig.
5, MGAT2 can acylate each of four monoacylglycerol isomers to form diacylglycerols. The radioactive bands
of diacylglycerols were quantitated, and the results were shown in Fig.
5B. The major products were sn-1,3-diacylglycerol and sn-1,2(2,3)-diacylglycerol when
rac-1-monoacylglycerol and 2-monoacylglycerol were employed,
respectively (Fig. 5). MGAT2 also can use
sn-3-stearoylglycerol as substrate (Fig. 5, lane 7 versus lane 3). The
chiral sn-1-monoacylglycerol was not included in the present
studies due to the lack of commercially available compound. However,
under the same concentration (50 µM), a similar amount of
diacylglycerol was produced when rac-1-stearoylglycerols were used as substrate in comparison with
sn-3-stearoylglycerol (Fig. 5, lane 8 versus lane 7), implying that
sn-1-stearoylglycerol can also be used as substrate of
MGAT2. In addition, MGAT2 utilized monoacylglycerol containing
unsaturated fatty acyls (rac-1-oleoylglycerol, C18:1) more
efficiently than that of saturated fatty acyls
(rac-1-stearoylglycerol, C18:0) (Fig. 5, lane
5 versus lane 8). mMGAT1
showed similar patterns of enzymatic activity to those of mMGAT2 (Fig.
5, lanes 9-12), which is consistent with a
previous report by Yen et al. (16).

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Fig. 5.
Comparison of the acylation of different
isomers of monoacylglycerols. A, MGAT activity assayed
by TLC. The reaction was conducted by incubating 50 µM
2-monooleoylglycerol and 25 µM of
[14C]oleoyl-coenzyme A for 20 min at room temperature in
the presence of 100 µg of cell homogenate, followed by lipid
extraction and TLC assay. B, quantitative analysis of
production of diacylglycerols as determined by the TLC system in
A. Note that rac-1-stearoylglycerol and
sn-3-stearoylglycerol, under the same concentration,
produced similar amounts of diacylglycerols (even more by the former),
implying that MGAT2 can acylate each of the 1-, 2-, and 3-positions of
monoacylglycerol. 1,3-diacylglycerols are the major products when
rac-1-monoacylglycerols or
sn-3-monoacylglycerol are used as substrate, and
1,2(2,3)-diacylglycerols are the main products when 2-monoacylglycerol
is used as substrate. Data are representative of two independent
experiments. For abbreviations, see the legend to Fig. 4.
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We next directly compared the pattern of enzymatic activity of
mMGAT2 with that of the human DGAT1 (hDGAT1) (14) under the same
experimental conditions. When assessed for the MGAT enzyme activity,
the murine MGAT2 demonstrated significantly higher MGAT activity than
the human DGAT1 (15-fold versus 2-fold) as measured by
incorporation of the [14C]oleoyl moiety into
1,2-diacylglycerol by cell homogenates from mMGAT2 and
hDGAT1-transfected cells, respectively, as compared with
mock-transfected cells (Fig. 6,
lanes 3 and 4 versus
lane 2, bottom bands). In
contrast, the production of labeled triacylglycerol by cell homogenates
from the hDGAT1-transfected cells was significantly higher than that
from the mMGAT2-transfected cells (7.6-fold versus 1.8-fold)
when compared with the negative controls (Fig. 6, lanes 3 and 4 versus lane
2, top bands). These data further
confirm that MGAT2 is a novel member of the MGAT family of enzymes
despite its weak DGAT activity. Furthermore, the MGAT activity of
mMGAT2 can be separated from its DGAT activity by treatment with
detergent. Solubilization of cell homogenates by Triton X-100 abolished
the DGAT activity but not the MGAT activity of mMGAT2 (Fig. 6,
lane 5 versus lane
4).

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Fig. 6.
Comparison of enzymatic activity
between DGAT1 and MGAT2. The reaction was conducted by incubating
200 µM 2-monooleoylglycerol and 25 µM
[14C]oleoyl-coenzyme A for 10 min at room temperature in
the absence of protein (Blank) or presence of 150 µg of
cell homogenate from empty vector (mock)-, human DGAT1
(hDGAT1)-, or mouse MGAT2 (mMGAT2)-transfected
COS-7 cells, followed by extraction of lipids and TLC assay. Cell
homogenate solubilized by 0.1% Triton X-100 was applied in the
last lane. Quantitative analysis of labeled
diacylglycerol (DAG) or triacylglycerol
(TAG) was performed by phosphorimaging, and the relative
values are shown below the bands. The same
experiments were repeated three times, and a representative TLC image
is shown.
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Kinetics Analysis of Product Formation by Protein Homogenate from
MGAT2-transfected COS-7 Cells--
We next examined the biochemical
characteristics of MGAT activity in cell homogenate from
MGAT2-transfected COS-7 cells. The dependence of the MGAT2 activity on
the protein concentration in the reaction as well as the incubation
time is shown in Fig. 7, A and
B, respectively. In Fig. 7A, increased amounts of
protein homogenate from MGAT2-transfected or 500 µg of that from
mock-transfected cells were incubated with 200 µM
Sn-2-monooleoylglycerol and 25 µM
[14C]oleoyl-CoA for 10 min, followed by lipid extraction
and TLC assay. In Fig. 7B, 50 µg of protein homogenate was
incubated with the above mentioned substrates for 0-60 min. MGAT
activity levels of MGAT2, as assessed by the formation of radioactive
1,2(2,3)-dioleoylglycerol, increased proportionately with incubation
times (Fig. 7B). The embedded values in Fig. 7, A
and B, represent the quantitative [14C]1,2(2,3)-diacylglycerol produced (arbitrary
units/min) in the reaction. Again, the homogenate protein from
mock-transfected cells showed only marginal activities (Fig.
7A, last lane; Fig. 7B,
last two lanes).

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Fig. 7.
Kinetics analysis of MGAT
activity determined with lipid products formed by membrane protein from
MGAT2-transfected COS-7 cells. A, dependence of MGAT
activity on protein concentration. Various amounts of protein
homogenates from MGAT2-transfected or mock-transfected cells were
incubated with 2-monooleoylglycerol and [14C]oleoyl-CoA
for 10 min, followed by extraction of lipids and TLC assay. With the
increased amount of protein in the reaction, labeled diacylglycerol
product showed a proportionate increase. B, time course of
MGAT activity. Fifty micrograms of protein was incubated with
2-monooleoylglycerol and [14C]oleoyl-CoA for the
indicated times (0-60 min), followed by extraction of lipids and TLC
assay. A time-dependent increase in MGAT activity was also
evident. This protein- and time-dependent manner was not
detected in control vector-transfected cells. The embedded values
below the 1,2-diacylglycerol bands represent the
quantitative [14C]1,2-diacylglycerol product formed in
the reaction (arbitrary units/min). For abbreviations, see the legend
to Figs. 4 and 6.
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We further examined the profile of MGAT2 activity at different time
points after transfection as well as the kinetics of MGAT activity. The
MGAT2 activity can be detected as early as 12 h post-transfection
period as judged by a 15-fold increase in the synthesis of radioactive
diacylglycerol as compared with the negative controls (Fig.
8A). The MGAT activity reached
a plateau at 24 h and began to decline at 72 h
post-transfection (Fig. 8A). In contrast, cell homogenates
from mock-transfected cells collected at 12 and 72 h
post-transfection did not display any increase of MGAT activity (1.2 versus 1.0 unit). We next analyzed the effect of substrates
concentration on the enzyme activity of MGAT2. When the concentration
of one component of MGAT substrates, either Sn-2-monooleoylglycerol or oleoyl-CoA, was increased while
the other was held constant (25 µM for oleoyl-CoA and 200 µM for Sn-2-monooleoylglycerol) in the
reaction, the formation of dioleoylglycerol also increased over a wide
range (0-66 µM for both substrates) (Fig.
8B). Further increases in substrate concentration resulted
in inhibition of the MGAT activity (Fig. 8B).

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Fig. 8.
A, time course of mouse MGAT2 activity
after transfection in COS-7 cells. Cell homogenates were prepared at
the indicated times after transfection with empty vector
(mock-transfected) or mouse MGAT2, followed by MGAT activity assay. The
embedded values below the diacylglycerol bands
represent the quantitative [14C]1,2-diacylglycerol
product formed. The same results were repeated three times, and a
representative experiment is shown. B, dependence of MGAT
activity of MGAT2 on substrate concentration. The values were obtained
by exposing the TLC plates with a PhosphorImager screen, which was
visualized with a Storm 860 PhosphorImager. Data represent the mean of
two separate experiments. For abbreviations, see the legend to Figs. 4
and 6.
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DISCUSSION |
It has been known for some time that MGAT enzyme activity is the
rate-limiting step in the synthesis of triacylglycerol in the small
intestine, which generates 80% of the triacylglycerol levels in
chylomicrons (21). However, a clear understanding of the biosynthesis
pathway of glycerolipids in intestinal mucosa had been hindered by the
absence of the purified enzymes. Although a mouse MGAT (MGAT1) enzyme
was recently identified by its sequence homology to the
mammalian DGAT2 gene (16), the mouse MGAT1 is not
expressed in small intestine, where dietary absorption takes place
(16), implying the existence of another MGAT gene.
In the present studies, we have identified and characterized a gene
encoding a MGAT enzyme from a mouse intestinal cDNA library by its
sequence homology to mouse MGAT1 and hence named MGAT2. In
addition to high sequence homology to MGAT1, the hydrophobicity profile
of the mouse MGAT2 protein also shares striking similarity with that of
the mouse MGAT1 but is quite different from that of the mouse DGAT2.
Evidence that the mouse MGAT2 is a MGAT enzyme is provided by the
findings that the MGAT2 expressed in three different mammalian cell
lines catalyzed significantly the monocylglycerol synthesis as measured
by a more than 30-fold increase in radiolabeled diacylglycerol than
that from mock-transfected cells. Furthermore, the increased MGAT
activity was dependent on the concentrations of either substrate,
monoacylglycerol or oleoyl-CoA. Protein homogenate from
MGAT2-transfected cells showed time- and dose-dependent
responses with incubation time and amount of protein, whereas this
phenomenon was not found in control cells under the same conditions.
The MGAT activity coincides with the expression profile of the
MGAT2 transgene, which can be detected as early as 12 h
after transfection and reached a plateau at 48 h
post-transfection.
As observed with MGAT1 (16), cell homogenates from three different
mammalian cell lines transiently expressing MGAT2 also displayed
significantly higher DGAT activity than that from mock-transfected cells. The DGAT activity is more pronounced with an extended incubation time. However, the DGAT activity is much weaker than the MGAT activity
as assessed by the rate of formation of radioactive products (700 pmol
of diacylglycerol/min/mg of protein versus 29 pmol of triacylglycerol/min/mg of protein in AV-12 cells) catalyzed by mouse
MGAT2 in the assay. This is consistent with our observation that no
significant levels of triacylglycerol over background were detected in
the MGAT assay from MGAT2-transfected cells, although MGAT2 produced a
significant amount of diacylglycerol (over 10 µM) during
10 min of incubation. Furthermore, when compared directly with the
human DGAT1 enzyme for substrate specificity under the same
experimental conditions, the murine MGAT2 demonstrated significantly
higher MGAT activity than the human DGAT1 enzyme that exhibited
primarily the DGAT activity, thus establishing the MGAT2 as a new
member of the MGAT family of enzymes.
A fatty acyl-CoA is required for the display of MGAT activity, since
the incorporation of [3H]oleic acid into diacylglycerol
by membrane protein from MGAT2-transfected cells is not significantly
higher than that by membrane from wild type cells (data not shown),
indicating that, similar to DGAT1, the activity of MGAT2 is
CoA-dependent (14). Tsujita et al. (20) reported
a relatively high CoA-independent MGAT activity due to pancreatic
lipase distributed within intestinal cells, which may serve as an
alternative pathway for diacylglycerol synthesis in intestinal mucosa.
The individual role of these two different pathways in intestinal fat
absorption remains unknown and awaits further investigation. It was
reported that MGAT and DGAT are integral membrane proteins, and
application of detergent severely inhibited the activity, which makes
it difficult to purify them, because tight lipid-protein interactions
are necessary for their optimal activities (13). Interestingly, our
result shows that solubilization of MGAT2-transfected cells
by Triton X-100, a nonionic detergent, resulted in a total loss of DGAT
activity but did not affect the MGAT activity possessed by MGAT2. Thus,
dependence on membrane integrity can be used to separate the DGAT
activity from the MGAT activity of the murine MGAT2 enzyme.
Using the intestinal mucosal microsomes, Paltauf and Johnston (22)
reported the utilization of a series of monoalkyl and monoacyl
glycerols as substrates for glyceride synthesis. In their studies, both
sn-1- and sn-2-alkyl glycerols are acylated,
whereas the sn-3-alkyl was a poor substrate. However, each
of the sn-acyl monoglycerols could be utilized as substrate
for the intestinal mucosa (22). Therefore, there were observed
differences between the sn-alkyl and acyl glycerols in their
studies. Our data show that MGAT2 expressed in mammalian cells can
catalyze the acylation of rac-1-, sn-2-, and
sn-3-monoacylglycerol (Fig. 5). Studies with everted sacs of
rat intestinal mucosa (23, 24) and a purified monoacylglycerol
acyltransferase from rat intestinal mucosa (25) indicated the acylation
in sn-1- and sn-3-positions of the
sn-2-monoacylglycerol occurred at about the same rate with a
slight preference at sn-1-position. In contrast, Johnston
et al. (26) reported that the intestinal microsomes greatly
preferred to acylate the sn-1-position, compared with the
sn-3-position. Further stereospecific analysis of
1,2(2,3)-diacylglycerol product would resolve the issue of whether
mMGAT2 has a preference at the sn-1- or
sn-3-position of 2-monoacylglycerol.
Manganaro and Kuksis (25) reported the purification and preliminary
characterization of a 2-monoacylglycerol acyltransferase from rat
intestinal villus cells, which gives a single band of 37,000 daltons on
SDS-polyacrylamide gel electrophoresis. This is very close to the
38,600 daltons predicted by the mouse MGAT2. Additionally,
both enzymes catalyzed efficient acylation of 2-monoacyglcerols. However, analysis of the amino acid composition of the mouse MGAT2 revealed significant differences between the two enzymes (data not
shown). Thus, the purified enzyme might represent a novel isoform/homologue of the MGAT enzyme that remains to be identified.
One of the unique features of MGAT2 is its selective tissue expression
pattern, which is quite different from the previously reported two
DGAT genes (DGAT1 and DGAT2) and
MGAT1 gene. The DGAT1 and DGAT2 genes
are ubiquitously expressed, whereas the MGAT1 expression is
absent in the small intestine (14-16). In contrast, the mouse
MGAT2 gene is most abundantly expressed in small intestine, which is consistent with the previous report that the mouse small intestine exhibits the highest MGAT activity among all of the tissues
examined (16). Although it remains elusive, the data suggest that the
mouse MGAT2 may play an important role in dietary fat absorption. A
high level of MGAT2 expression was also detected in mouse kidney, a
tissue where triacylglycerol synthesis and MGAT activity are reported
(27-30). Additionally, MGAT2 expression is detected in the white
adipocytes, implicating a role in fat storage. In contrast to abundant
MGAT1 expression in the stomach (16), only a low level of
MGAT2 mRNA is detected in this tissue, which is
consistent with the pattern of dietary fat absorption that starts in
stomach and mainly occurs in small intestine (1).
Consumption of a western diet enriched with a high fat content has been
cited as one of the common causes of the ongoing epidemic of obesity.
In a recent study conducted in Otsuka Long-Evans Tokushima fatty rat,
which is an animal model of diabetes and obesity, increased small
intestine MGAT activities were associated with the onset of the
metabolic diseases (31). In Zucker obese rats, although the specific
activities of MGAT in the small intestine were not different from those
in lean rats, the total activity of MGAT increased significantly in the
obese rats because of the heavy and large intestinal segments (32). The
importance of triacylglycerol synthesis and storage is further
underlined by recent characterization of mice deficient in one of the
DGAT genes (8). Inactivation of DGAT1 results in mice that
are resistant to diet-induced obesity and other related metabolic
abnormalities (8), although a recent report indicates that the DGAT1 is
not required for dietary fat absorption (33). Our current study on
MGAT2 has provided basic information for future work on the potential
physiological and pathological roles of MGAT2 in dietary fat absorption
and thus may present MGAT2 as an attractive drug target for obesity intervention.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Dod Michael and Craig
Hammond for critically reading the manuscript and Dr. Jose Caro for
encouragement and support of the studies.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Endocrine Research, DC
0545, Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly
and Company, Indianapolis, IN 46285. Tel.: 317-276-6753; Fax:
317-276-9574; E-mail: shi_yuguang@lilly.com.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M300139200
 |
ABBREVIATIONS |
The abbreviations used are:
MGAT, acyl-CoA:monoacylglycerol acyltransferase;
mMGAT, murine MGAT;
DGAT, acyl-CoA:diacylglycerol acyltransferase;
hDGAT, human DGAT.
 |
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