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Endocrinology 146(4):1752–1763
Copyright © 2005 by The Endocrine Society
doi: 10.1210/en.2004-1082
Role of Meltrin ␣ (ADAM12) in Obesity Induced by HighFat Diet
Megumi Masaki, Tomohiro Kurisaki, Kamon Shirakawa, and Atsuko Sehara-Fujisawa
Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University (M.M., T.K., A.S.-F.), Sakyo-ku,
Kyoto 606-8507, Japan; Mochida Pharmaceutical Co. Ltd. (K.S.), Shizuoka 412-8524, Japan; and Japan Society for the
Promotion of Science (M.M.), Tokyo 102-8471, Japan
Meltrin ␣ is a member of the metalloprotease-disintegrin
(ADAM) family. In this paper we demonstrate that meltrin ␣
is involved in the development of white adipose tissue. Compared with wild-type mice, meltrin ␣ⴚ/ⴚ mice displayed moderate resistance to weight gain induced by a high-fat diet,
mainly because of an impaired increase in the number of adipocytes. There was no obvious difference in adipocyte size
between wild-type and meltrin ␣ⴚ/ⴚ mice, suggesting normal
maturation of adipocytes of the latter under a high-fat diet.
T
HERE ARE TWO types of adipose tissue in the mammalian body. White adipose tissue (WAT) is distributed in many locations, stores excess energy as triglycerides,
and releases fatty acids in response to energy requirements.
The second type of adipose tissue is brown adipose tissue
(BAT), which is the main thermogenic tissue in rodents. In
BAT, fatty acid oxidation stimulated by the sympathetic nervous system generates heat through the induction of uncoupling protein-1. The developmental patterns of these tissues
are quite different. BAT develops during fetal stages and
essentially acquires all the features of mature tissue at birth,
when nonshivering thermogenesis is required (1). In contrast, the development of WAT continues after birth, and its
mass increases during postnatal life (2).
In this study we report the roles of meltrin ␣ in obesity, a
pathological development of WAT. Meltrin ␣ (ADAM12) is
a member of the metalloprotease-disintegrin (ADAM) family
of proteins that contain metalloprotease and disintegrin domains (3). ADAMs play important roles in fertilization (4 – 6)
and various aspects of morphogenesis (3, 7, 8) and are implicated in certain pathogenetic processes (9, 10). Genetic and
biochemical studies have revealed that some ADAMs participate in the ectodomain shedding of various membraneanchored proteins (by proteolysis of these proteins at the
First Published Online January 6, 2005
Abbreviations: BAT, Brown adipose tissue; DAPI, 4⬘,6-diamidino-2phenylindole; DEX, dexamethasone; DIM, differentiation induction medium; EGF, epidermal growth factor; FBS, fetal bovine serum; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HB-EGF, heparin-binding
epidermal growth factor; IGFBP-3, IGF-binding protein-3; INS, insulin;
MEF, mouse embryonic fibroblast; MIX, 3-isobutyl-1-methylxanthine;
NEFA, nonesterified fatty acid; PFA, paraformaldehyde; PMA, phorbol
12-myristate 13-acetate; S-V, stromal-vascular; WAT, white adipose
tissue.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
Embryonic fibroblasts and stromal-vascular cells lacking meltrin ␣ exhibited impaired cell proliferation upon adipogenic
stimulation, which was accompanied by moderate defects in
adipose differentiation. Addition of culture medium conditioned with wild-type cells in an early phase of adipose differentiation did not restore the defects in the meltrin ␣ⴚ/ⴚ
cells. These results uncover the involvement of meltrin ␣ in
the development of obesity and in adipogenic cell proliferation. (Endocrinology 146: 1752–1763, 2005)
extracellular juxtamembrane region), including growth
factors, intercellular signaling molecules, and adhesion
molecules (11–13). Meltrin ␣ modulates myotube formation
in vitro (3) and in vivo (14, 15). Two candidate substrates
for meltrin ␣ protease have been reported to date: heparinbinding epidermal growth factor (HB-EGF) (14) and IGFbinding protein-3 (IGFBP-3) (16, 17), which regulates the
activation of IGF-I. Evidence suggests that different ADAM
proteases participate in the phorbol 12-myristate 13-acetate
(PMA)-induced ectodomain shedding of membrane-anchored ErbB ligands, including HB-EGF (9, 18), TGF-␣ (8),
and neuregulin (19). In addition to TNF-␣-converting enzyme (TACE/ADAM17), meltrin ␣ and meltrin ␤ (ADAM19)
are examples of such proteases, although their expression
patterns, ligand specificity, and regulation of protease activity differ (9, 20, 21).
We reported previously that during embryogenesis, some
meltrin ␣⫺/⫺ mice display impaired development of interscapular BAT and of the skeletal muscles that surround BAT
(14). The increase in ectodomain shedding of HB-EGF in
response to PMA was markedly reduced in meltrin ␣⫺/⫺
embryonic fibroblasts. In contrast, Kawaguchi et al. (22) reported that transgenic mice overexpressing a placental isoform of human meltrin ␣ exhibit increased adipogenesis.
Although the lack of meltrin ␣ also affects WAT formation
in some meltrin ␣⫺/⫺ mouse embryos, decreased formation
of WAT is not as prominent as that of BAT and recovers after
birth, suggesting that compensatory mechanisms restore the
roles of meltrin ␣ in the surviving population of meltrin ␣⫺/⫺
mice during WAT formation in utero or before weaning
(Kurisaki, T., and A. Sehara-Fujisawa, unpublished observations). Alternatively, the slight decrease in WAT formation
in meltrin ␣⫺/⫺ mice and the enhanced formation of WAT in
transgenic mice expressing a placental isoform of human
meltrin ␣ could be secondary effects of other phenotypes,
such as systemic growth retardation or aberrant placental
1752
Masaki et al. • Role of Meltrin ␣ in Obesity
development. To determine whether meltrin ␣ plays a direct
role in WAT development and to reveal its role in adipogenesis, we examined the effects of meltrin ␣ deficiency on
the induction of obesity by a high-fat diet and on adipogenesis of stromal-vascular (S-V) cells or embryonic fibroblasts
cultured in vitro, both of which eliminate maternal factors
and other compensatory factors in utero and before weaning.
As a result, we demonstrated the involvement of meltrin ␣
in obesity induced by a high-fat diet. Meltrin ␣ participates
mainly in increasing the number of adipogenic cells during
the progression of obesity and in cell proliferation at an early
stage of adipogenesis in S-V cells and embryonic fibroblasts,
which are critical for the expansion of adipocytes in vivo and
in vitro.
Materials and Methods
Animal experiments
The meltrin ␣⫺/⫺ mouse line was generated as described previously
(14). The initial chimeras were backcrossed to C57BL/6J more than 12
times. The numbers of animals used in experiments are mentioned in
Results and the figure legends. Animals were maintained in a temperature-controlled facility with a 12-h light, 12-h dark cycle. When male
mice were 4 wk of age, C57BL/6J wild-type and meltrin ␣⫺/⫺ mice were
divided into two groups. One group was given a high-fat diet containing
60% fat (Oriental Yeast Co. Ltd., Tokyo, Japan), and the second group
was given a normal (10% fat) diet (Research Diets, Inc., New Brunswick,
NJ). The diets contained 5.2 and 3.8 kcal/g. Body weight was recorded
every week, and food intake for the high-fat diet was determined every
second day. For the measurement of metabolic parameters, mice were
fed the normal or high-fat diet for 12 wk. Blood was collected from the
heart after an overnight fast. Plasma triglycerides, total cholesterol, and
nonesterified fatty acids (NEFA) were measured using enzymatic assays: the triglyceride E test (Wako Pure Chemical Industries Ltd., Osaka,
Japan), the cholesterol E test (Wako Pure Chemical Industries Ltd.), and
the NEFA test (Wako Pure Chemical Industries Ltd.). For the determination of plasma leptin, adiponectin, and insulin (INS) levels, ELISA kits
were purchased from Morinaga (Kanagawa, Japan), Otsuka (Tokyo,
Japan), and Shibayagi (Gunma, Japan), respectively. Glucose tolerance
testing and INS tolerance testing were performed with the C57BL/6J and
meltrin ␣⫺/⫺ mice fed the normal diet at 25–35 wk of age or with the
C57BL/6J and meltrin ␣⫺/⫺ mice fed the high-fat diet for 10 wk. Glucose
at 1 g/kg body weight or INS at 0.75 U/kg was injected ip after an
overnight fast. Blood was collected from the tail vein. Glucose quantification was performed with the One Touch Ultra blood glucose-monitoring system (Johnson & Johnson, Tokyo, Japan). For measurement of
INS concentrations before and after glucose injection, glucose at 1 g/kg
body weight was injected ip into the C57BL/6J and meltrin ␣⫺/⫺ mice
fed the normal diet after overnight fasting. At each time point, blood was
collected from an intraorbital vein. The serum INS concentration was
determined by ELISA.
Histological analysis of adipose tissues and liver
Pieces of adipose tissues and livers were fixed in 4% paraformaldehyde (PFA), dehydrated in ethanol, embedded in paraffin, and sectioned
at a thickness of 4 ␮m. Sections were deparaffinized, rehydrated, and
stained with hematoxylin and eosin. The number of adipocytes was
determined as described previously (23). Briefly, adipocytes in the nine
slices for each animal (n ⫽ 3) were counted, and the average cell volume
was determined. Relative cell numbers were calculated based on the
average cell volume and tissue weights. For Oil Red-O staining, livers
were immediately embedded in tissue-freezing medium (Tissue-Tek
OCT compound, Miles, Inc., Elkhart, IN), and sections 7 ␮m thick were
stained with Oil Red-O.
RNA preparation and real-time PCR
Total RNA was prepared from tissues of adult male mice with
RNeasy Lipid Tissue Mini (Qiagen, Valencia, CA) in accordance with the
Endocrinology, April 2005, 146(4):1752–1763
1753
manufacturer’s instructions. For the isolation of total RNA from mouse
embryonic fibroblasts (MEFs), RNeasy mini (Qiagen) was used. Meltrin
␣ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
expression were determined by RT, followed by real-time TaqMan PCR
analysis. Primers and probes of meltrin ␣ and GAPDH were purchased
from Applied Biosystems (Foster City, CA; Mm00475719_m1and P/N
4308313, respectively). The primers used to detect mRNA of IGFBP-3
were 5⬘-GACACCCAGAACTTCTCCTCC-3⬘ and 5⬘-CATACTTGTCCACACACCAGC-3⬘.
Preparation of S-V cells and immunofluorescent staining
Inguinal fat pads were harvested from 4- to 5-wk-old, wild-type or
meltrin ␣⫺/⫺ male mice. After blood was washed out of the tissues, they
were minced and digested with 1 mg/ml collagenase type I (Worthington Biochemical Corp., Freehold, NJ) for 30 min at 37 C. Cells were
filtered through 200-␮m pore size nylon meshes. The S-V cells were
separated from adipocytes by centrifugation and washed with DMEM
(Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS). S-V cells were plated and propagated
to confluence in DMEM supplemented with 10% heat-inactivated FBS,
50 U/ml penicillin, and 50 ␮g/ml streptomycin. Two days later, the
medium was replaced with differentiation induction medium (DIM)
containing 1 ␮m dexamethasone (DEX; Sigma-Aldrich Corp., St. Louis,
MO), 10 ␮g/ml INS (Sigma-Aldrich Corp.), and 0.5 mm 3-isobutyl-1methylxanthine (MIX; Nacalai Tesque, Inc.) with 10% heat-inactivated
FBS. After 24 h, cells were washed with PBS twice and fixed with 4% PFA
for 30 min, followed by treatment with ice-cold 0.2% Triton X-20 for 5
min, then stained with anti-Ki-67 (BD PharMingen, San Diego, CA; 1:100
dilution) as a first antibody and antimouse IgG-Alexa 488-conjugated
antibody (Molecular Probes, Eugene, OR; 1:400 dilution) as a secondary
antibody. 4⬘,6-Diamidino-2-phenylindole (DAPI; 0.2 ␮g/ml; Molecular
Probes) was used for staining of all nuclei. Photographs were taken on
a Zeiss fluorescent microscope (New York, NY) with MetaMorph software (Universal Imaging Corp., Brock and Michelsen, Brikerød, Denmark). The ratio of Ki-67-positive cells to DAPI-positive cells was
determined.
Preparation of MEFs and induction of adipogenesis
Primary embryonic fibroblasts were prepared from 14.5 d postcoitus
embryos. Cells (8 ⫻ 105) were plated on 12-well plastic dishes and
cultured at 37 C in standard medium: ␣-MEM (Nacalai Tesque, Inc.)
supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, and
50 ␮g/ml streptomycin. Cells were propagated to confluence. Two days
later, the medium was replaced with DIM with 10% heat-inactivated
FBS. After 2 d, this medium was replaced with maturation medium,
which contains 10 ␮g/ml INS. After another 3 d, the maturation medium
was replaced with standard medium. For evaluation of the effect of
HB-EGF, wild-type MEFs were cultured in DIM or ␣-MEM containing
10 ␮g/ml INS with or without 100 ng/ml HB-EGF (R&D Systems, Inc.,
Minneapolis, MN). After 2 d, the medium was replaced with a maturation medium with or without HB-EGF. After another 3 d, the maturation medium was replaced with standard medium. In the medium
exchange experiment, we inducted meltrin ␣⫺/⫺ and wild-type MEFs
with DIM as described above (d 0). Then, culture medium of wild-type
MEFs was replaced with meltrin ␣⫺/⫺-conditioned medium and vice
versa on d 1, 3, and 6. The medium was replaced with fresh maturation
medium on d 2 and with standard medium on d 5. After 8 d, cytoplasmic
lipid accumulation was observed by brightfield microscopy with Oil
Red-O staining. The composition of each medium is shown in Tables 1
and 2 in detail. Oil Red-O staining was performed as follows. Cells were
washed with PBS, fixed with 4% PFA for 10 min, then stained with 60%
filtered Oil Red-O stock solution (0.15 g Oil Red-O in 50 ml isopropanol)
for 30 min, washed with 60% isopropanol, then briefly washed with PBS
twice before being visualized. To quantify the amount of lipid, stained
oil was eluted with isopropanol, and the absorbance at a wavelength of
510 nm was read after dilution of the eluate to a linear range. To
determine the number of cells, on d 0 (before induction with DIM) and
d 3 (after induction), cells were trypsinized and well suspended with
␣-MEM with 10% heat-inactivated FBS. Cells were counted using
Neubauer’s hemocytometer (Erma, Tokyo, Japan) under light micros-
1754
Masaki et al. • Role of Meltrin ␣ in Obesity
Endocrinology, April 2005, 146(4):1752–1763
TABLE 1. Composition of each culture medium
Standard medium
␣-MEM with 10% FBS, 50 U/ml penicillin,
and 50 ␮g/ml streptomycine
DIM
Standard medium ⫹ 1 ␮M DEX, 10 ␮g/ml
INS, and 0.5 mM MIX
Maturation medium
Standard medium ⫹ 10 ␮g/ml INS
copy. Experiments were performed in triplicate with four different litters
of mice, and values are shown as the mean ⫾ sem.
Western blot
In preparation for Western blotting, 1.5 ⫻ 106 MEFs were placed in
six-well dishes and cultured in a standard medium. Two days later, the
medium was replaced with Opti-MEM (Invitrogen Life Technologies,
Inc., Carlsbad, CA) reduced serum with either DIM or 10 ␮g/ml INS.
After 18 or 36 h, the conditioned medium was concentrated 10-fold using
a centrifuge tube filtration unit (Amicon Ultra-4 10,000 MWCO, Millipore Corp., Bedford, MA). Western blotting was performed as described
previously (19, 21). Goat polyclonal antibody against IGFBP-3 (sc-6004)
was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Statistical analysis
Data are expressed as the mean ⫾ sem. A two-tailed t test was used
to calculate P values.
⫺/⫺
Meltrin ␣
high-fat diet
Results
mice are resistant to induction of obesity by a
To explore the role of meltrin ␣ in the development of
WAT in adult mice, we examined the body weight gain of
meltrin ␣⫺/⫺ mice on either a high-fat diet or a normal diet.
After weaning, meltrin ␣⫺/⫺ mice fed a normal diet gained
weight similarly to wild-type mice. However, meltrin ␣⫺/⫺
mice on a high-fat diet were protected against weight gain to
some extent, despite having the same food intake as their
wild-type counterparts (Fig. 1, A and B). Representative
specimens of wild-type and meltrin ␣⫺/⫺ mice given the
high-fat diet for 12 wk are shown in Fig. 1C. The decreased
weight gain of meltrin ␣⫺/⫺ mice fed the high-fat diet was
mainly reflected by the smaller mass and lower weight of
their WAT compared with those of wild-type mice (Fig. 1, D
and E). Resistance of meltrin ␣⫺/⫺ mice to high-fat dietinduced obesity was also shown by the normal appearance
of their livers compared with those of the wild-type mice,
which displayed hepatic steatosis. Furthermore, the interscapular BAT of meltrin ␣⫺/⫺ mice on the high-fat diet was
darker and had less mass of WAT surrounding it than that
of wild-type mice (Fig. 1D). These results indicate that mel-
trin ␣⫺/⫺ mice were resistant to the obesity induced by a
high-fat diet.
Glucose homeostasis of meltrin ␣⫺/⫺ mice fed a highfat diet
Defects in glucose homeostasis are important factors causing obesity. To evaluate the metabolic difference between
wild-type and meltrin ␣⫺/⫺ mice, we performed INS tolerance and glucose tolerance tests on these mice fed a normal
diet or a high-fat diet. In a normal diet condition, increased
INS sensitivity was observed in meltrin ␣⫺/⫺ mice (Fig. 2A).
Hypoglycemia was evident 30 and 60 min after INS injection
in both groups of mice. Wild-type mice more or less recovered their plasma glucose concentrations by 120 min after
injection, whereas meltrin ␣⫺/⫺ mice remained hypoglycemic (P ⬍ 0.05). Plasma triglyceride and NEFA levels were
also significantly lower in meltrin ␣⫺/⫺ mice fed a normal
diet than in wild-type (Table 3). Thus, such altered metabolic
profiles of meltrin ␣⫺/⫺ mice fed a normal diet might be
associated with the resistance of meltrin ␣⫺/⫺ mice to highfat diet-induced weight gain. In contrast, no statistically significant difference was found between wild-type and meltrin
␣⫺/⫺ mice 10 wk after a high-fat diet. The glucose clearance
rates after ip glucose injection in wild-type and meltrin ␣⫺/⫺
mice were almost the same whether animals were fed a
normal diet or a high-fat diet (Fig. 2B). There was no difference in serum INS concentrations before and after glucose
injection between wild-type and meltrin ␣⫺/⫺ mice (Fig. 2C).
Histological analysis of meltrin ␣⫺/⫺ mice fed a high
fat diet
We performed a histological analysis of BAT, liver, and inguinal, epididymal, and retrorenal WAT of wild-type and meltrin ␣⫺/⫺ mice fed a normal or a high-fat diet. In the inguinal
WAT, the adipocytes of wild-type and meltrin ␣⫺/⫺ mice on the
normal diet were roughly the same size. On the high-fat diet,
the adipocytes in inguinal WAT of both types of mice increased
in size due to intracellular lipid accumulation (Fig. 3A). These
lipid accumulation patterns were the same in retrorenal and
epididymal WAT (data not shown). This indicates that fat storage in adipocytes was normal in meltrin ␣⫺/⫺ mice.
The adipocytes in the BAT of wild-type mice on a high-fat
diet accumulated lipid in large droplets. However, the BAT
adipocytes of meltrin ␣⫺/⫺ mice accumulated less lipid in
smaller droplets after being fed a high-fat diet (Fig. 3A),
accounting in part for the macroscopically darker color of the
BAT of meltrin ␣⫺/⫺ mice. Hematoxylin and eosin staining
and Oil Red-O staining of liver sections confirmed the pres-
TABLE 2. Cell culture conditions in the medium exchange experiment
WT MEF
Day
Day
Day
Day
Day
Day
Day
0
1
2
3
5
6
8
Mel ␣⫺/⫺, Metrin ␣⫺/⫺.
Mel ␣⫺/⫺ MEF
Freshly prepared DIM
WT MEF-conditioned medium on d 1
Mel ␣⫺/⫺ MEF-conditioned medium on d 1
Freshly prepared maturation medium
Mel ␣⫺/⫺ MEF-conditioned medium on d 3
WT MEF-conditioned medium on d 3
Freshly prepared standard medium
⫺/⫺
Mel ␣
WT MEF-conditioned medium on d 6
MEF-conditioned medium on d 6
Oil Red-O staining
Masaki et al. • Role of Meltrin ␣ in Obesity
Endocrinology, April 2005, 146(4):1752–1763
1755
FIG. 1. Decreased body weight gain of meltrin ␣⫺/⫺ mice on a high-fat diet. A, Body weight curves for male mice on normal or high-fat diets.
Body weights were recorded weekly in wild-type (WT) and meltrin ␣⫺/⫺ (Mel.␣⫺/⫺) mice fed either a normal diet (ND) or a high-fat diet (HFD).
Only on the high-fat diet did meltrin ␣⫺/⫺ mice have lower body weights than WT mice. Values are expressed as the mean ⫾ SEM. WT-HFD,
n ⫽ 8; WT-ND, n ⫽ 6; Mel.␣⫺/⫺ ND, n ⫽ 6; Mel.␣⫺/⫺ NFD, n ⫽ 6. Asterisks indicate a statistically significant difference (P ⬍ 0.05) between
high fat-fed WT and meltrin ␣⫺/⫺ mice. B, Food intake was recorded every second day and was the same in WT and meltrin ␣⫺/⫺ mice (10.5 ⫾
0.1 and 10.7 ⫾ 0.2 kcal/d, respectively). C, Morphology of 15-wk-old mice fed a high-fat diet. D, Morphology of interscapular BAT, liver, and
inguinal WAT in 16-wk-old mice fed a high-fat diet. Scale bar, 1 cm. E, Tissue weights in mice after 12 wk on a high fat or a normal diet, expressed
as the mean ⫾ SEM (n ⫽ 6). Asterisks indicate statistically significant difference (P ⬍ 0.05) between two experimental groups.
1756
Masaki et al. • Role of Meltrin ␣ in Obesity
Endocrinology, April 2005, 146(4):1752–1763
FIG. 2. A, INS tolerance test in wild-type (WT)
and meltrin ␣⫺/⫺ (Mel.␣⫺/⫺) mice fed a normal or
a high-fat diet. INS (0.75 U/kg) was injected into
wild-type and meltrin ␣⫺/⫺ 25- to 35-wk-old mice
fed a normal diet or 15-wk-old mice fed a high-fat
diet for 10 wk after overnight fasting. In each
experiment, values are expressed as the mean ⫾
⫺/⫺
SEM. ND, Normal diet (WT, n ⫽ 14; meltrin ␣
,
n ⫽ 9); HFD, high-fat diet (WT, n ⫽ 7; meltrin
⫺/⫺
␣ , n ⫽ 6). B, Glucose tolerance test in wild-type
(WT) and meltrin ␣⫺/⫺ (Mel.␣⫺/⫺) mice fed a
normal or a high-fat diet. Glucose (1 g/kg) was
injected ip into wild-type and meltrin ␣⫺/⫺ 25- to
35-wk-old mice fed a normal diet or 15-wk-old
mice fed a high-fat diet for 10 wk after overnight
fasting. In each experiment, values are expressed
as the mean ⫾ SEM. ND, Normal diet (WT, n ⫽ 9;
meltrin ␣⫺/⫺, n ⫽ 5); HFD, high-fat diet (WT, n ⫽
7; meltrin ␣⫺/⫺, n ⫽ 6). C, INS concentrations preand postinjection of glucose. Glucose (1 g/kg) was
injected ip into wild-type and meltrin ␣⫺/⫺ 25- to
35-wk-old mice fed a normal diet after overnight
fasting. Before and after 15-min glucose injection,
blood was collected from an intraorbital vein. The
serum INS concentration was determined. In
each experiment, values are expressed as the
mean ⫾ SEM. WT, n ⫽ 5; meltrin ␣⫺/⫺, n ⫽ 4.
than in wild-type mice on the same diet (Fig. 3C), but cell
numbers were approximately the same in mice fed a normal
diet. These data indicate that meltrin ␣ is involved in increasing
the number of adipocytes in mice fed a high-fat diet.
ence of steatosis in the wild-type mice fed a high-fat diet,
whereas there was no sign of steatosis in the livers of meltrin
␣⫺/⫺ mice on the same diet (Fig. 3B).
Because the fat pads of meltrin ␣⫺/⫺ mice on a high-fat diet
were smaller than those of wild-type mice despite the similar
size of individual adipocytes (Figs. 1E and 3A), we counted the
number of cells in representative populations of white adipocytes from the inguinal, epididymal, and retrorenal WAT.
There were fewer cells in meltrin ␣⫺/⫺ mice fed a high-fat diet
Meltrin ␣ is expressed in various adipose tissues
We used quantitative real-time PCR to examine relative
meltrin ␣ expression in several tissues of wild-type mice fed
TABLE 3. Metabolic parameters in wild-type (WT) and meltrin ␣⫺/⫺ (Mel.␣⫺/⫺) mice
Normal diet
WT (n ⫽ 6)
Leptin (ng/ml)
INS (ng/ml)
Adiponectin (␮g/ml)
Cholesterol (mg/dl)
Triglyceride (mg/dl)
NEFA (mE/liter)
2.22 ⫾ 0.65
0.57 ⫾ 0.09
22.37 ⫾ 1.59
68.09 ⫾ 4.90
126.20 ⫾ 7.01
0.98 ⫾ 0.03
Values are expressed as the mean ⫾
a
P ⬍ 0.05.
SEM.
High-fat diet
Mel.␣
⫺/⫺
(n ⫽ 6)
3.55 ⫾ 1.50
0.67 ⫾ 0.11
27.72 ⫾ 2.36
55.74 ⫾ 6.67
97.08 ⫾ 8.07a
0.62 ⫾ 0.09a
WT (n ⫽ 6)
Mel.␣⫺/⫺ (n ⫽ 6)
33.00 ⫾ 1.65
3.11 ⫾ 0.33
33.38 ⫾ 3.54
186.34 ⫾ 30.30
108.97 ⫾ 7.98
1.10 ⫾ 0.26
24.84 ⫾ 5.21a
2.64 ⫾ 0.61
32.34 ⫾ 2.89
135.25 ⫾ 29.61
96.34 ⫾ 8.84
0.86 ⫾ 0.07
Masaki et al. • Role of Meltrin ␣ in Obesity
Endocrinology, April 2005, 146(4):1752–1763
1757
FIG. 3. A, Histological analysis of adipose tissues. Inguinal WAT and interscapular BAT sections were stained with hematoxylin and eosin.
Magnifications to show morphology of inguinal WAT and BAT are ⫻10 and ⫻20, respectively. Scale bar, 100 ␮m. WT, Wild-type mice; Mel.␣⫺/⫺,
meltrin ␣⫺/⫺ mice; HFD, high-fat diet; ND, normal diet. Average cell volumes for WT ND and Mel.␣⫺/⫺ ND iguinal WAT were 1.51 ⫻ 104 ⫾
0.12 ⫻ 104 and 1.90 ⫻ 104 ⫾ 0.44 ⫻ 104 ␮m3, respectively. Average cell volumes for WT HFD and Mel.␣⫺/⫺ HFD inguinal WAT were 2.37 ⫻
105 ⫾ 0.30 ⫻ 105 and 2.07 ⫻ 105 ⫾ 0.46 ⫻ 105 ␮m3, respectively. Data from nine slices for each animal (n ⫽ 3) were analyzed. B, Histological
analysis of liver. Liver sections were stained with either hematoxylin and eosin (H & E) or Oil Red-O. Magnification for morphological
examination, 10⫻ and 20⫻, respectively. Scale bar, 100 ␮m. Abbreviations are explained in A. C, Relative cell numbers in wild-type and meltrin
␣⫺/⫺ WATs. The cell numbers in wild-type and meltrin ␣⫺/⫺ WATs were estimated as described in Materials and Methods. Data from nine slices
for each animal (n ⫽ 3) were analyzed. Asterisks indicate a statistically significant difference (P ⬍ 0.05) between two experimental groups.
Abbreviations are explained in A. D, Meltrin ␣ mRNA expression in WAT and liver of mice fed a normal diet. Total RNA was prepared from
tissues of adult male mice, and the mRNA level of meltrin ␣ was determined by real-time PCR. Levels of mRNA were normalized to that of
GAPDH. Values from epididymal WAT were set at 1. In each experiment, n ⫽ 3, and values are expressed as the mean ⫾ SEM. E, High-fat
diet-induced expression of meltrin ␣ mRNA in liver and epididymal WAT. Total RNA was prepared from tissues of adult male mice fed either
a normal diet (䡺) or a high-fat diet (f), and the mRNA level of meltrin ␣ was determined by real-time PCR. Levels of mRNA were normalized
to that of GAPDH. Values from epididymal WAT of mice fed a normal diet were set at 1. In each experiment, n ⫽ 3, and values are expressed
as the mean ⫾ SEM. TA, Tibialis anterior muscle. Asterisks indicate a statistically significant difference (P ⬍ 0.05) between two experimental
groups.
1758
Endocrinology, April 2005, 146(4):1752–1763
a normal diet. The level of meltrin ␣ mRNA was almost the
same among epididymal and retrorenal WAT and interscapular BAT. The meltrin ␣ mRNA level in inguinal WAT was
12 times higher than that in other adipose tissues. Meltrin ␣
mRNA expression was much lower or was not recorded in
the liver and tibialis anterior muscle (Fig. 3, D and E). This
suggests that meltrin ␣ is expressed in adipose tissues and is
required for the increase in cell number induced by a high-fat
diet.
Although meltrin ␣ mRNA expression was low in the liver
in the normal diet condition, meltrin ␣⫺/⫺ mice were resistant to hepatic steatosis, which was observed in wild-type
mice fed a high-fat diet (Figs. 1D and 3B). We examined the
influence of a high-fat diet on the expression of meltrin ␣
mRNA in the liver, tibialis anterior muscle, and epididymal
WAT. As a result, meltrin ␣ mRNA expression was upregulated in epididymal WAT (2-fold) and liver (12-fold), but
not in muscle, when mice were fed a high-fat diet (Fig. 3E).
Up-regulation of meltrin ␣ mRNA in the liver on a high-fat
diet might be involved in hepatic steatosis.
S-V cells lacking meltrin ␣ have a defect(s) in cell
proliferation during adipogenesis
We next asked whether decreased cell number in meltrin
␣⫺/⫺ adipose tissue after a high-fat diet is due to impaired
proliferation of adipogenic cells in meltrin ␣⫺/⫺ mice. We
found the most prominent difference in the development of
inguinal WAT between wild-type and meltrin ␣⫺/⫺ mice
after a high-fat diet. Previous studies indicate that inguinal
WAT has a greater proliferation capacity than other WAT,
such as epididymal fat pads (2). To analyze cell proliferation
FIG. 4. A, Ki-67-positive cells in S-V cells after
adipogenic induction. S-V cells from wild-type
(WT) or meltrin ␣⫺/⫺ (Mel.␣⫺/⫺) mice were incubated with or without DIM for 24 h, and Ki67-positive cells were visualized. DAPI stain was
used for nuclei. The microscopic images show representative fields of individual treatments for
three independent experiments. B, The rate of
proliferating cells in S-V cells. Data from five
fields for each well were analyzed. The ratios of
Ki-67-positive cells to DAPI-positive cells were
determined. Experiments were performed in duplicate (three independent experiments were carried out), and values are shown as the mean ⫾
SEM. An asterisk indicates a statistically significant difference (P ⬍ 0.05) between two experimental groups.
Masaki et al. • Role of Meltrin ␣ in Obesity
under adipogenic conditions in vitro, we isolated S-V cells,
which contain preadipocytes and precursors of them, from
inguinal WAT of wild-type and meltrin ␣⫺/⫺ mice. The S-V
cells were propagated to confluence and induced to differentiate into adipocytes in DIM 2 d after confluence. After
24 h, cellular proliferation was assessed by immunofluorescent staining with an antibody against Ki-67 nuclear antigen,
a marker of cellular proliferation, followed by quantification
of the rate of Ki-67-positive cells. The proliferation rates of
cells from wild-type and meltrin ␣⫺/⫺ mice were relatively
low (⬃30%) without DIM. Upon treatment with DIM, the
proliferation rates of wild-type and meltrin ␣⫺/⫺ S-V cells
were enhanced to 65% and 53%, respectively (Fig. 4). This
modestly decreased proliferation rate of meltrin ␣⫺/⫺ S-V
cells after DIM treatment suggests a role for meltrin ␣ in cell
proliferation in vitro.
MEFs lacking meltrin ␣ have a defect(s) in cell proliferation
during adipogenesis
The conversion of MEFs to adipocytes after hormonal
stimulation has been extensively studied as a means of identifying key regulatory factors in adipogenesis (24, 25). We
prepared primary embryonic fibroblasts from wild-type and
meltrin ␣⫺/⫺ mice to examine their adipogenic properties.
The MEFs were induced to differentiate into adipocytes in
DIM. Meltrin ␣⫺/⫺ MEFs showed a modestly reduced capacity to differentiate into adipocytes compared with wildtype MEFs. It is noteworthy that Oil Red-O-negative space
was more pronounced in the culture of meltrin ␣⫺/⫺ MEFs
than in that of wild-type cells (Fig. 6A). Quantification of
triglyceride production confirmed a decreased differentia-
Masaki et al. • Role of Meltrin ␣ in Obesity
tion in meltrin ␣⫺/⫺ MEFs (Fig. 6B). We performed quantitative real-time PCR to examine meltrin ␣ mRNA expression
in each step of adipogenesis. Meltrin ␣ mRNA expression
was transiently increased after induction of adipogenesis
(highest 2 d after adipogenic differentiation) and decreased
during the process of terminal differentiation (Fig. 6C). These
results suggested that meltrin ␣ might have an essential role
in the early steps of adipogenesis, including the production
of adipogenic precursors and their mitotic clonal expansion,
that precede terminal differentiation. To test this idea, we
evaluated the increase in cell numbers during differentiation
in wild-type and meltrin ␣⫺/⫺ MEFs. We found a significant
increase in cell numbers in wild-type, but not meltrin ␣⫺/⫺,
MEFs 3 d after adipogenic differentiation (Fig. 6D). This
impaired increase in the number of cells during adipogenic
induction also suggests a role for meltrin ␣ in cell proliferation, which occurs in the early stages of differentiation and
would affect the number of differentiated adipocytes
produced.
Requirement for meltrin ␣ in autocrine or
juxtacrine signaling
Meltrin ␣ encodes a membrane-anchored metalloprotease.
Some ADAMs participate in the limited proteolysis of various membrane proteins and extracellular matrix proteins.
Meltrin ␣ expressed in MEFs, therefore, may stimulate the
FIG. 5. A, The effect of HB-EGF on adipogenesis.
Primary MEFs from wild-type (WT) mice were
incubated in the absence (⫺) or presence of DIM
or INS with or without HB-EGF. After 8 d of
differentiation, cells were fixed and stained with
Oil Red-O. B, IGFBP-3 expression in MEFs. Endogenous IGFBP-3 mRNA expression in wildtype and meltrin ␣⫺/⫺ was confirmed by RT-PCR.
C, IGFBP-3 cleavage in response to culture in
DIM. Primary MEFs from wild-type or meltrin
␣⫺/⫺ mice were cultured in Opti-MEM reduced
serum medium containing DIM or INS. After 18
or 36 h, the conditioned media were collected, and
full-length IGFBP-3 and degraded IGFBP-3 were
detected by immunoblotting.
Endocrinology, April 2005, 146(4):1752–1763
1759
secretion of some soluble factors into the culture medium
that enhance adipogenesis. Alternatively, meltrin ␣ may degrade inhibitory factors for adipogenesis, thus activating
adipogenesis. We previously showed reduced ectodomain
shedding of HB-EGF in response to phorbol ester stimulation
in meltrin ␣⫺/⫺ MEFs (14). Because HB-EGF is a growth
factor, it might enhance adipogenesis by stimulating cell
proliferation. Addition of soluble HB-EGF to the culture medium of wild-type cells, however, tended to inhibit adipogenesis (Fig. 5A). We also added soluble HB-EGF for the
initial 6 h. In this case, HB-EGF also inhibited adipogenesis
(data not-shown). IGFBP-3 is another candidate for a meltrin
␣ substrate. Proteolysis of IGFBP-3 can enhance adipogenesis
through activation of IGF, which is kept in a latent form when
it is associated with members of the IGFBP family. To test this
model, we next examined IGFBP-3 behavior after adipogenic
induction with DIM or with INS alone. Endogenous IGFBP-3
expression in wild-type MEFs was confirmed by RT-PCR and
Western blot (Fig. 5, B and C). Although IGFBP-3 secreted
into the culture medium was intact during the first 18 h after
adipogenic induction with DIM, both wild-type and meltrin
␣⫺/⫺ MEFs completely cleaved IGFBP-3 within the next 18 h.
IGFBP-3 was not cleaved when adipogenesis was induced by
INS alone (Fig. 5C), suggesting that one or more components
of DIM may play a role in the cleavage of IGFBP-3. We did
not observe the cleavage of IGFBP-5 or IGFBP-6 under the
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Endocrinology, April 2005, 146(4):1752–1763
same conditions (data not shown). Thus, meltrin ␣⫺/⫺ MEFs
cleave IGFBPs as efficiently as do wild-type MEFs, suggesting that meltrin ␣ is not likely to be a key regulator of IGFBP-3
cleavage in response to DIM.
Finally, we asked whether meltrin ␣ plays a regulatory role
in paracrine signaling in adipogenesis. If meltrin ␣ does
modulate paracrine signaling, through either the secretion of
activating factors or the degradation of inhibitory factors,
then wild-type conditioned medium might compensate for
the defect in meltrin ␣⫺/⫺ MEFs. To examine this possibility,
we exchanged the conditioned medium of meltrin ␣⫺/⫺ and
wild-type MEFs every day, except on d 2 and 5, when the
medium was replaced with fresh medium. (Precise protocols
are described in Materials and Methods and summarized in
Table 2.) Surprisingly, medium conditioned by wild-type
cells did not enhance differentiation in meltrin ␣⫺/⫺ MEFs,
FIG. 6. A, Adipogenic differentiation of
primary MEFs prepared from wild-type
and meltrin ␣⫺/⫺ mice. Primary MEFs
from wild-type (WT) or meltrin ␣⫺/⫺
(Mel.␣ ⫺/⫺) mice were incubated in the
absence (⫺) or presence of DIM. (⫺).ME
and DIM.ME, Cultures in conditioned
medium prepared from meltrin ␣⫺/⫺
and wild-type MEFs, respectively. After
8 d of differentiation, cells were fixed
and stained with Oil Red-O. B, Triglyceride accumulation in wild-type and
meltrin ␣⫺/⫺ MEFs after adipogenic induction. Triglyceride accumulation was
expressed as absorbance at an OD of
510 nm. In each experiment, n ⫽ 3, and
values were expressed as the mean ⫾
SEM. Three independent experiments
were carried out, and representative
data are shown. Asterisks indicate a statistically significant difference (P ⬍
0.05) between two experimental groups.
C, Meltrin ␣ mRNA expression in MEFs
during adipogenesis. Total RNA was
prepared from wild-type MEFs, and the
mRNA level of meltrin ␣ was determined by real-time PCR. Levels of
mRNA were normalized to that of
GAPDH. Values from growing cells
were set at 1. In each experiment, n ⫽
3, and values are expressed as the
mean ⫾ SEM. G, Growing cells; GA,
growth-arrested cells; d1, d2, d3, d4, d6,
and d8, 1, 2, 3, 4, 6, and 8 d after adipogenic induction. D, Cell proliferation
of MEFs after adipogenic induction. On
d 0 and 3 after induction with DIM,
primary MEFs from WT or meltrin ␣⫺/⫺
mice were counted. Four independent
experiments were carried out, and values are shown as the mean ⫾ SEM. An
asterisk indicates a statistically significant difference (P ⬍ 0.05) between two
experimental groups.
Masaki et al. • Role of Meltrin ␣ in Obesity
but medium conditioned by meltrin ␣⫺/⫺ MEFs enhanced
adipocyte differentiation in wild-type MEFs (Fig. 6A). This
suggests that although meltrin ␣⫺/⫺ cells secreted some stimulatory factors for adipogenesis into the conditioned medium, they could not use them because of the lack of protease
activity. The implication is that meltrin ␣ mediates autocrine
or juxtacrine signaling for adipogenesis, rather than paracrine signaling.
Discussion
One of the fundamental questions concerning adipogenesis is how adipogenic cell proliferation is regulated during
adipogenesis in the processes of both development and the
progression of obesity. In cell culture, growth-arrested preadipocytes undergo several rounds of mitotic clonal ex-
Masaki et al. • Role of Meltrin ␣ in Obesity
pansion upon adipogenic stimulation and differentiate to
express various adipocyte-specific genes, such as those required for lipid accumulation (26, 27). Evidence suggests that
both cell growth arrest and proliferation are required for
adipogenesis in vitro. The importance of proliferation of preadipocytes and/or mesenchymal stem cells, however, has
tended to be underestimated in studies of obesity. That is
because proliferation proceeds together with differentiation
and hypertrophy during increases in adipose tissue mass in
obesity. Dissection of the individual steps in terms of genes
and molecules will be required to evaluate the contribution
of each step to obesity, and such a dissection will shed new
light on the regulatory mechanisms of weight gain in obesity.
In the present study we demonstrated the role of meltrin
␣ in obesity. Meltrin ␣⫺/⫺ mice were moderately resistant to
the weight gain induced by a high-fat diet. Although the lack
of meltrin ␣ in these mice did not prevent an increase in the
size of adipocytes in their WAT under a high-fat diet, the
high-fat diet did prevent a significant increase in the number
of adipocytes, unlike the situation in wild-type mice. This
inhibition of increase in the number of adipocytes was therefore the gross mechanism of moderate weight gain resistance
in the knockout mice. In contrast, there was no obvious
difference in adipocyte size and number between wild-type
and meltrin ␣⫺/⫺ mice on a normal diet, suggesting normal
formation of adipose tissues. Thus, meltrin ␣ preferentially
regulates adipose cell mass by adding new adipocytes to
WAT, rather than by stimulating differentiation and/or maturation of preexisting adipocytes during obesity. The involvement of meltrin ␣ in the proliferation of preadipocytes
or mesenchymal stem cells during a high-fat diet is supported by the results obtained with S-V cells and MEFs
prepared from meltrin ␣⫺/⫺ mice. Meltrin ␣-lacking cells do
not proliferate as efficiently as wild-type cells in response to
adipogenic stimuli, although they can differentiate into adipocytes. The moderate defects in the proliferation of S-V cells
in meltrin ␣⫺/⫺ adipose tissues suggest that meltrin ␣ participates in the proliferation of subpopulations of S-V cells in
response to DIM treatment. We believe that these cells are
probably DIM-responsive cells in S-V cells. However, we
have not determined what cell types are affected in meltrin
␣⫺/⫺ S-V cells and MEFs, because preadipocytes, which are
determined or destined to give rise to adipocytes, cannot be
distinguished from precursors of preadipocytes and mesenchymal stem cells by the surface markers suitable for FACS
analyses currently available. We would like to continue our
study to determine what kind of cell proliferation is affected
during adipogenic induction in vitro and during high-fat
diet-induced obesity. It is also possible that FBS in growth
medium compensates to some extent for the role of meltrin
␣ in the growth of S-V cells.
To determine whether meltrin ␣⫺/⫺ mice suffered from
any energy imbalance that might explain their resistance to
the development of obesity, we measured their food intake
and glucose metabolism. Meltrin ␣⫺/⫺ mice showed glucose
tolerance similar to that in wild-type mice under either a
normal diet or a high-fat diet. Interestingly, in contrast, increased INS sensitivity was observed in meltrin ␣⫺/⫺ mice
in the normal diet condition, although a slight difference in
INS sensitivity between these mice during a high-fat diet was
Endocrinology, April 2005, 146(4):1752–1763
1761
not statistically significant. Plasma triglyceride and NEFA
levels were also significantly lower in meltrin ␣⫺/⫺ mice than
in wild-type mice only when they were fed a normal diet.
More precise investigation will be necessary to determine
whether the altered metabolic profile of meltrin ␣⫺/⫺ mice
fed a normal diet is associated with the resistance of meltrin
␣⫺/⫺ mice to high-fat diet-induced weight gain and their
decreased proliferation of preadipocytes.
Although we are currently in the process of investigating
metabolic alterations in meltrin ␣⫺/⫺ mice, the marked reductions in intracellular lipid accumulation in their BAT and
livers under a high-fat diet suggest that energy expenditure
might be up-regulated in these tissues. It is especially noteworthy that the liver steatosis seen in wild-type mice after a
high-fat diet was not observed in meltrin ␣⫺/⫺ mice. Meltrin
␣ mRNA expression was up-regulated when animals were
fed a high-fat diet. Thus, meltrin ␣ induced in the liver may
play a critical role in the steatotic liver. Alternatively, the
decreased lipid accumulation in the livers of meltrin ␣⫺/⫺
mice may be caused by a systemic increase in energy expenditure or may reflect the reduced amount of visceral fat
close to the liver in these mice.
Meltrin ␣ is a metalloprotease that belongs to the ADAM
family. The evidence suggests that some of the ADAM proteases are involved in ectodomain shedding of membrane
proteins and limited proteolysis of extracellular matrix proteins. We previously showed impaired ectodomain shedding
of HB-EGF induced by PMA in meltrin ␣⫺/⫺ MEFs (14). The
possible involvement of HB-EGF in adipogenesis has been
reported: for example, the abundant expression of HB-EGF
mRNA in human and mice adipose tissues (28, 29), the induction of HB-EGF with IGF-I during adipogenesis in vitro
(28, 29), and the close link between HB-EGF and vascular
disease in obesity (28). The inhibitory effects of HB-EGF on
adipogenesis of wild-type MEFs excluded the possibility of
involvement of meltrin ␣ in the release of mature soluble
HB-EGF ligands into the culture medium during adipogenesis. These inhibitory effects of HB-EGF are similar to those
of TGF-␣ on adipogenesis (30). In contrast, IGFBP-3 has also
been reported as a substrate of meltrin ␣ in vitro (16, 17).
IGFBP-3 is the most abundant of the six IGFBPs in plasma (31,
32) that transport and modulate the biological actions of
IGF-1, one of the key regulators of adipogenesis and myogenesis (33). Comparison of IGFBP-3 cleavage after adipogenic induction in meltrin ␣⫺/⫺ MEFs and wild-type MEFs
showed no significant difference in cleavage. Therefore, meltrin ␣ is not the major protease that cleaves IGFBP-3 after the
addition of DIM. Many metalloproteases, including matrix
metalloproteases and ADAM28, degrade IGFBP-3 (34 –36),
and some matrix metalloproteases are up-regulated during
adipocyte differentiation (37). These metalloproteases might
contribute to the cleavage of IGFBP-3. Nevertheless, we cannot exclude the possibility that local cleavage of IGFBPs by
meltrin ␣ is necessary for correct signal transduction.
There are different types of intercellular signaling mediated by growth and differentiation factors: paracrine, juxtacrine, and autocrine signaling. In an attempt to determine
which type(s) of signaling is mediated by meltrin ␣, we
performed medium exchange experiments in which wildtype and meltrin ␣⫺/⫺ MEFs were incubated with DIM/
1762
Endocrinology, April 2005, 146(4):1752–1763
maturation medium conditioned by each other’s MEFs. If
meltrin ␣ modulates paracrine signaling, through either the
secretion of activating factors or the degradation of inhibitory factors in the culture medium, we would expect that
wild-type MEF-conditioned medium would enhance the adipogenesis of meltrin ␣⫺/⫺ MEFs and that meltrin ␣⫺/⫺conditioned medium would not enhance the adipogenesis of
wild-type MEFs. Instead, the reverse occurred. These data
suggest that meltrin ␣ is involved in adipogenesis in a cellautonomous process or a process dependent on cell-cell contacts. It is plausible that soluble factors stimulating adipogenesis are not available in meltrin ␣⫺/⫺ MEFs. Meltrin ␣
might mediate the cell-autonomous activation of such soluble factors or the activation of their receptors. In this sense,
we still cannot exclude the possibility that cell-autonomous
and transient activation of HB-EGF or IGF by meltrin ␣ are
necessary for obesity and adipogenesis.
Previous reports suggest that the expression of meltrin ␣
enhances myoblast aggregation (3) or causes morphological
changes in 3T3L1 adipocytes (38). However, we did not observe cell morphological differences between wild-type and
meltrin ␣⫺/⫺ MEFs. Additional investigations are required
to determine whether disintegrin- and cystein-rich domains
play regulatory roles in adipogenesis.
In conclusion, we demonstrated that meltrin ␣ is one of the
key regulators of obesity and adipogenesis. Because meltrin
␣ participates mainly in increasing adipocyte numbers and
not in adipocyte hypertrophy, meltrin ␣⫺/⫺ mice showed
mild resistance to high-fat diet-induced obesity, in which
adipocyte hypertrophy is prominent. Moderate defects in the
proliferation of S-V cells and adipogenesis in MEFs in meltrin
␣⫺/⫺ mice also suggest that meltrin ␣ mainly plays a role in
the proliferation of preadipocytes or mesenchymal stem cells
that give rise to preadipocytes during high-fat diet-induced
obesity and does not play a major role in the differentiation
or cellular hypertrophy of preexisting adipocytes. Interestingly, meltrin ␣⫺/⫺ knockout mice develop WAT similarly to
wild-type mice on a normal diet. It is plausible that the
proliferation of preadipocytes in high-fat diet-induced obesity involves distinct pathways for cells in embryos and
nurslings. Meltrin ␣ might participate mainly in a pathway(s)
critical for high-fat diet-induced adipogenesis in adults, but
not for adipogenesis during development. Alternatively, maternal factors provided in utero and during nursing may
compensate for the defects in WAT development in meltrin
␣⫺/⫺ mice. The roles of meltrin ␣ in adult adipogenesis
suggest novel directions in the development of therapies for
obesity and lipoatrophy.
Acknowledgments
We thank Nobuyuki Itoh, Toshiyuki Asaki, Kazuwa Nakao, and
Hiroaki Masuzaki (Kyoto University) for technical advice and helpful
discussions.
Received August 18, 2004. Accepted December 29, 2004.
Address all correspondence and requests for reprints to: Dr. Atsuko
Sehara-Fujisawa, Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: [email protected].
This work was supported in part by Grants-in-Aid for Scientific
Research (B) from the Ministry of Education, Culture, Sports, Science,
Masaki et al. • Role of Meltrin ␣ in Obesity
and Technology of Japan and research grants from the Ministry of
Health and Welfare of Japan. M.M. was supported by a research fellowship from the Japan Society for the Promotion of Science.
References
1. Moulin K, Truel N, Andre M, Arnauld E, Nibbelink M, Cousin B, Dani C,
Penicaud L, Casteilla L 2001 Emergence during development of the whiteadipocyte cell phenotype is independent of the brown-adipocyte cell phenotype. Biochem J 356:659 – 664
2. Hausman DB, DiGirolamo M, Bartness TJ, Hausman GJ, Martin RJ 2001 The
biology of white adipocyte proliferation. Obes Rev 2:239 –254
3. Yagami-Hiromasa T, Sato T, Kurisaki T, Kamijo K, Nabeshima Y, FujisawaSehara A 1995 A metalloprotease-disintegrin participating in myoblast fusion.
Nature 377:652– 656
4. Blobel CP, Wolfsberg TG, Turck CW, Myles DG, Primakoff P, White JM 1992
A potential fusion peptide and an integrin ligand domain in a protein active
in sperm-egg fusion. Nature 356:248 –252
5. Cho C, Bunch DO, Faure JE, Goulding EH, Eddy EM, Primakoff P, Myles
DG 1998 Fertilization defects in sperm from mice lacking fertilin ␤. Science
281:1857–1859
6. Nishimura H, Cho C, Branciforte DR, Myles DG, Primakoff P 2001 Analysis
of loss of adhesive function in sperm lacking cyritestin or fertilin ␤. Dev Biol
233:204 –213
7. Rooke J, Pan D, Xu T, Rubin GM 1996 KUZ, a conserved metalloproteasedisintegrin protein with two roles in Drosophila neurogenesis. Science 273:
1227–1231
8. Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell
WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ,
Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, Black RA 1998 An
essential role for ectodomain shedding in mammalian development. Science
282:1281–1284
9. Asakura M, Kitakaze M, Takashima S, Liao Y, Ishikura F, Yoshinaka T,
Ohmoto H, Node K, Yoshino K, Ishiguro H, Asanuma H, Sanada S, Matsumura Y, Takeda H, Beppu S, Tada M, Hori M, Higashiyama S 2002 Cardiac
hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF:
metaloprotease inhibitors as a new therapy. Nat Med 8:35– 40
10. Van Eerdewegh P, Little RD, Dupuis J, Del Mastro RG, Falls K, Simon J,
Torrey D, Pandit S, McKenny J, Braunschweiger K, Walsh A, Liu Z, Hayward
B, Folz C, Manning SP, Bawa A, Saracino L, Thackston M, Benchekroun Y,
Capparell N, Wang M, Adair R, Feng Y, Dubois J, FitzGerald MG, Huang
H, Gibson R, Allen KM, Pedan A, Danzig MR, Umland SP, Egan RW, Cuss
FM, Rorke S, Clough JB, Holloway JW, Holgate ST, Keith TP 2002 Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness.
Nature 418:426 – 430
11. Black R, White JM 1998 ADAMs: focus on the protease domain. Curr Opin
Cell Biol 10:654 – 659
12. Seals DF, Courtneidge SA 2003 The ADAMs family of metalloproteases:
multidomain proteins with multiple functions. Genes Dev 17:7–30
13. Werb Z, Yan Y 1998 A cellular striptease act. Science 282:1279 –1280
14. Kurisaki T, Masuda A, Sudo K, Sakagami J, Higashiyama S, Matsuda Y,
Nagabukuro A, Tsuji A, Nabeshima Y, Asano M, Iwakura Y, FujisawaSehara A 2003 Phenotypic analysis of meltrin ␣ (ADAM12)-deficient mice:
involvement of meltrin ␣ in adipogenesis and myogenesis. Mol Cell Biol
23:55– 61
15. Kronqvist P, Kawaguchi N, Albrechtsen R, Xu X, Schroder HD, Moghadaszadeh B, Nielsen FC, Frohlich C, Engvall E, Wewer UM 2002 ADAM12
alleviates the skeletal muscle pathology in mdx dystrophic mice. Am J Pathol
161:1535–1540
16. Loechel F, Fox JW, Murphy G, Albrechtsen R, Wewer UM 2000 ADAM 12-S
cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3. Biochem Biophys Res
Commun 278:511–515
17. Shi Z, Xu W, Loechel F, Wewer UM, Murphy LJ 2000 ADAM12, a disintegrin
metalloprotease, interacts with insulin-like growth factor-binding protein-3.
J Biol Chem 275:18574 –18580
18. Hinkle CL, Sunnarborg SW, Loiselle D, Parker CE, Stevenson M, Russell
WE, Lee DC 2004 Selective roles for TACE/ADAM17 in the shedding of the
epidermal growth factor receptor ligand family. The juxtamembrane stalk
determines cleavage efficiency. J Biol Chem 279:24179 –24188
19. Shirakabe K, Wakatsuki S, Kurisaki T, Sehara-Fujisawa A 2001 Roles of
meltrin ␤ (ADAM19) in the processing of neuregulin. J Biol Chem 276:9352–
9358
20. Mori S, Tanaka M, Nanba D, Nishiwaki E, Ishiguro H, Higashiyama S,
Matsuura N 2003 PACSIN3 binds ADAM12/meltrin ␣ and up-regulates
ectodomain shedding of heparin-binding epidermal growth factor-like growth
factor. J Biol Chem 278:46029 – 46034
21. Kurohara K, Komatsu K, Kurisaki T, Masuda A, Irie N, Asano M, Sudo K,
Nabeshima Y, Iwakura Y, Sehara-Fujisawa A 2004 Essential roles of meltrin
␤ (ADAM19) in heart development. Dev Biol 267:14 –28
22. Kawaguchi N, Xu X, Tajima R, Kronqvist P, Sundberg C, Loechel F, Albrechtsen R, Wewer UM 2003 ADAM12 protease induces adipogenesis in transgenic mice. Am J Pathol 160:1895–1903
Masaki et al. • Role of Meltrin ␣ in Obesity
23. Picard F, Gehin M, Annicotte J, Rocchi S, Champy MF, O’Malley BW,
Chambon P, Auwerx J 2002 SRC-1 and TIF2 control energy balance between
white and brown adipose tissues. Cell 111:931–941
24. Tanaka T, Yoshida N, Kishimoto T, Akira S 1997 Defective adipocyte differentiation in mice lacking the C/EBP␤ and/or C/EBP␦ gene. EMBO J 16:
7432–7443
25. Sakaue H, Konishi M, Ogawa W, Asaki T, Mori T, Yamasaki M, Takata M,
Ueno H, kato S, Kasuga M, Itoh N 2002 Requirement of fibroblast growth
factor 10 in development of white adipose tissue. Genes Dev 16:908 –912
26. Tang Q-Q, Otto TC, Lane MD 2002 Mitotic clonal expansion: A synchronouse
process required for adipogenesis. Proc Natl Acad Sci USA 100:44 – 49
27. Tang Q-Q, Otto TC, Lane MD 2003 CCAAT/enhancer-binding protein ␤ is
required for mitotic clonal expansion during adipogenesis. Proc Natl Acad Sci
USA 100:850 – 855
28. Matsumoto S, Kishida K, Shimomura I, Maeda N, Nagaretani H, Matsuda M,
Nishizawa H, Kihara S, Funahashi T, Matsuzawa Y, Yamada A, Yamashita S,
Tamura S, Kawata S 2002 Increased plasma HB-EGF associated with obesity and
coronary artery disease. Biochem Biophys Res Commun 292:781–786
29. Mulligan C, Rochford J, Denyer G, Stephens R, Yeo G, Freeman T, Siddle
K, O’Rahilly S 2002 Microarray analysis of insulin and insulin-like growth
factor-1 (IGF-1) receptor signaling reveals the selective up-regulation of the
mitogen heparin-binding EGF-like growth factor by IGF-1. J Biol Chem 277:
42480 – 42487
30. Luetteke NC, Lee DC, Palmiter RD, Brinster RL, Sandgren EP 1993 Regulation of fat and muscle development by transforming growth factor ␣ in
transgenic mice and in cultured cells. Cell Growth Differ 4:203–213
31. Baxter RC 2001 Signalling pathways involved in antiproliferative effects of
IGFBP-3: a review. Mol Pathol 54:145–148
Endocrinology, April 2005, 146(4):1752–1763
1763
32. Modric T, Silha JV, Shi Z, Gui Y, Suwanichkul A, Durham SK, Powell DR,
Murphy LJ 2001 Phenotypic manifestations of insulin-like growth factorbinding protein-3 overexpression in transgenic mice. Endocrinology 142:1958 –
1967
33. Sordella R, Jiang W, Chen GC, Curto M, Settleman J 2003 Modulation of Rho
GTPase signaling regulates a switch between adipogenesis and myogenesis.
Cell 113:147–158
34. Sadowski T, Dietrich S, Koschinsky F, Sedlacek R 2003 Matrix metalloproteinase 19 regulates insulin-like growth factor-mediated proliferation, migration, and adhesion in human keratinocytes through proteolysis of insulin-like
growth factor binding protein-3. Mol Biol Cell 14:4569 – 4580
35. Manes S, Llorente M, Lacalle RA, Gomez-Mouton C, Kremer L, Mira E,
Martinez AC 1999 The matrix metalloproteinase-9 regulates the insulin-like
growth factor-triggered autocrine response in DU-145 carcinoma cells. J Biol
Chem 274:6935– 6945
36. Mochizuki S, Shimoda M, Shiomi T, Fujii Y, Okada Y 2004 ADAM28 is
activated by MMP-7 (matrilysin-1) and cleaves insulin-like growth factor binding protein-3. Biochem Biophys Res Commun 315:79 – 84
37. Chavey C, Mari B, Monthouel MN, Bonnafous S, Anglard P, Van Obberghen
E, Tartare-Deckert S 2003 Matrix metalloproteinases are differentially expressed in adipose tissue during obesity and modulate adipocyte differentiation. J Biol Chem 278:11888 –11896
38. Kawaguchi N, Sundberg C, Kveiborg M, Moghadaszadeh B, Asmar M,
Dietrich N, Thodeti CK, Nielsen FC, Moller P, Mercurio AM, Albrechtsen
R, Wewer UM 2003 ADAM12 induces actin cytoskeleton and extracellular
matrix reorganization during early adipocyte differentiation by regulating ␤1
integrin function. J Cell Sci 116:3893–3904
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