From the a Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan, b CREST of Japan Science and Technology Corporation, Japan, d Biological Research Laboratories, Nissan Chemical Industries, Saitama 349-0294, Japan, the e Central Institute for Experimental Animals, Kanagawa 216-0001, Japan, the f Division of Laboratory Animal Science, Animal Research Center, Tokyo Medical University, Tokyo 160-8402, Japan, g Central Research Laboratories, Kyorin Pharmaceutical, Tochigi 329-0114, Japan, the h Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan, and the i Institute of Biology-CNRS, Pasteur Institute of Lille, UPRES A8090, 59000 Lille, France
Received for publication, September 4, 2002, and in revised form, November 5, 2002
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ABSTRACT |
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The adipocyte-derived hormone adiponectin has
been shown to play important roles in the regulation of energy
homeostasis and insulin sensitivity. In this study, we analyzed
globular domain adiponectin (gAd) transgenic (Tg) mice crossed with
leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite
an unexpected similar body weight, gAd Tg ob/ob mice showed
amelioration of insulin resistance and Obesity is defined as increased mass of adipose tissue, conferring
a higher risk of cardiovascular and metabolic disorders such as
diabetes, hyperlipidemia, and coronary heart disease (1, 2). However,
the molecular basis for that association remains to be elucidated. The
adipose tissue itself serves as the site of triglyceride
(TG)1 storage and free fatty
acid (FFA)/glycerol release in response to changing energy demands (1).
It also participates in the regulation of a wide variety of energy
homeostasis as an important endocrine organ that secretes a number of
biologically active substances (1, 3) called adipokines (4) such
as FFA (5), adipsin (6), leptin (7), plasminogen activator inhibitor-1 (PAI-1) (8), resistin (9), and tumor necrosis factor- Adiponectin or Acrp30 (11-14) is an adipocyte-derived hormone with
multiple biological functions. Dr. Matsuzawa and co-workers (15, 16)
have reported that adiponectin may have putative anti-atherogenic
properties in vitro. Dr. Scherer and co-workers (17, 18)
have reported that an acute increase in circulating Acrp30 levels
lowers hepatic glucose production. Dr. Lodish and co-workers (19) have
reported that a globular Acrp30 increases fatty acid oxidation in
muscle and causes weight loss in mice. We have also shown that
treatment with recombinant adiponectin increased fatty acid oxidation
in muscle, thereby ameliorating insulin resistance in obese mice (20).
Moreover, we have shown that insulin resistance in lipoatrophic mice
was completely reversed by the combination of physiological doses of
adiponectin and leptin, but only partially by either adiponectin or
leptin alone (20). These observations suggested that leptin and
adiponectin may be two major insulin-sensitizing hormones secreted from
adipose tissue; however, it has not yet been clarified whether leptin
and adiponectin have distinct or overlapping functions in the
regulation of insulin sensitivity. Moreover, in relation to regulation
of body weight, leptin has been shown to play a major role in the
regulation of body weight, whereas the role of adiponectin in this
process has been controversial (19, 20). Most recently,
adiponectin-deficient mice were reported to display insulin resistance,
glucose intolerance (21, 22), and increased neointimal formation (21).
However, it remains to be determined whether overexpression of
adiponectin could indeed ameliorate diabetes and atherosclerosis
in vivo.
To examine whether overexpression of adiponectin is protective
against diabetes and atherosclerosis in vivo, we analyzed
globular adiponectin (gAd) transgenic (Tg) mice crossed with
leptin-deficient ob/ob mice (7) or with a well established animal model
of atherosclerosis, apoE-deficient mice (23, 24), since treatment with
gAd for 2 weeks had been previously shown to ameliorate insulin
resistance more potently than full-length (20). gAd Tg ob/ob mice
showed amelioration of diabetes and insulin resistance. Moreover, gAd Tg ob/ob mice showed increased plasma insulin levels in response to
glucose despite amelioration of insulin resistance, which appeared to
be due to the amelioration of Thus, adiponectin could compensate for leptin deficiency at least in
part in insulin resistance and Generation of Transgenic Mice Expressing Adiponectin--
A
fusion gene was designed, comprising the human serum amyloid P
component (SAP) promoter (25) and mouse globular adiponectin cDNA coding sequences (Fig. 1A), so that the hormone
expression be targeted to the liver (Fig. 1C), since there
is the possibility that overexpression of a hormone such as leptin can
induce complete disappearance of its original site of production, the
adipose tissue (25). The purified HindIII-XhoI
fragment was microinjected into the pronucleus of fertilized C57BL6
mice (Nippon CREA, Tokyo, Japan) eggs. Transgenic founder mice were
identified by Southern blot analysis of tail DNAs using the cDNA
probe to the BglII/HincII site in globular
adiponectin. Transgenic mice were used as heterozygotes.
Generation of gAd Tg ob/ob or ApoE-deficient Mice--
The gAd
Tg, ob/ob (7) and apoE-deficient mice (23, 24) used in this study were
all on a B6 background. To generate gAd Tg ob/+ or
apoE+/ Animals, Blood Sample Assays, and in Vivo Glucose
Homeostasis--
All the experiments in this study were performed
using male mice unless otherwise stated. Male mice 8 weeks of age were
fed an indicated powdered diet for indicated time periods according to
previously described methods (20). For example, our high fat diet
contains oil, 1152 g (from Benibana, Japan; safflower oil (high
oleic type) contained 46% oleic acid (18:1n-9) and 45% linoleic acid
(18:2n-6) from total fatty acids); casein, 1191.6 g (from Oriental
Yeast, No. 19); sucrose, 633.6 g (from Oriental Yeast, No. 13); vitamin
mixture, 50.4 g (from Oriental Yeast, No. 20 (AIN76); mineral
mixture, 352.8 g (from Oriental Yeast, No. 25 (AIN76); cellulose
powder, 201.6 g (from Oriental Yeast, No. 19);
DL-methionine, 18 g (from Wako Pure Chemicals); water, 360 ml; Total, 3600 g. Cumulative food intake was measured using transgenic and nontransgenic littermates daily over a 2-week period. For some experiments, the same amounts of food were given to the pair-fed group of gAd Tg ob/ob mice as to nontransgenic ob/ob littermates. The Tokyo University Graduate School of Medicine Committee
on Animal Research approved all experimental procedures. The glucose
tolerance and insulin tolerance tests were carried out according to
previously described methods (20). Plasma glucose, serum FFA, TChol,
and TG levels were determined by the glucose B-test, NEFA C-test, TChol
E-type, and TG L-type (Wako Pure Chemical Industries), respectively.
For analysis of lipoprotein distribution, pooled serum samples from
five mice per group were subjected to high performance liquid
chromatography (HPLC) (SRL). Plasma insulin was measured by an insulin
immunoassay (Morinaga Institute of Biological Science, Yokohama, Japan)
(20). Plasma leptin and adiponectin levels were determined by a
Quintikine M kit (R & D Systems Inc.) and mouse adiponectin
radioimmunoassay (RIA) kit (LINCO Research Inc.), respectively
(21).
Northern Blot Analysis and Immunoblotting--
Total RNA was
subjected to Northern blot analysis with the probes for rat ACO (Dr. T. Hashimoto) or mouse UCP2 (Dr. K. Motojima), or mouse UCP3 or
adiponectin cDNA (20). The radioactivity in each band was
quantified as described (20), and the fold change in each mRNA was
calculated after correction for loading differences by measuring the
amount of 28 S rRNA. Plasma adiponectin levels were determined by
immunoblotting as described (20). Representative data from one of more
than three independent experiments are shown.
Histological and Immunohistochemical Analysis of Islets--
20
sections of islets were evaluated for morphometry. The isolated
pancreas was immersion-fixed in Bouin's solution at 4 °C overnight.
Tissues were routinely processed for paraffin-embedding, and 4-µm
sections were cut and mounted on silanized slides. Pancreatic sections
were double-stained with anti-insulin (brown) and cocktails of
anti-glucagon, anti-somatostatin, and anti-pancreatic polypeptide antibodies (red). The amounts of Islet Isolation, Lipid Metabolism and Measurement of Tissue TG
Content--
Measurements of [14C]CO2
production from [1-14C]palmitic acid were performed using
liver and muscle slices, as described (20). Liver and muscle
homogenates were extracted, and their TG content was determined as
described previously (20).
Measurement of PPAR Quantitative Analyses of Aortic Atherosclerotic Lesions--
En
face Sudan IV staining of the excised aortas from the arch to the
common iliac levels was performed after fixation in phosphate-buffered 10% formaldehyde (28). Percentages of en face Sudan IV-positive areas
to total aortic areas were calculated. Quantitative analyses were
performed by computer-assisted planimetry using NIH Image software.
Lesion Analysis in the Aortic Valve and
Immunohistochemistry--
Atherosclerotic lesions were quantified in
the aortic valve of each mouse as described previously (29). Briefly,
the OCT-embedded, frozen aortic valves were sectioned serially at
10-µm thickness for a total of 300 µm beginning at the base of the
aortic valve, where all three leaflets are first visible. Every fourth
section for a total of five sections from each animal was stained with Oil-Red O to identify the lipid-rich lesions. The mouse aortic valve
lesions were analyzed immunohistochemically with the following antibodies: anti-mouse macrophage Mac-3 (PharMingen) and anti-mouse SRA
2F8 (Serotec). To determine the proportion of SRA-positive macrophages
for each animal, the total number of cells positive for Mac-3 or SRA in
atherosclerotic plaques of the aorta was counted for each section. To
avoid bias, two investigators who were unaware of the type of staining
or assignment of group were asked to determine the proportion of
SRA-positive macrophages.
Generation of Transgenic Mice Expressing Globular
Adiponectin--
To elucidate the metabolic consequences of increased
effects of adiponectin on a long-term basis in vivo, we
produced transgenic mice with elevated plasma concentrations of
globular adiponectin (gAd). A fusion gene was designed, comprising the
human SAP promoter (25) and mouse globular adiponectin
cDNA coding sequences, so that the hormone expression might be
targeted to the liver (Fig. 1A). Several transgenic lines
on a C57BL6 background (B6) with different copy numbers of the
transgene were obtained (Fig. 1B). The 4.5-kb band
corresponded to the endogenous gene and the 0.5-kb band to the gAd
transgene (Fig. 1B). Northern blot analysis identified a
single mRNA species found in the liver from gAd (Fig.
1C), but not in other tissues we studied (data not shown).
Plasma gAd concentrations were elevated significantly in transgenic
mice in proportion to the transgene copy number (Fig. 1D).
In transgenic mice carrying 10 copies of the gAd transgene (Fig.
1B), plasma gAd levels in transgenic mice were approximately
one-tenth of plasma full-length adiponectin levels in nontransgenic
littermates (data not shown). Transgenic mice overexpressing gAd were
viable throughout adulthood with no appreciable complications.
gAd Tg Mice Showed Amelioration of Insulin Resistance and
Hyperglycemia under the HF Diet--
During postnatal development, no
significant differences in body weight (wild-type: 20.8 ± 1.4 g; gAd Tg: 20.1 ± 1.1 g, male mice 8 weeks of age),
linear growth and histology (data not shown) were observed between
nontransgenic control B6 and gAd Tg mice. The plasma adiponectin
(wild-type: 11.4 ± 1.7 µg/ml; gAd Tg: 12.8 ± 1.2 µg/ml)
and leptin levels (wild-type: 4.5 ± 0.7 ng/ml; gAd Tg: 4.1 ± 0.9 ng/ml) were not significantly different. To elucidate the long
term effects of globular adiponectin on glucose metabolism, we measured
plasma glucose and insulin concentrations in transgenic mice
overexpressing globular adiponectin. No significant differences in
plasma glucose and insulin concentrations were noted between gAd Tg
mice and nontransgenic littermates on a high carbohydrate (HC) diet
(data not shown).
Even on an HF diet, there were no significant differences in body
weight between gAd Tg mice and nontransgenic littermates (data not
shown). Glucose and insulin tolerance tests were performed using gAd Tg
and nontransgenic littermates on the HF diet. After intraperitoneal
glucose injection, plasma glucose and insulin levels were significantly
lower in gAd Tg mice than those in nontransgenic littermates (Fig. 1,
E and F). When mice were injected with insulin, the hypoglycemic response was significantly exaggerated in gAd Tg mice
as compared with nontransgenic littermates on the HF diet (Fig.
1G). gAd Tg mice showed reduced TG content (5) in skeletal muscle (Fig. 1H) and in the liver (Fig. 1I),
which was associated with improved insulin resistance (Fig.
1G). These observations suggest that overexpression of
globular adiponectin increased glucose tolerance and insulin
sensitivity under the HF diet.
gAd Tg ob/ob Mice Showed Almost the Same Body Weight as ob/ob
Mice--
To examine whether there is any functional redundancy
between leptin and adiponectin, we analyzed gAd Tg mice crossed with leptin-deficient ob/ob mice on a B6 background (Fig.
2A). Overexpression of gAd had
no effect on obesity observed in ob/ob mice allowed free access to food
(Fig. 2B); however, food intake was markedly increased in
gAd Tg ob/ob mice (~130% of that in nontransgenic ob/ob littermates)
(Fig. 2C). Pair-feeding revealed that overexpression of
globular adiponectin indeed markedly reduced body weight gain of ob/ob
mice (Fig. 2D). These data suggest that overexpression of
globular adiponectin primarily increased energy expenditure, and
consequently increased food intake.
We next studied whether overexpression of globular adiponectin could
compensate for leptin deficiency in hyperlipidemia. gAd Tg ob/ob mice
showed significantly decreased serum FFA (Fig. 2E) and TG
levels (Fig. 2F) as compared with ob/ob mice.
gAd Tg ob/ob Mice Were Partially Protected from Diabetes, Which Was
Associated with Increased Insulin Sensitivity and Secretion--
We
studied whether overexpression of adiponectin could compensate for
leptin deficiency in insulin resistance and gAd Tg ob/ob Mice Showed Increased Expression of Molecules Involved
in Fatty Acid Oxidation and Energy Dissipation and Increased Fatty Acid
Oxidation in Skeletal Muscle--
To determine the mechanisms by which
hypolipidemic and anti-diabetic effects can be achieved by
overexpression of gAd in ob/ob mice on the HF diet, we examined its
effects in individual target organs. Overexpression of gAd in ob/ob
mice significantly increased expression of molecules involved in fatty
acid oxidation such as ACO (Fig.
4E), and molecules involved in
energy dissipation such as UCP2 (Fig. 4F) and UCP3 (Fig.
4G), and indeed increased fatty acid oxidation in skeletal
muscle (Fig. 4H), but not in the liver (Fig. 4,
A-C). These alterations in skeletal muscle significantly
decreased tissue TG content (5) in skeletal muscle (Fig. 4I)
associated with decreased serum FFA (Fig. 2E) and TG levels
(Fig. 2F), leading to decreased tissue TG content in the liver (Fig. 4D) and decreased insulin resistance (Fig.
3A) in gAd Tg ob/ob mice. Although decrease of tissue TG
content can be explained by the decrease of TG synthesis and/or the
increase of fatty acid oxidation, there were no significant differences in expression levels of SREBP1c between gAd Tg mice and nontransgenic littermates (data not shown), suggesting that the latter mechanism may
be dominant in the action of adiponectin.
gAd Increased PPAR gAd Tg ApoE-deficient Mice Were Partially Protected from the
Atherosclerosis Associated with ApoE-deficient
Mice--
ApoE-deficient mice are hypercholesterolemic and
spontaneously develop severe atherosclerosis (23, 24). To examine the effect of overexpression of globular adiponectin on atherosclerotic lesion development in vivo, we crossed gAd Tg mice with
apoE-deficient mice on a B6 background, and compared the extent of
resultant atherosclerotic lesions to that in control apoE-deficient
mice (Fig. 5, A-C). The en
face Sudan IV-positive lesion areas of the arch and the descending
aorta in gAd Tg apoE-deficient mice were significantly smaller than in
nontransgenic apoE-deficient littermates (Fig. 5, A-C).
Thus, overexpression of globular adiponectin resulted in marked
reduction of atherosclerotic lesion formation.
Although overexpression of globular adiponectin significantly
ameliorated hyperlipidemia, insulin resistance and diabetes induced by
HF diet or leptin deficiency, on the other hand, body weight, plasma
glucose (Table I), lipoprotein profiles
(Fig. 5D), and serum total cholesterol, HDL cholesterol,
FFA, TG (Table I), and plasma apoB concentrations (data not shown) were
not significantly different between gAd Tg apoE-deficient and their control apoE-deficient mice. These data raised the possibility that the
protective effect of globular adiponectin may be a direct consequence
of adiponectin action on the vascular wall and/or macrophages rather
than an indirect consequence of altered conventional atherosclerotic
risk factors in vivo.
Globular Adiponectin Reduced Lipid Accumulation and Inhibited the
Progression of Atherosclerosis, Which Was Associated with Decreased
Expressions of SRA and TNF Adiponectin Can Ameliorate Insulin Resistance and
The major difference between this study and the previous studies is
that the previous studies showed the effects of recombinant globular
adiponectin administration only for 2 weeks, whereas this study showed
the effects of chronic elevation of plasma globular adiponectin levels
for up to 20 weeks by establishing the globular adiponectin transgenic
mice. By using these transgenic mice, we show two new observations on
the function of globular adiponectin in this study. First, this study
provide the first demonstration that gAd Tg ob/ob mice unexpectedly
failed to show amelioration of obesity presumably due to compensatory
increase in food intake for increased energy expenditure. Leptin has
been shown to reduce food intake and increase energy expenditure (7).
Our study clearly revealed that adiponectin could not suppress food
intake, however, pair-feeding experiments revealed that adiponectin may increase energy expenditure. It is a very important issue where the
excess energy goes in the gAd Tg mice allowed free access to food,
which are relatively hyperphagic, yet have similar body weights to the
control mice. These may be explained at least in part by the
significantly increased UCP2 (Fig. 4F) and UCP3 (Fig. 4G) expressions in skeletal muscle of gAd Tg mice. These
observations suggested that leptin and adiponectin may have both
overlapping and distinct functions. Second, this study provide the
first demonstration that gAd Tg ob/ob mice showed not only increased
insulin sensitivity but increased plasma insulin levels during glucose
tolerance test, which was associated with increased insulin content. In
contrast, treatment of ob/ob mice with PPAR
We have recently reported that decreased adiponectin levels, whether
due to genetic factors such as variations of adiponectin gene itself
(32) or environmental factors such as HF diet (20), can significantly
contribute to the development of type 2 diabetes. Conversely, we have
now demonstrated that adiponectin treatment could reverse type 2 diabetes due to amelioration of insulin resistance and impaired insulin
secretion. Our study indicated that adiponectin itself or an activator
or inducer of adiponectin has the potential to be used as an
anti-diabetic agent. In this context, the PPAR gAd May Stimulate Fatty Acid Oxidation Directly in Skeletal Muscle
but Not in the Liver--
The results of this study also suggest that
gAd increases molecules involved in fatty acid oxidation such as ACO
and stimulates fatty acid oxidation in skeletal muscle but not in the
liver (Fig. 4) and that this effect may be due to the direct action of
globular adiponectin to increase in PPAR Adiponectin Can Protect from Atherosclerosis in Vivo Independent of
Conventional Atherogenic Risk Factors--
In this study, we provided
the first direct evidence that overexpression of globular adiponectin
could inhibit the progression of atherosclerosis. gAd Tg apoE-deficient
mice failed to show any significant differences in glucose and lipid
levels under a hypercholesterolemic state (Table I and Fig.
5D), strongly suggesting that the protective effect of
globular adiponectin may be a direct consequence of adiponectin action
on the vascular wall and/or macrophages rather than an indirect
consequence of altered conventional atherosclerotic risk factors
in vivo. In this respect, Dr. Matsuzawa and co-workers (15,
16) has reported that adiponectin may have putative anti-atherogenic
properties in vitro. In this study, we showed the potential
molecular mechanisms by which globular adiponectin attenuated
atherosclerosis in apoE-deficient mice in vivo. Although
globular adiponectin had little effect on expression of ICAM-1, we
found that it suppressed expressions of class A scavenger receptor and
TNF
In conclusion, replenishment of globular adiponectin may provide a
novel treatment modality for both type 2 diabetes and atherosclerosis in that this agent has a direct anti-atherogenic effect, in addition to
anti-diabetic and anti-hyperlipidemic effects.
-cell degranulation as well
as diabetes, indicating that globular adiponectin and leptin appeared
to have both distinct and overlapping functions. Amelioration of
diabetes and insulin resistance was associated with increased
expression of molecules involved in fatty acid oxidation such as
acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in
skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma
glucose and lipid levels on an apoE-deficient background, gAd Tg
apoE-deficient mice showed amelioration of atherosclerosis, which was
associated with decreased expression of class A scavenger receptor and
tumor necrosis factor
. This is the first demonstration that
globular adiponectin can protect against atherosclerosis in
vivo. In conclusion, replenishment of globular adiponectin may
provide a novel treatment modality for both type 2 diabetes and atherosclerosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF
) (10).
-cell degranulation. Amelioration of
diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as
acyl-CoA oxidase (ACO) and molecules involved in energy dissipation such as uncoupling protein (UCP) 2 and 3, and increased fatty acid
oxidation in skeletal muscle of gAd Tg ob/ob mice. Unexpectedly, they
did not show amelioration of obesity presumably due to compensatory increase in food intake for increased energy expenditure. Furthermore, despite similar plasma glucose and lipid levels on an apoE-deficient background, we showed that gAd Tg apoE-deficient mice showed
amelioration of atherosclerosis, which was associated with decreased
expression of class A scavenger receptor and TNF
.
-cell degranulation, but not in
obesity, indicating that adiponectin and leptin appeared to have both
distinct and overlapping functions. Moreover, we showed that gAd may
stimulate fatty acid oxidation directly only in skeletal muscle but not
in the liver. Furthermore, we provide the first evidence that
adiponectin has anti-atherogenic properties in vivo
independent of conventional atherogenic risk factors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mice, conceptuses that were obtained by in
vitro fertilization of ova from ob/+ or apoE
/
female mice and sperm from gAd Tg male mice were implanted into pseudopregnant foster mothers, as previously described (26). No
significant differences in the body weight, glucose and lipid metabolism, and atherosclerosis were observed between wild-type mice
and ob/+ or apoE+/
mice (data not shown). Then, to
generate gAd Tg ob/ob or apoE
/
mice, conceptuses that
were obtained by in vitro fertilization of ova from ob/+ or
apoE+/
female mice and sperm from the resultant gAd Tg
ob/+ or apoE+/
male mice were implanted into
pseudopregnant foster mothers.
-cells and non-
-cells were calculated as the proportions of the area of
-cells or
non-
-cells, assessed by immunostaining, to the area of the whole
pancreas. More than 50 islets were analyzed per mouse in each group.
-Cell Preparation, and Analysis of
Insulin Content--
Isolation of islets from mice was carried out as
described previously (27). In brief, after clamping the common bile
duct at a point close to the duodenum outlet, 2.5 ml of Krebs-Ringer bicarbonate buffer (27) containing 10 mg of collagenase (Sigma) was
injected into the duct. The swollen pancreas was taken out and
incubated at 37 °C for 3 min. The pancreas was dispersed by pipetting and washed twice with Krebs-Ringer bicarbonate buffer. Islets
were collected by manual picking. Single cells were isolated with
trypsin/EDTA (Invitrogen) as previously described (27) with some
modification. Isolated islets were extracted in acid ethanol at
20 °C, and their insulin content was measured by RIA.
Ligand Activity--
Bacterially
expressed murine globular adiponectin (gAd) were purified as previously
described (20). gAd produced in a mammalian expression system were
isolated and purified from NIH-3T3 cells that were stably expressing
gAd, as previously described (17). ActiClean Etox affinity columns
(Sterogene Bioseparations) were used to remove potential endotoxin
contaminations. No significant differences in the PPAR
ligands
activities in C2C12 myocytes and isolated hepatocytes were observed
between the bacterially expressed gAd and the gAd produced by the
mammalian expression system (data not shown). Differentiated C2C12
myocytes (19) or isolated hepatocytes (17) were treated with the
indicated concentrations of adiponectin. PPAR
ligands activities
were quantitatively determined using a (UAS)x4-tk-LUC reporter plasmid,
a GAL4-rat PPAR
ligand-binding domain expression plasmid, and a
-galactosidase expression plasmid as the internal control as
described previously (26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Generation of transgenic mice overexpressing
adiponectin. A, schematic representation of the human
SAP promoter/mouse gAd cDNA fusion gene. Stippled
blocks, polyadenylation signal. The DNA fragment used as probe A
for Southern blotting is also shown under A. B,
Southern blot analysis of BamHI/EcoRI-digested
mouse genomic DNA from WT, gAd Tg mice hybridized with probe A. The
4.5-kb band corresponds to the endogenous gene and the 0.5-kb band to
the gAd transgene. C, Northern blot analyses of total RNA
from the liver of each genotype. Aliquots of total RNA (10 µg) were
hybridized with the cDNA probe to the
BglII/HincII site in globular adiponectin.
D, immunoblot of plasma from WT and gAd Tg mice using the
anti-C-terminal portion of adiponectin antibody (20). E-G,
plasma glucose levels (E) and plasma insulin levels
(F) during the glucose tolerance test (1.5 g of glucose/kg
of body weight); plasma glucose levels during ITT (G); TG
content in skeletal muscle (H) or in the liver
(I) of WT and gAd Tg mice on the HF diet. Male mice 8 weeks
of age were fed the HF diet for 4 weeks and then analyzed. The results
are expressed as the percentage of the value of untreated mice
(G). Each bar represents the mean ± S.E.
(n = 5-10). *, p < 0.05; **,
p < 0.01; transgenic versus
nontransgenic.
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Fig. 2.
Adiponectin could partially compensate for
leptin deficiency for hyperlipidemia, but not for obesity.
A, immunoblot of plasma from WT, ob/ob, and gAd Tg ob/ob
using the anti-C-terminal portion of adiponectin antibody (20).
B-F, body weight (B), food intake
(C), body weight gain (D), serum FFA levels
(E), and serum TG levels (F) of WT and gAd Tg
ob/ob mice and nontransgenic ob/ob littermates allowed free access to
food (A-C, E, F) on the HF diet. Male
mice 8 weeks of age were fed the HF diet for 4 weeks and then analyzed.
The same amounts of food were given to the pair-fed group of gAd Tg
ob/ob mice as to nontransgenic ob/ob littermates (D). Each
bar represents the mean ± S.E. (n = 5-14). *, p < 0.05; **, p < 0.01;
transgenic versus nontransgenic.
-cell degranulation. gAd
Tg ob/ob mice showed significantly increased insulin sensitivity (Fig.
3A) and increased glucose
tolerance (Fig. 3B) as compared with ob/ob mice on the HF
diet. Unexpectedly, gAd Tg ob/ob mice showed increased plasma insulin
levels during the glucose tolerance test (GTT) as compared with
nontransgenic ob/ob littermates (Fig. 3C). Interestingly,
gAd Tg ob/ob mice showed increased insulin immunoreactivity (Fig.
3D) and increased insulin content (Fig. 3, E and
F) as compared with nontransgenic ob/ob littermates. Protection from
-cell degranulation may be a consequence of the decreased insulin requirement caused by increased insulin sensitivity in gAd Tg ob/ob mice as well as direct effect of adiponectin on
-cell. The effect of overexpression of globular adiponectin on the
amelioration of diabetes in ob/ob mice was more pronounced on the HF
diet than on the HC diet, although there was a tendency for an
amelioration of diabetes even on the HC diet (data not shown).
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Fig. 3.
Adiponectin could partially compensate for
leptin deficiency for diabetes, which was associated with increased
insulin sensitivity and secretion. A-F, plasma
glucose levels during ITT (A), plasma glucose levels
(B), and plasma insulin levels (C) during the
glucose tolerance test (0.5 g of glucose/kg of body weight), insulin
immunoreactivity (brown) (D), and insulin content
(ng/islet) (E), insulin content (ng/ng of DNA)
(F) of WT, gAd Tg ob/ob, and nontransgenic ob/ob littermates
on the HF diet. Male mice 8 weeks of age were fed the HF diet for 4 weeks and then analyzed. Bars indicate 100 µm
(D). The results are expressed as the percentage of the
value of untreated mice, respectively (A). The basal glucose
levels (time = 0 of ITT) of untreated ob/ob and gAd Tg ob/ob mice
on the HF diet were 398.6 ± 36.1 and 311.2 ± 30.8 mg/dl,
respectively (A). Each bar represents the
mean ± S.E. (n = 5-14). *,
p < 0.05; **, p < 0.01; transgenic
versus nontransgenic.
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Fig. 4.
Overexpression of globular adiponectin
increased expression of molecules involved in fatty acid oxidation and
molecules involved in energy dissipation and increased fatty acid
oxidation in skeletal muscle. A-F, amounts of the
mRNAs of ACO (A and E), uncoupling protein
(UCP) 2 (B and F) and UCP3 (G), fatty
acid oxidation (C and H), tissue TG content
(D and I) in the liver (A-D) and in
skeletal muscle (E-I) of gAd Tg ob/ob mice and
nontransgenic ob/ob littermates on the HF diet. Male mice 8 weeks of
age were fed the HF diet for 4 weeks and then analyzed. The results are
expressed as the ratio of the value of nontransgenic ob/ob littermates
(A-C, E-H). C and H,
measurements of [14C]CO2 production from
[1-14C]palmitic acid were performed using liver and
muscle slices, as described (20). J and K,
PPAR ligand activities in C2C12 myocytes (J) and in
primary hepatocytes (K) incubated with indicated
concentrations of gAd. Each bar represents the mean ± S.E. (n = 5-14). *, p < 0.05; **,
p < 0.01; transgenic versus nontransgenic,
or compared with untreated C2C12 myocytes.
Ligands Activities Only in Myocytes but Not
in Hepatocytes in Vitro--
To clarify the mechanisms by which
overexpression of gAd increased the expressed levels of ACO only in
skeletal muscle but not in the liver, we measured endogenous PPAR
ligands activities in skeletal muscle and the liver in
vitro, since the ACO gene possesses the peroxisome
proliferator response element (PPRE) in its promoter regions (30).
Interestingly, treatment of C2C12 myocytes (Fig. 4J) but not
primary hepatocytes (Fig. 4K) with gAd increased PPAR
ligands activities in a dose-dependent manner. These
observations may at least partly explain the increased fatty acid
oxidation only in skeletal muscle but not in the liver of gAd Tg ob/ob
mice (Fig. 4, C and H).
View larger version (36K):
[in a new window]
Fig. 5.
Overexpression of globular adiponectin
inhibited the progression of atherosclerosis observed in ApoE-deficient
mice. A-F, representative cases of the en face
Sudan IV- positive lesion areas of more than five apoE-deficient
( /
) mice (A) or gAd Tg apoE-deficient (
/
) mice
(B) are presented. Male mice 8 weeks of age were fed a
normal diet for 12 weeks and then atherosclerotic lesion areas were
quantified by en face Sudan IV staining of the arch and the descending
aorta (C). HPLC analysis of lipoprotein distribution of
pooled serum samples from five apoE-deficient (
/
) mice or five gAd
Tg apoE-deficient (
/
) mice are presented (D).
Representative examples of Oil-Red O staining (left) or
staining of Mac-3 (middle) and class A scavenger receptor
(SRA) (right) (×400) of atherosclerotic lesions in the
aortic valve are presented (E), and the results of
quantification of Oil-Red O staining or staining of Mac-3, SRA, TNF
,
and intracellular adhesion molecule (ICAM)-1 are expressed as the
percentage of the value of nontransgenic apoE-deficient (
/
) mice
(F). Each bar represents the mean ± S.E.
(n = 6-7). Serum was separated and pooled for HPLC
analyses as described under "Experimental Procedures"
(D). *, p < 0.05; **, p < 0.01; transgenic versus nontransgenic littermates, or
between two groups as indicated.
gAd had no significant effect on plasma glucose and lipid levels of
apoE/
mice
in the Vascular Wall--
In order to
address this point, we carried out Oil-Red O staining and
immunohistochemistry using macrophage-specific marker Mac-3,
intercellular adhesion molecule (ICAM)-1, TNF
, and scavenger receptor antibodies (Fig. 5, E and F). As
compared with number of macrophages in atherosclerotic lesion (Fig.
5E, middle and F), we found that
globular adiponectin suppressed expression of class A scavenger
receptor (SRA) in vivo (Fig. 5E, right
and F), which may lead to reduced lipid accumulation in
macrophages in the vascular wall (Fig. 5E, left
and F). Although globular adiponectin had little effect on
ICAM-1, we also found that it suppressed expression of an inflammatory
cytokine TNF
in atherosclerotic lesion in vivo (Fig.
5F). Thus, it appears that adiponectin can protect from
atherosclerosis in vivo independent of conventional atherogenic risk factors. Our study suggested that adiponectin suppressed expressions of SRA and TNF
, which may lead to reduced lipid accumulation and inflammation in macrophages in the vascular wall, thereby inhibiting the progression of atherosclerosis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cell
Degranulations as Well as Diabetes--
In this study, we showed that
overexpression of globular adiponectin could prevent diabetes in
vivo. These data may be consistent with the previous observations
that globular adiponectin increased fatty acid oxidation in skeletal
muscle and protected against accumulation of excess tissue TG (19, 20),
thereby ameliorating insulin resistance in obese mice (20).
agonist has been
reported to show increased insulin sensitivity associated with
decreased insulin levels during glucose tolerance test (31). These
observations suggested that adiponectin, unlike PPAR
agonists, may
have direct protective effects on
-cells.
agonist increases
plasma adiponectin levels (20, 33), raising the possibility that the
PPAR
agonist may exert its anti-diabetic effects in vivo.
However, we speculate that a specific adiponectin secretagogue or
activator has a clear therapeutic advantage over the PPAR
agonist;
adiponectin exerts its effects without increasing body weight, whereas
the PPAR
agonist increases body weight.
ligands in myocytes but not
in hepatocytes (Fig. 4, J and K). Interestingly,
full Ad increases ACO and stimulates fatty acid oxidation both in
skeletal muscle and the liver, which may be explained by the data that
full Ad can increase PPAR
ligands activities both in myocytes and
hepatocytes.2 In this
context, decreases in hepatic TG content in gAd Tg ob/ob mice may be
independent of direct hepatic action of gAd, and it might be a
secondary effect. Whether the putative receptors for adiponectin in the
membrane fractions of skeletal muscle and the liver are structurally
and functionally distinct is now under investigation.
, which may result in reduced lipid accumulation and inflammation
in macrophages in the vascular wall.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Y. Ogawa and Dr. K. Nakao at Kyoto University, Dr. N. Kubota, Dr. R. Suzuki, Dr. K. Tobe, Dr. M. Noda, and Dr. K. Izumi for helpful suggestions; Dr. T. Hashimoto for the generous gift of a DNA probe for ACO; and Dr. K. Motojima for kindly providing UCP2 cDNA probe. We are grateful to K. Kirii, M. Shibata, A. Okano, and T. Nagano for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by a grant from the Human Science Foundation (to T. K.), a grant-in-aid for the Development of Innovative Technology from the Ministry of Education, Culture, Sports, Science and Technology (to T. K.), Grant-in-aid for Creative Scientific Research 10NP0201 from the Japan Society for the Promotion of Science (to T. K.), and by Health Science Research grants (Research on Human Genome and Gene Therapy) from the Ministry of Health and Welfare (to T. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
c These authors contributed equally to this work.
j To whom correspondence should be addressed: Dept. of Internal Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: 81-3-5800-8818; Fax: 81-3-5689-7209; E-mail: kadowaki-3im@h.u-tokyo.ac.jp.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M209033200
2 J. Kamon, T. Yamauchi, and T. Kadowaki, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: TG, triglycerides; RIA, radioimmunoassay; HC, high carbohydrates; HF, high fat; PPAR, peroxisome proliferator-activated receptor; gAd, globular adiponectin; ITT, insulin tolerance test; Tg, transgenic; ACO, acyl-CoA oxidase; SRA, class A scavenger receptor; SAP, serum amyloid P component; FFA, free fatty acids; TNF, tumor necrosis factor; WT, wild type.
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