Original Article
Identification of a Novel Member of the Carboxylesterase
Family That Hydrolyzes Triacylglycerol
A Potential Role in Adipocyte Lipolysis
Hiroaki Okazaki,1 Masaki Igarashi,1 Makiko Nishi,1 Makiko Tajima,1 Motohiro Sekiya,1Sachiko Okazaki,1 Naoya Yahagi,1 Ken Ohashi,1 Kazuhisa Tsukamoto,1 Michiyo Amemiya-Kudo,2Takashi Matsuzaka,3 Hitoshi Shimano,3 Nobuhiro Yamada,3 Junken Aoki,4 Rei Morikawa,4Yasukazu Takanezawa,4 Hiroyuki Arai,4 Ryozo Nagai,5 Takashi Kadowaki,1 Jun-ichi Osuga,1and Shun Ishibashi1,6

Molecular mechanisms underlying lipolysis, as defined bymobilization of fatty acids from adipose tissue, are notfully understood. A database search for enzymes with ␣/␤hydrolase folds, the GXSXG motif for serine esterase andthe His-Gly dipeptide motif, has provided a previously
unannotated gene that is induced during 3T3-L1 adipocytic
differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4%
identity, we have tentatively designated this enzyme as
TGH-2 and the original TGH as TGH-1. TGH-2 is alsosimilar to TGH-1 in terms of tissue distribution, subcellular
localization, substrate specificity, and regulation. Both
enzymes are predominantly expressed in liver, adipose
tissue, and kidney. In adipocytes, they are localized in
microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein,cholesteryl oleate, or phospholipids; hydrolysis of shortchain fatty acid ester was 30,000-fold more efficient thanthat of long-chain fatty acid triacylglycerol. Fasting increased the expression of both genes in white adipose
tissue, whereas refeeding suppressed their expression.
RNA silencing of TGH-2 reduced isoproterenol-stimulated
glycerol release by 10% in 3T3-L1 adipocytes, while its
overexpression increased the glycerol release by 20%.
Thus, TGH-2 may make a contribution to adipocyte lipoly-

From the 1Department of Metabolic Diseases, Graduate School of Medicine,
University of Tokyo, Bunkyo-ku, Tokyo, Japan; the 2Okinaka Memorial
Institute for Medical Research and Toranomon Hospital, Tokyo, Japan;
3
Metabolism, Endocrinology and Atherosclerosis, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan; the 4Department of Health
Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo,
Bunkyo-ku, Tokyo, Japan; the 5Department of Cardiovascular Diseases,
Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan;
and the 6Division of Endocrinology and Metabolism, Department of Medicine,
Jichi Medical University, Tochigi, Japan.
Address correspondence and reprint requests to Shun Ishibashi, Division of
Endocrinology and Metabolism, Department of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail:ishibash@jichi.ac.jp.
Received for publication 6 May 2005 and accepted in revised form 17 April
2006.
H.O. and M.I. contributed equally to this work.
ATGL, adipose triglyceride lipase; BAT, brown adipose tissue; DMEM,
Dulbecco’s modified Eagle’s medium; FFA, free fatty acid; HSL, hormonesensitive lipase; iPLA2, Ca2⫹-independent PLA2; PNPB, p-nitrophenyl butyrate; TGH, triacylglycerol hydrolase; WAT, white adipose tissue.
DOI: 10.2337/db05-0585© 2006 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

DIABETES, VOL. 55, JULY 2006

sis during period of increased energy demand. Diabetes 55:
2091–2097, 2006

C

ellular accumulation of excessive neutral lipids
underlies many diseases such as obesity, type 2
diabetes, and atherosclerosis, all of which are
epidemic in industrialized countries. Therefore,
elucidating the metabolic pathways that degrade excessive
neutral lipids is of extreme importance in the preventionof the diseases caused by lipotoxicity (1).
Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that catalyzes the hydrolysis of cellular triacylglycerol, diacylglycerol, monoacylglycerol, and cholesterylester, as well as other lipids (2,3). HSL is expressed in awide variety of organs and cells including adipose tissues,
heart, skeletal muscle, adrenal glands, testes, ovaries, andpancreatic ␤-cells and is under neural and hormonal
control. To define the role of HSL in the hydrolysis ofneutral lipids in vivo, several laboratories, including ours,
have generated HSL-deficient (HSL⫺/⫺) mice by targeted
gene disruption (4 – 6). Although HSL⫺/⫺ mice had adecreased ability to release free fatty acid (FFA) from
adipocytes in response to the ␤-adrenergic stimulation
both in vivo and in vitro, they were neither obese nor cold
sensitive (4,5). The attenuated development of adipocyteassociated phenotypes may result from the presence of aresidual triacylglycerol lipase activity in adipocytes thatmediates lipolysis stimulated by both ␤-adrenergic agonist
and tumor necrosis factor-␣ (7). These findings resulted in
the use of a bioinformatic approach to search for a proteinthat shares several motifs with HSL. Of 33 genes that havenot been annotated, only 1 shows hydrolytic activity
toward triacylglycerol and is expressed in adipose tissue.
Here we report the structure, substrate specificity, subcellular localization, tissue distribution, and regulation of
expression in adipose tissue of this enzyme, which wehave designated as triacylglycerol hydrolase (TGH)-2 because of its striking sequence similarity to microsomal
liver/adipocyte TGH originally reported by Lehner andcolleagues (8,9).
RESEARCH DESIGN AND METHODS
Triolein, lecithin, BSA fraction V (BSA), leupeptin, pentadecanoic acid,p-nitrophenyl butyrate (PNPB), 2-monoolein, dioleoylglycerol, dioleoyl-phos2091

A NOVEL PARALOGUE OF TRIACYLGLYCEROL HYDROLASE

phatidylserine, dioleoyl-phosphatidylcholine, dioleoyl-phosphatidic acid, dioleoyl-phosphatidylethanolamine, and phosphatidylinositol (18:0, 24:0) werepurchased from Sigma. Dexamethasone, 3-isobutyl-2-methylxanthine, fatty
acid–free BSA, isoproterenol, and sodium taurocholate were purchased from
Wako Pure Chemicals (Osaka). Pioglitazone was provided by Takeda Pharmaceutical (Osaka). Tri[3H]oleoylglycerol and cholesteryl [1-14C]oleate werepurchased from Applied Biosystems (Foster City, CA). Adenovirus plasmidcarrying ␤-galactosidase (pAd-LacZ) has been described previously (10).
Database search and amino acid sequence analysis. We used the MOTIF
search program in the GENES protein database of Kyoto Encyclopedia of
Genes and Genomes (11) to search for proteins containing lipase consensus
motifs and ␣/␤ hydrolase fold. We predicted the secondary structures ofproteins with the programs PSIPRED and PHDsec and searched for theirorthologue or paralogue of TGH-2 by SSDB in the Kyoto Encyclopedia of
Genes and Genomes database (11). Alignment of the deduced protein sequence to other lipases, and calculation of the percent identity, were performed using the CLUSTAL W program.
Mice. C57BL/6 male mice were purchased from Clea (Tokyo, Japan). Mice
were caged separately with 12-h light/dark cycles and given free access tostandard chow diet containing 0.075% cholesterol (MF; Oriental Yeast, Osaka,
Japan) in accordance with the regulations of the animal care committee of the
University of Tokyo. For refeeding, animals were fasted for 24 h (from 2200 to2200) and then refed for 12 h (2200 –1000). For fasting, animals were fasted for
24 h (1000 –1000).
Cell culture. HEK293 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% (vol/vol) fetal bovine serum. 3T3-L1 cells
were cultured in medium A (DMEM containing 10% calf serum, supplemented
with Ca-pantothenate and biotin). To induce differentiation of 3T3-L1 cells
into mature adipocytes, 2-day postconfluent preadipocytes (day 0) wereincubated with 1 ␮mol/l dexamethasone and 0.5 mmol/l 3-isobutyl-2-methylxanthine, 5 ␮g/ml insulin, and 1 ␮mol/l pioglitazone for 48 h, followed bytreatment with 5 ␮g/ml insulin and 1 ␮mol/l pioglitazone for 48 h. After the
incubation period, cells were switched to medium A, and the medium wasrenewed every other day.
Northern blot analyses. Ten micrograms of total RNA, which was isolatedby TRIzol reagent (Invitrogen), were used for Northern blot analysis as
described (12). Probes for murine TGH-1 (mouse orthologue of TGH) andTGH-2 were constructed from cDNA fragments amplified by RT-PCR using
cDNA obtained from mouse liver as a template. Because TGH-1 and -2 arehighly homologous proteins, the region that is specific to each mRNA andshares no sequence homology with the other was chosen for the constructionof cDNA probe, i.e., from 1,130 to 1,260 bp for TGH-1 and from 1,130 to 1,260
bp for TGH-2. The cDNA fragment of HSL encoded by exon 8 was amplified by
RT-PCR using cDNA obtained from mouse adipose tissue as a template andused as a probe (12).
Preparation of polyclonal antibody. To prepare polyclonal anti-mouse
TGH-1 and -2 antisera, amino acid residues containing the catalytic domain of
TGH-1 (amino acids 196 –332) and TGH-2 (amino acids 196 –333) wereexpressed in Escherichia coli as a glutathione S-transferase fusion protein,which was subsequently purified by glutathione affinity chromatography andused for immunization of rabbits according to the standard protocols (7).
Cross-reactivities of the antisera against TGH-1 and -2 were tested using celllysates overexpressing TGH-1 or -2 by adenovirus-mediated gene delivery.
Western blot analysis. Cells or tissues were homogenized in buffer A (50mmol/l Tris-HCl, pH 7.0, 250 mmol/l sucrose, 1 mmol/l EDTA, and 2 ␮g/ml
leupeptin) and centrifuged at 100,000g for 45 min at 4°C. The supernatant wasused as S-100 cytosolic fraction, and the precipitates were resuspended andused as microsomal fraction (12). For Western blot analysis of fatcake
proteins, white adipose tissue (WAT) was homogenized in buffer A andcentrifuged at 100,000g for 45 min at 4°C, followed by extraction of fatcake
proteins from the floating fat layer by the acetone precipitation method,essentially as described (7). Ten micrograms of protein from each fraction
were subjected to Western blot analysis, as previously described, using ananti–TGH-1, anti–TGH-2, anti-HSL, or anti-perilipin antibody that was raised
according to the described method (7). NIH Image (http://rsb.info.nih.gov/nihimage/) was used to compare the intensity of the bands.
Expression in mammalian cells. The expression vector was constructed byligating the cDNA fragments, which were amplified by RT-PCR from mouse
liver, into the TA cloning site of pTARGET vector (Promega). DNA transfection was performed with HEK293 cells plated on a six-well plate using
SuperFect reagent (Qiagen). Expression vector (1.5 ␮g) was simultaneouslytransfected with pSV-␤gal (0.5 ␮g) to measure transfection efficiency. The
total amount of DNA in each transfection was adjusted to 1.5 ␮g/well with themock vector. After a 48-h incubation, the cells were harvested and assayed for
enzyme activity.
Construction of recombinant adenoviruses. Recombinant adenoviruses
that carried murine TGH-1 and -2 cDNA under the control of cytomegalovirus
2092

promoter, designated as Ad-TGH-1 and Ad-TGH-2, respectively, were constructed as previously described (12). The recombinant adenoviruses wereexpanded in HEK293 cells and purified by cesium chloride ultracentrifugation(13). The purified viruses were stored in 10% (vol/vol) glycerol/PBS at ⫺80°C.
In our preparations, 1 m.o.i. (multiplicity of infection) corresponded to 25
particles of adenovirus per cell, and cells were infected at 1 m.o.i. for HEK293
cells and 1,000 m.o.i. for 3T3-L1 adipocytes. Seventy-two hours after the
infection, the cells were used for the experiments.
RNA silencing. The following 21-mer sense strands of RNA oligonucleotides
were designed as described (14): scrambled, 5⬘-CAGUCGCGUUUGCGACUG
GdTdT-3⬘; HSL, 5⬘-GCCAAAGAUGAAGUGAGACdTdT-3⬘; TGH-1, 5⬘-AGAACAGCAGAGACUACCAGUdTdT-3⬘; and TGH-2, 5-GAAUGUAGUAGGAGACCAU
dTdT-3⬘. The sense and antisense strands were synthesized, mixed, andannealed. At 5 days of differentiation, 3T3-L1 adipocytes were transfected withsiRNA duplexes by electroporation as described (15). After incubation in
medium A for 48 h, the cells were used for the experiments.
Enzyme assays. Cells were sonicated in buffer A and centrifuged at 15,000g
for 10 min at 4°C. The supernatant was used for the enzyme assays. Neutralcholesteryl ester hydrolase or triacylglycerol lipase activities were measured
using radiolabeled triolein or cholesterol oleate, respectively, as previously
described (4). PNPB esterase activity was measured as previously described(16,17). Hydrolytic activities toward monoacylglycerol, diacylglycerol, andphospholipids were determined essentially as previously described (18,19). In
brief, 2-monoolein, dioleoylglycerol, dioleoyl-phosphatidylserine, dioleoylphosphatidylcholine, dioleoyl-phosphatidic acid, and dioleoyl-phosphatidylethanolamine were incubated with cell extracts or tissue homogenates at
37°C for 1 h. Immediately thereafter, standard pentadecanoic acid (C15:0) was
added to the reaction mixture and total lipids were extracted by Bligh andDyer’s method and reconstituted in chloroform/methanol (2:1 [vol/vol]). Mass
spectrometry spectra were obtained by introducing the samples into a Quattro
Micro tandem quadrupole mass spectrometer (Micromass, Manchester, U.K.)
equipped with an ESI source at the flow rate of 30 ␮l/min. The eluting solvent
used was acetonitrile/methanol/water (2:3:1 [vol/vol/vol]) containing 0.1%
ammonium formate (pH 6.4). The mass spectrometer was operated in thenegative ion scan mode. The nitrogen drying gas flow rate was 12 l/min and thetemperature 80°C. The capillary voltage was set at 3.7 kV and cone voltage at
50 V. Amounts of oleic acid were deduced from its mass spectrometry spectra
relative to that of standard pentadecanoic acid.
Lipolysis assay. The 3T3-L1 adipocytes either infected with the recombinantadenovirus or transfected with siRNA duplexes were cultured in 12-wellplates. After incubation in medium A supplemented with 2% (wt/vol) fatty
acid–free BSA in the absence of serum for 12 h, cells were treated with orwithout 10 mmol/l isoproterenol for the indicated amounts of time and theconcentrations of glycerol and FFA in the media were determined by the
Triglyceride-G kit and NEFA-C kit, respectively (Wako Pure Chemicals).
Cellular protein contents were measured by bincinchoninic acid protein assay(Pierce) after solubulization in 0.1 N NaOH. Amounts of glycerol or FFA in themedium were normalized to cellular protein.
Statistical analyses. Results are presented as means ⫾ SE. Student’s t test
was used to compare the means. All calculations were performed with
StatView (version 5.0; SAS Institute) for Macintosh.

RESULTSTo search for enzymes with triacylglycerol lipase activity,
we screened the gene database for murine and humanproteins with structural homologies to known lipases, i.e.,
␣/␤ hydrolase folds (20), the GXSXG active serine motif
for serine esterases, and the His-Gly dipeptide motif that ispresent in 70 –100 amino acids’ NH2-terminal of the catalytic site serine in many lipases (21), yielding 53 candidates that include HSL, lipoprotein lipase, hepatic lipase,endothelial lipase, pancreatic lipase, carboxyl ester lipase,gastric lipase, lysosomal acid lipase, monoacylglycerol
lipase, and TGH. We selected 33 candidates that had notbeen previously annotated, and expressed them in
HEK293 cells for measurement of triacylglycerol hydrolase activity. Northern blot analysis was used to verify theexpression in mouse adipose tissue. Only one previously
unannotated enzyme fulfilled these requirements, and wetentatively designated it as TGH-2 because it has sequencehomology to TGH (22).
The murine gene for TGH-2 (National Center for Biotechnology Information nucleotide entry BC013479) enDIABETES, VOL. 55, JULY 2006

H. OKAZAKI AND ASSOCIATES

FIG. 1. Primary sequence of murine TGH-2 in comparison with humanand murine TGH. The deduced amino acid sequence of murine TGH-1(mTGH-1) and TGH-2 (mTGH-2) proteins are aligned and comparedwith human TGH (hTGH). The first Met residue is numbered as 1. The
mature TGH proteins have been highly conserved in evolution, including 1) conserved catalytic triad, Ser, Glu, His residues (shaded box); 2)
the His-Gly dipeptide motif (shaded box); 3) the NH2-glycosylation site(solid circle); and 4) the signal peptide cleavage site (vertical arrowhead), all of which are determined from the functional analysis of
hTGH protein. Identical amino acids are shown by an asterisk (*).
Amino acids considered strongly conserved are STA, NEQK, NHQK,
NDEQ, QHRK, MILV, MILF, HY, and FYW and indicated by a colon (:).
Amino acids considered weakly conserved are CSA, ATV, SAG, STNK,
STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY andindicated by dots. The putative lipid binding domain is shown inboldface. Conserved cysteine residues that putatively form disulfide
bonds are labeled with an open circle. A putative endoplasmic reticulum retrieval sequence (COOH-terminal HXEL) is indicated by outlined letters.

codes a 561–amino acid protein (NP_659179) with acalculated molecular mass of 61.6 kDa. Figure 1 showsalignments of amino acid sequences of murine TGH-2; its
paralogue, TGH (22,23), which has also been designated as
mouse carboxylesterase 3 and carboxylesterase MH1 (23);and its human orthologue (human carboxylesterase 1)(24,25). TGH-2 might be identical to carboxyesterase-ML1,
based on the available partial amino acid sequence (26).
TGH-2 shows 70.4% identity to murine TGH-1, which
encodes a 565–amino acid protein. The human TGH geneencodes a 566 –amino acid protein (NP_001257) with 77.9
and 67.0% identity to the murine TGH-1 and -2, respectively. The NH2-terminal regions of 18 residues in both themurine and the human enzyme contain a predicted signal
sequence that is cleaved in the mature human TGH protein(25). The Ser 221, Glu 353, and His 466 of the murine
TGH-2 protein form a putative catalytic triad; this triad ishighly conserved in human TGH, in which mutation of any
DIABETES, VOL. 55, JULY 2006of these triad residues to Ala abolishes TGH esterase
activity (25). A potential NH2-glycosylation site could be
deduced at Asn 79 from the alignment with human TGH(25). The sequence contains four conserved Cys residues
that are predicted to form disulfide bonds: Cys 87 to 116
and Cys 273 to Cys 284 (25). The protein sequence also
contains a hydrophobic stretch of amino acids that may be
involved in lipid binding: Gly 414 to Val 429. A COOHterminal HXEL sequence possibly functions as an endoplasmic reticulum retrieval signal. In contrast to HSL,
neither TGH-1 nor -2 contains putative phosphorylationsite for protein kinase A.
A homology search revealed the presence of related
proteins in a wide range of organisms, including animals,
plants, and bacteria. In humans, two proteins, LOC390732(XP_372639) and carboxylesterase 4 –like (NP_057364),
have a high degree of homology to human TGH (67.7 and92.9%, respectively). These proteins appear to be distinct
proteins and have been implicated as carboxylesterase. In
mice, three other proteins, 2310039D24Rik (XP_134476),
LOC244595 (XP_146438), and LOC382044 (XP_356117),
have a high sequence similarity to murine TGH-2 (75.4,
67.5, and 63.8%, respectively). Homologous proteins arealso found in Drosophila melanogaster and Caenorhabditis elegans. Interestingly, additional putative proteins sharing some degree of homology are present in Arabidopsis
thaliana, Cyanidioschyzon merolae, and others in prokaryotes, including Xanthomonas campestris, Bacillus
subtilis, and Mycobacteriumavium paratuberculosis. The
presence of TGH-1 and -2, as well as other related proteins,in such a wide variety of organisms suggests that TGH-1 and-2 comprise a subfamily of carboxyesterases mediating basic
cellular functions that is not exclusive of higher organisms.
Northern blot analysis was performed to compare themRNA expression of TGH-2 with that of HSL and TGH-1 invarious tissues. The tissue distribution of TGH-2 mRNA
expression is remarkably similar to that of TGH-1 except
in intestine: they are expressed at high levels in WAT,
brown adipose tissue (BAT), liver, and kidney but to alesser degree in adrenal, ovary, and heart (Fig. 2). TGH-2,
but not TGH-1, is highly expressed in intestine. To determine whether they are expressed in adipocytes in adifferentiation-dependent manner, we performed Northernblot analysis of TGH-1, TGH-2, and HSL at 0, 2, 5, and 8
days postinduction of 3T3-L1 adipocyte differentiation(Fig. 2B). Like HSL, mRNA expression of both TGH-1 and-2 was induced during differentiation of 3T3-L1 adipocytes.
Because of the high sequence similarities between the
peptides that were used for immunization, it is possible thatthe antibodies cross-react with TGH-1 and -2. To exclude thispossibility, we performed Western blot analysis using theantibodies against TGH-1 or -2 (Fig. 3A). The antibody
against TGH-1 detected TGH-1 but not TGH-2, which wereboth overexpressed in HEK293 cells over the backgroundlevel. On the other hand, the antibody against TGH-2 detected TGH-2 but not TGH-1, which were both overexpressed
in HEK293 cells over the background level. These results
indicate that cross-reaction of the antibodies is negligible.
To determine subcellular localization of TGH-1, TGH-2,
and HSL, we performed subcellular fractionation of WAT
and BAT by ultracentrifugation. Substantial amounts of
TGH-1, TGH-2, and HSL were present in fatcake fraction(Fig. 3B), corroborating the idea that TGH-1 and -2 mediate hydrolysis of neutral lipids in fat droplets whereperilipin colocalizes. Unlike fatcake-specific perilipin,
TGH-1, TGH-2, and HSL were also present in cytosolic and2093

A NOVEL PARALOGUE OF TRIACYLGLYCEROL HYDROLASE

FIG. 2. Northern blot analysis of HSL, TGH-1,
and TGH-2. Total RNA (10 ␮g) from tissues andmouse peritoneal macrophages (MPM) of
C57BL/6J mice (A and B) and 3T3-L1 adipocytes
at various stages of differentiation (B) were
subjected to Northern blot analysis. SpecificmRNAs were detected with a radiolabeled cDNA
probes for HSL, TGH-1, or TGH-2. Ethidium
bromide staining of gels is shown as a loading
control. ed, epididymal; sc, subcutaneous.

microsomal fraction. There were no obvious differences intheir distribution between subcutaneous and epididymal
WAT and BAT (Fig. 3C).
To determine the types of lipids that TGH-2 hydrolyzes,
we infected HEK293 cells with recombinant adenoviruses
carrying expression cassettes of LacZ, HSL, TGH-1, or
TGH-2. Whole-cell extracts of cells infected with AdTGH-1 or Ad-TGH-2 exhibited substantial amounts ofhydrolytic activities toward PNPB, triacylglycerol, or
monoacylglycerol (Table 1). Both TGH-1 and -2 hydrolyzed
PNPB, a short-chain fatty acid ester, more preferentiallythan triolein, a long-chain fatty acid ester. Their preference
was ⬃50- to 60-fold higher than that of HSL. However,neither TGH-1 nor TGH-2 showed detectable hydrolytic
activities toward diacylglycerol, cholesteryl ester, or phospholipids. Thus, TGH-2 is indistinguishable from TGH-1 intheir substrate specificity. The substrate specificity of HSL
is different from TGH-1 and -2 in that it prefers triacylglycerol, diacylglycerol, and cholesteryl ester compared with
TGH-1 and -2.

FIG. 3. Western blot analysis of HSL, TGH-1, and TGH-2. A: Crossreaction of the antibodies. We overexpressed TGH-1 or -2 in HEK293
cells by plasmid transfection and used these cell lysate for Western
blot analysis. Anti–TGH-1 and anti–TGH-2 detected the overexpressed
TGH-1 and -2, respectively, but did not cross-react with TGH-2 and -1
over the background levels, respectively. B and C: Subcellular distribution of the lipases. Tissues of C57BL/6J mice were homogenized andcentrifuged at 100,000g. Fatcake proteins (FC), which were extracted
from floating fat layers, and supernatant (C) were subjected to Western blot analysis (B). Supernatant or microsomal (Ms) fractions
were subjected to Western blot analysis (C). ed, epididymal; sc,
subcutaneous.
2094

To determine whether TGH-2 is a lipolytic enzyme that
contributes to the lipolysis in adipocytes, we examined theeffects of knockdown of TGH-2 by RNA silencing on the
lipolysis in 3T3-L1 adiocytes (Fig. 4). Western blot analyses revealed that transfection of siRNA duplexes for HSL,
TGH-1, or TGH-2 inhibited the expression of respective
lipases by 82, 86, and 86%, respectively (Fig. 4A). When thecells were not stimulated with isoproterenol, the RNAinterference for HSL, TGH-1, or TGH-2 did not significantly
inhibit the amounts of either glycerol or FFA release at 3 hof the incubation (n ⫽ 3). When the cells were stimulated
by isoproterenol, the RNA interference for HSL, TGH-1, or
TGH-2 inhibited the amounts of glycerol release by 30%(P ⬍ 0.05), ⫺2%, and 10% (P ⬍ 0.05), respectively (Fig. 4B),
whereas it inhibited the amounts of FFA release by 36%(P ⬍ 0.05), 3%, and 6%, respectively, at 3 h after the
stimulation with isoproterenol (n ⫽ 3).
To determine whether TGH-2 is involved in lipolysis in
adipocytes, we infected 3T3-L1 adipocytes with Ad-TGH-2
and measured the amounts of FFA and glycerol released
with or without isoproterenol (Fig. 5). In the absence of
isoproterenol, we could not detect any differences inlipolysis. Isoproterenol stimulated basal lipolysis, as
shown in the cells infected with Ad-LacZ. Overexpressionof TGH-2 resulted in 3.6- and 2.0-fold increases in theprotein expression in cytosolic and microsomal fractions,
respectively. These changes were associated with further
increases in the release of glycerol by 20% (P ⬍ 0.05) (Fig.
5B) and FFA by 26% (Fig. 5C) at 3 h after the stimulation
with isoproterenol, supporting the idea that TGH-2 isinvolved in lipolysis.
To explore the possibility that mRNA expression of
TGH-1 and -2 is regulated in response to feeding cycle, we
examined changes in the mRNA expression of TGH-1 and-2 in various tissues, including WAT and BAT, after fastingor refeeding in comparison with the expression of HSL(Fig. 6). In contrast to HSL, whose expression was relatively stable during the fasting/refeeding cycle, mRNAlevels of both TGH-1 and -2 were relatively low in a refedstate and increased by fasting in WAT and BAT. In theliver, intestine, and kidney, TGH-2 was upregulated in therefed state, whereas this did not occur in WAT.
DISCUSSION

Here we report identification and characterization of apreviously undescribed TGH that is predominantly expressed in adipose tissue. Because this enzyme has high
DIABETES, VOL. 55, JULY 2006

H. OKAZAKI AND ASSOCIATESTABLE 1Comparison of substrate specificity of HSL, TGH-1, and TGH-2
Substrates⫺1⫺1

PNPB (nmol 䡠 min 䡠 mg )Triolein (pmol 䡠 min⫺1 䡠 mg⫺1)
Diolein (pmol 䡠 min⫺1 䡠 mg⫺1)
Monoolein (pmol 䡠 min⫺1 䡠 mg⫺1)
Cholesteryl oleate (pmol 䡠 min⫺1 䡠 mg⫺1)
Phospholipid (pmol 䡠 min⫺1 䡠 mg⫺1)

LacZ

HSL

TGH-1

TGH-2

59
78 ⫾ 20
10.3
40.5
8.3 ⫾ 4.5
7.3

5,258
9,581
113.3
184.5
1,184 ⫾ 22
15.2

9,494 ⫾ 1,268
429 ⫾ 1510.5
190.0
3.7 ⫾ 0.5
9.0

14,349 ⫾ 2,289
506 ⫾ 3510.0
217.7
2.6 ⫾ 0.3
9.0

Data are means (duplicate measurements) or means ⫾ SE (triplicate measurements). We overexpressed LacZ, HSL, TGH-1, or TGH-2 byadenovirus-mediated gene delivery and used whole-cell extracts for measurements of hydrolytic activities. Hydrolytic activities for PNPB
were measured by colorimetric assay, hydrolytic activities for triolein and cholesteryl oleate were measured by determining the release ofradiolabeled oleate, and hydrolytic activities for monoolein, diolein, and phospholipids were measured by determining the release of oleic
acid by mass spectrometry. Since different buffers were used for different assays, care should be taken when making comparison between
different methods.

sequence homology to TGH, we have designated theoriginal TGH as TGH-1 and the newly identified one as
TGH-2. These enzymes belong to a carboxylesterase family and have remarkable similarity in terms of tissue
distribution, subcellular localization, substrate specificity,and regulation.
Recently, three laboratories have independently reported the cloning of a novel triacylglycerol lipase expressed in adipocytes (27–29). This protein, designated as
desnutrin and adipose triglyceride lipase (ATGL) in miceand Ca2⫹-independent PLA2 (iPLA2)␨ in humans according to each laboratory, contains ␣/␤ hydrolase fold andGXSXG active serine motif, is expressed in adipose tissue,
is localized in both cytosol and lipid droplets, and mediates hydrolysis of triacylglycerol but not cholesteryl ester
or diacylglycerol, thereby contributing to adipocyte lipolysis (28) and acylglycerol transacylation to synthesize
diacylglycerol or triacylglycerol (29). The Drosophila orthologue Brummer lipase has also been reported (30).
There are two other paralogues of ATGL: adiponutrin inmice (31), which is also called iPLA2ε in humans (29), andiPLA2␩ in humans (29). Desnutrin/ATGL/iPLA2␨ was notfound in our database search because it does not containan oxyanion His-Gly dipeptide motif. Instead, it contains apatatin domain that is conserved from plants to mammals(28). According to Zimmermann et al. (28) and Kershaw etal. (32), silencing ATGL gene by siRNA or antisense RNA
markedly decreased lipolysis in stimulated and nonstimulated 3T3-L1 adipocytes, supporting the notion that desnutrin/ATGL/iPLA2␨ is the major non-HSL lipase mediating
lipolysis in adipocytes.
Despite these substantial evidences for the involvementof desnutrin/ATGL/iPLA2␨ in the lipolysis, TGH-2 as well

as TGH-1 may contribute to the residual triacylglycerol
lipase activity in HSL-deficient adipocytes (4,6,7). First, thesubstrate specificity of TGH-1 and -2 is consistent with thecharacteristics of the residual lipase activity in HSLdeficient WAT: substantial triacylglycerol lipase activity
with almost complete absence of neutral cholesteryl esterhydrolase and diacylglycerol lipase activity. When overexpressed in HEK293 cells, TGH-1 and -2 were both able tohydrolyze triacylglycerol and monoacylglycerol but notdiacylglycerol, cholesteryl ester, or phospholipids (Table
1). Second, expression of TGH-1 and -2 was increased
during the differentiation of 3T3-L1 adipocytes (Fig. 2B).
Third, both fatcake and cytosolic fractions contain TGH-1
and -2 , which is consistent with the residual triacylglycerol lipase activity present in HSL-deficient WAT (Fig. 3)(7). Indeed, Soni et al. (17) have recently reported that
TGH, carboxylesterase 3, is the major protein present in
the second peak, with substantial PNPB esterase activity
that was eluted from oleic acid–agarose chromatographyof infranatant as well as fatcake fractions from mouseadipose tissue. However, our studies on the inhibition oroverexpression of TGH-1 and -2 (Figs. 4 and 5) indicatethat TGH-1 and -2 appear to make a relatively minor
contribution to net triacylglycerol hydrolysis in adipose
tissues compared with other adipocyte lipases such as
desnutrin/ATGL/iPLA2␨ and HSL.
The robust induction of the expression of TGH-1 and -2
during fasting is intriguing from the point of view of energy
homeostasis. Fasting also induces the expression of desnutrin/ATGL/iPLA2␨ (27,32). Induction of these multiple
lipases is reasonable physiological response to fasting/starvation if these enzymes mediate lipolysis that may
have evolved for adaptation to fasting/starvation. Desnu-

FIG. 4. Inhibition of TGH-2 by RNA silencing
and its effect on lipolysis in 3T3-L1 adipocytes. At 5 days of differentiation, 3T3-L1
adipocytes were transfected with siRNA duplexes by electroporation. After 48 h, thecells were treated with isoproterenol for 3 h.
Cell lysates were used for Western blot analysis (A), and the amounts of glycerol in themedium were measured and divided by thevalues for scramble siRNA in each experiment after normalizing to the cellular protein (B). Data are expressed as means ⴞ SE
of four independent experiments. *P < 0.05
vs. control scramble siRNA.
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A NOVEL PARALOGUE OF TRIACYLGLYCEROL HYDROLASE

FIG. 5. Overexpression of TGH-2 by recombinant adenovirus and its
effect on lipolysis in 3T3-L1 adipocytes. Recombinant adenoviruses
carrying LacZ (Ad-LacZ) or TGH-2 (Ad-TGH2) were used to infect
3T3-L1 adipocytes on day 8 of differentiation, and experiments
were performed 3 days after infection. A: Overexpression of TGH-2
protein was verified by Western blot analysis using cytosolic (S100) and microsomal (Ms) fraction of 3T3-L1 adipocytes infectedwith Ad-TGH2 or Ad-LacZ for 72 h. B and C: Cells overexpressing
TGH-2 (circle) or LacZ (square) were incubated in DMEM/2% fatty
acid–free BSA in the absence (䡺 and E) or presence (f and F) of 10
␮mol/l isoproterenol for the indicated times, and glycerol (B) andFFA (C) released into the media were measured. Data are expressed as means ⴞ SE of triplicate measurements. *P < 0.05, vs.
control Ad-LacZ–infected cells. C, control; Ms, microsomal fraction.

trin/ATGL/iPLA2␨ is primarily localized in cytosol andfatcake (28), while TGH-1 and -2 are mainly localized in
fatcake and microsome (Fig. 3B and C). This difference insubcellular localization suggests that these lipases play
distinct roles in response to fasting.
In addition to adipose tissues, TGH-1 and -2 are expressed in a wide variety of nonadipose tissues. Liver isthe organ where both TGH-1 and -2 are most abundantly
expressed. Lehner and colleagues (9,33,34) have previously assigned the function of hepatic TGH to VLDL
assembly. This may be the case with TGH-2, since theexpression of TGH-2 was increased in a refed state after
VLDL production was stimulated. A similar role in lipoprotein assembly can be assigned to TGH-2, which is
uniquely expressed in intestine where chylomicron is
produced. Clearly, these intriguing possibilities await further investigation.
It is also noteworthy that TGH-1 and -2 have a muchhigher substrate specificity for short-chain carbonic acid
esters than long-chain triacylglycerol (Table 1). Indeed,murine TGH is identical to carboxylesterase 3, also designated carboxylesterase MH1, which has been identified asa carboxylesterase isozyme induced by di-(2-ethylhexyl)phthalate, a peroxisome proliferator (23). Furthermore,
based on the available partial amino acid sequence, TGH-2
itself might be identical to carboxylesterase-ML1 (26,35).
From these considerations, both enzymes might be better
designated by their original names, carboxylesterase-3 for
TGH-1 and carboxylesterase-ML1 for TGH-2.
Our results in Table 1 do not appear to be consistent
with the previous findings that HSL hydrolyzes diacylglycerol better than triacylglycerol (36,37). These contradictions might result from the difference in the sensitivity of
two different methods used in our study: measurements ofthe end products, FFAs, by radioactivity versus mass
spectrometry. The method that used mass spectrometry
omitted phospholipids from the reaction mixture to avoid
generation of FFAs from the hydrolysis of phospholipids;
this could profoundly impair the sensitivity of the assay.
When the triacylglycerol lipase activities of HSL weredirectly measured by the method that used mass spectrometry, its specific triacylglycerol lipase activity wascalculated to be 6.9 pmol 䡠 min⫺1 䡠 mg⫺1. This value was
15-fold smaller than the diacylglycerol lipase activity mea-

FIG. 6. Changes in the expressions of TGH-1 and -2
mRNA during fasting/refeeding. Total RNA (10 ␮g)from tissues of mice fasted for 24 h (F) or fasted
and then refed for 12 h (R) were subjected to
Northern blot analysis. Specific mRNAs were detected with a radiolabeled cDNA probes for HSL,
TGH-1, TGH-2, or 36B4. ed, epididymal; EDL, extensor digitorum longus; sc, subcutaneous.
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H. OKAZAKI AND ASSOCIATES

sured by the same method (103 pmol 䡠 min⫺1 䡠 mg⫺1)(Table 1), roughly corroborating the ratios in the literature(36,37).
In conclusion, we have identified a novel member of the
carboxylesterase family, TGH-2, with structural, catalytic,
and expressional similarity to TGH or TGH-1. TGH-1 and -2
are highly expressed in adipocytes and are transcriptionally regulated in response to changes in nutritional conditions, suggesting their potential role in adipocyte lipolysis.
The elucidation of the true function of these “redundant”
lipases awaits further in vivo studies of the gain or loss offunction by pharmacologic and/or genetic manipulation.
ACKNOWLEDGMENTS

This work was supported in part by Grant-in-Aid for
Scientific Research from the Ministry of Education, Science and Culture.
REFERENCES
1. Unger RH: Lipotoxic diseases. Annu Rev Med 53:319 –336, 2002
2. Holm C, Osterlund T, Laurell H, Contreras JA: Molecular mechanisms
regulating hormone-sensitive lipase and lipolysis. Annu Rev Nutr 20:365–
393, 2000
3. Kraemer FB, Shen WJ: Hormone-sensitive lipase: control of intracellulartri-(di-)acylglycerol and cholesteryl ester hydrolysis. J Lipid Res 43:1585–
1594, 2002
4. Osuga J, Ishibashi S, Oka T, Yagyu H, Tozawa R, Fujimoto A, Shionoiri F,
Yahagi N, Kraemer FB, Tsutsumi O, Yamada N: Targeted disruption of
hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc Natl Acad Sci U S A 97:787–792, 2000
5. Wang SP, Laurin N, Himms-Hagen J, Rudnicki MA, Levy E, Robert MF, Pan
L, Oligny L, Mitchell GA: The adipose tissue phenotype of hormonesensitive lipase deficiency in mice. Obes Res 9:119 –128, 2001
6. Haemmerle G, Zimmermann R, Hayn M, Theussl C, Waeg G, Wagner E,
Sattler W, Magin TM, Wagner EF, Zechner R: Hormone-sensitive lipase
deficiency in mice causes diglyceride accumulation in adipose tissue,
muscle, and testis. J Biol Chem 277:4806 – 4815, 2002
7. Okazaki H, Osuga J, Tamura Y, Yahagi N, Tomita S, Shionoiri F, Iizuka Y,
Ohashi K, Harada K, Kimura S, Gotoda T, Shimano H, Yamada N, Ishibashi
S: Lipolysis in the absence of hormone-sensitive lipase: evidence for acommon mechanism regulating distinct lipases. Diabetes 51:3368–3375, 2002
8. Lehner R, Verger R: Purification and characterization of a porcine liver
microsomal triacylglycerol hydrolase. Biochemistry 36:1861–1868, 1997
9. Lehner R, Vance DE: Cloning and expression of a cDNA encoding a hepatic
microsomal lipase that mobilizes stored triacylglycerol. Biochem J 343:1–
10, 1999
10. Ye X, Robinson MB, Batshaw ML, Furth EE, Smith I, Wilson JM: Prolonged
metabolic correction in adult ornithine transcarbamylase-deficient micewith adenoviral vectors. J Biol Chem 271:3639 –3646, 1996
11. Kanehisa M, Goto S, Kawashima S, Nakaya A: The KEGG databases at
GenomeNet. Nucleic Acids Res 30:42– 46, 2002
12. Okazaki H, Osuga J, Tsukamoto K, Isoo N, Kitamine T, Tamura Y, Tomita
S, Sekiya M, Yahagi N, Iizuka Y, Ohashi K, Harada K, Gotoda T, Shimano
H, Kimura S, Nagai R, Yamada N, Ishibashi S: Elimination of cholesterolester from macrophage foam cells by adenovirus-mediated gene transfer ofhormone-sensitive lipase. J Biol Chem 277:31893–31899, 2002
13. Tsukamoto K, Smith P, Glick JM, Rader DJ: Liver-directed gene transfer
and prolonged expression of three major human ApoE isoforms in
ApoE-deficient mice. J Clin Invest 100:107–114, 1997
14. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T:
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature 411:494 – 498, 2001
15. Jiang ZY, Zhou QL, Coleman KA, Chouinard M, Boese Q, Czech MP: Insulin
signaling through Akt/protein kinase B analyzed by small interfering
RNA-mediated gene silencing. Proc Natl Acad Sci U S A 100:7569 –7574,
2003
16. Shirai K, Jackson RL: Lipoprotein lipase-catalyzed hydrolysis of p-nitrophenyl butyrate: interfacial activation by phospholipid vesicles. J Biol
Chem 257:1253–1258, 1982

DIABETES, VOL. 55, JULY 2006

17. Soni KG, Lehner R, Metalnikov P, O’Donnell P, Semache M, Gao W,
Ashman K, Pshezhetsky AV, Mitchell GA: Carboxylesterase 3 (EC 3.1.1.1)
is a major adipocyte lipase. J Biol Chem 279:40683– 40689, 2004
18. Hosono H, Aoki J, Nagai Y, Bandoh K, Ishida M, Taguchi R, Arai H, Inoue
K: Phosphatidylserine-specific phospholipase A1 stimulates histamine release from rat peritoneal mast cells through production of 2-acyl-1lysophosphatidylserine. J Biol Chem 276:29664 –29670, 2001
19. Hiramatsu T, Sonoda H, Takanezawa Y, Morikawa R, Ishida M, Kasahara K,
Sanai Y, Taguchi R, Aoki J, Arai H: Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPAPLA1alpha and mPA-PLA1beta. J Biol Chem 278:49438 – 49447, 2003
20. Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M,
Remington SJ, Silman I, Schrag J, Sussman JL, Verschueren KHG, Goldman
A: The alpha/beta hydrolase fold. Protein Eng 5:197–211, 1992
21. Feller G, Thiry M, Gerday C: Nucleotide sequence of the lipase gene lip2
from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues. DNA Cell Biol
10:381–388, 1991
22. Dolinsky VW, Sipione S, Lehner R, Vance DE: The cloning and expressionof a murine triacylglycerol hydrolase cDNA and the structure of its
corresponding gene. Biochim Biophys Acta 1532:162–172, 2001
23. Furihata T, Hosokawa M, Koyano N, Nakamura T, Satoh T, Chiba K:
Identification of di-(2-ethylhexyl) phthalate-induced carboxylesterase 1 in
C57BL/6 mouse liver microsomes: purification, cDNA cloning, and baculovirus-mediated expression. Drug Metab Dispos 32:1170 –1177, 2004
24. Alam M, Ho S, Vance DE, Lehner R: Heterologous expression, purification,and characterization of human triacylglycerol hydrolase. Protein Expr
Purif 24:33– 42, 2002
25. Alam M, Vance DE, Lehner R: Structure-function analysis of humantriacylglycerol hydrolase by site-directed mutagenesis: identification of thecatalytic triad and a glycosylation site. Biochemistry 41:6679 – 6687, 2002
26. Satoh T, Hosokawa M: The mammalian carboxylesterases: from molecules
to functions. Annu Rev Pharmacol Toxicol 38:257–288, 1998
27. Villena JA, Roy S, Sarkadi-Nagy E, Kim KH, Sul HS: Desnutrin, an
adipocyte gene encoding a novel patatin domain-containing protein, is
induced by fasting and glucocorticoids: ectopic expression of desnutrin
increases triglyceride hydrolysis. J Biol Chem 279:47066 – 47075, 2004
28. Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A,
Zechner R: Fat mobilization in adipose tissue is promoted by adipose
triglyceride lipase. Science 306:1383–1386, 2004
29. Jenkins CM, Mancuso DJ, Yan W, Sims HF, Gibson B, Gross RW:
Identification, cloning, expression, and purification of three novel humancalcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. J Biol Chem
279:48968 – 48975, 2004
30. Gronke S, Mildner A, Fellert S, Tennagels N, Petry S, Muller G, Jackle H,
Kuhnlein RP: Brummer lipase is an evolutionary conserved fat storage
regulator in Drosophila. Cell Metab 1:323–330, 2005
31. Baulande S, Lasnier F, Lucas M, Pairault J: Adiponutrin, a transmembraneprotein corresponding to a novel dietary- and obesity-linked mRNA
specifically expressed in the adipose lineage. J Biol Chem 276:33336 –
33344, 2001
32. Kershaw EE, Hamm JK, Verhagen LA, Peroni O, Katic M, Flier JS: Adipose
triglyceride lipase: function, regulation by insulin, and comparison withadiponutrin. Diabetes 55:148 –157, 2006
33. Gilham D, Ho S, Rasouli M, Martres P, Vance DE, Lehner R: Inhibitors ofhepatic microsomal triacylglycerol hydrolase decrease very low density
lipoprotein secretion. FASEB J 17:1685–1687, 2003
34. Gilham D, Alam M, Gao W, Vance DE, Lehner R: Triacylglycerol hydrolase
is localized to the endoplasmic reticulum by an unusual retrieval sequence
where it participates in VLDL assembly without utilizing VLDL lipids as
substrates. Mol Biol Cell 16:984 –996, 2005
35. Satoh T, Hosokawa M: Molecular aspects of carboxylesterase isoforms incomparison with other esterases. Toxicol Lett 82- 83:439 – 445, 1995
36. Fredrikson G, Stralfors P, Nilsson NO, Belfrage P: Hormone-sensitive
lipase of rat adipose tissue. Purification and some properties. J Biol Chem
256:6311– 6320, 1981
37. Ben Ali Y, Chahinian H, Petry S, Muller G, Carriere F, Verger R, Abousalham A: Might the kinetic behavior of hormone-sensitive lipase reflect theabsence of the lid domain? Biochemistry 43:9298 –9306, 2004

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