Globular Adiponectin Protected ob/ob Mice from Diabetes and ApoE-deficient Mice from Atherosclerosis*

Toshimasa Yamauchiabc, Junji Kamonacd, Hironori Wakiab, Yasushi Imaia, Nobuhiro Shimozawae, Kyouji Hiokie, Shoko Uchidaa, Yusuke Itoad, Keisuke Takakuwaa, Junji Matsuia, Makoto Takataa, Kazuhiro Etoab, Yasuo Terauchiab, Kajuro Komedaf, Masaki Tsunodag, Koji Murakamig, Yasuyuki Ohnishie, Takeshi Naitohd, Kenichi Yamamurah, Yoshito Ueyamae, Philippe Frogueli, Satoshi Kimuraa, Ryozo Nagaia, and Takashi Kadowakiabj

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 alpha . 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

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-alpha (TNFalpha ) (10).

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 beta -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 TNFalpha .

Thus, adiponectin could compensate for leptin deficiency at least in part in insulin resistance and beta -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

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+/-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.

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 beta -cells and non-beta -cells were calculated as the proportions of the area of beta -cells or non-beta -cells, assessed by immunostaining, to the area of the whole pancreas. More than 50 islets were analyzed per mouse in each group.

Islet Isolation, beta -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.

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 PPARalpha 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 PPARalpha 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. PPARalpha ligands activities were quantitatively determined using a (UAS)x4-tk-LUC reporter plasmid, a GAL4-rat PPARalpha ligand-binding domain expression plasmid, and a beta -galactosidase expression plasmid as the internal control as described previously (26).

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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.

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 beta -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 beta -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 beta -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.

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.


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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, PPARalpha 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.

gAd Increased PPARalpha 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 PPARalpha 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 PPARalpha 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).

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.


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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, TNFalpha , 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.

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.

                              
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Table I
gAd had no significant effect on plasma glucose and lipid levels of apoE-/- mice
Male mice 8 weeks of age were fed a normal diet for 12 weeks and then analyzed. Each data point represents the mean ± S.E. (n = 5).

Globular Adiponectin Reduced Lipid Accumulation and Inhibited the Progression of Atherosclerosis, Which Was Associated with Decreased Expressions of SRA and TNFalpha 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, TNFalpha , 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 TNFalpha 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 TNFalpha , 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

Adiponectin Can Ameliorate Insulin Resistance and beta -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).

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 PPARgamma 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 PPARgamma agonists, may have direct protective effects on beta -cells.

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 PPARgamma agonist increases plasma adiponectin levels (20, 33), raising the possibility that the PPARgamma 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 PPARgamma agonist; adiponectin exerts its effects without increasing body weight, whereas the PPARgamma agonist increases body weight.

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 PPARalpha 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 PPARalpha 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.

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 TNFalpha , which may result in reduced lipid accumulation and inflammation in macrophages in the vascular wall.

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.

    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.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Spiegelman, B. M., and Flier, J. S. (1996) Cell 87, 377-389[Medline] [Order article via Infotrieve]
2. Reaven, G. M. (1995) Diabetologia 38, 3-13[CrossRef][Medline] [Order article via Infotrieve]
3. Ailhaud, G., Grimaldi, P., and Negrel, R. (1992) Annu. Rev. Nutr. 12, 207-233[CrossRef][Medline] [Order article via Infotrieve]
4. Matsuzawa, Y., Funahashi, T., and Nakamura, T. (1999) Ann. N. Y. Acad. Sci. 892, 146-154[Abstract/Free Full Text]
5. Shulman, G. I. (2000) J. Clin. Invest. 106, 171-176[Free Full Text]
6. White, R. T., Damm, D., Hancock, N., Rosen, B. S., Lowell, B. B., Usher, P., Flier, J. S., and Spiegelman, B. M. (1992) J. Biol. Chem. 267, 9210-9213[Abstract/Free Full Text]
7. Friedman, J. M. (2000) Nature 404, 632-634[Medline] [Order article via Infotrieve]
8. Shimomura, I., Hammer, R. E., Ikemoto, S., Brown, M. S., and Goldstein, J. L. (1999) Nature 401, 73-76[CrossRef][Medline] [Order article via Infotrieve]
9. Steppan, C. M., Bailey, S. T., Bhat, S., Brown, E. J., Banerjee, R. R., Wright, C. M., Patel, H. R., Ahima, R. S., and Lazar, M. A. (2001) Nature 409, 307-312[CrossRef][Medline] [Order article via Infotrieve]
10. Hotamisligil, G. S. (1999) J. Intern. Med. 245, 621-625[CrossRef][Medline] [Order article via Infotrieve]
11. Maeda, K., Okubo, K., Shimomura, I., Funahashi, T., Matsuzawa, Y., and Matsubara, K. (1996) Biochem. Biophys. Res. Commun. 221, 286-296[CrossRef][Medline] [Order article via Infotrieve]
12. Scherer, P. E., Williams, S., Fogliano, M., Baldini, G., and Lodish, H. F. (1995) J. Biol. Chem. 270, 26746-26749[Abstract/Free Full Text]
13. Hu, E., Liang, P., and Spiegelman, B. M. (1996) J. Biol. Chem. 271, 10697-10703[Abstract/Free Full Text]
14. Nakano, Y., Tobe, T., Choi-Miura, N. H., Mazda, T., and Tomita, M. (1996) J. Biochem. (Tokyo) 120, 802-812
15. Yokota, T., Oritani, K., Takahashi, I., Ishikawa, J., Matsuyama, A., Ouchi, N., Kihara, S., Funahashi, T., Tenner, A. J., Tomiyama, Y., and Matsuzawa, Y. (2000) Blood 96, 1723-1732[Abstract/Free Full Text]
16. Ouchi, N., Kihara, S., Arita, Y., Nishida, M., Matsuyama, A., Okamoto, Y., Ishigami, M., Kuriyama, H., Kishida, K., Nishizawa, H., Hotta, K., Muraguchi, M., Ohmoto, Y., Yamashita, S., Funahashi, T., and Matsuzawa, Y. (2001) Circulation 103, 1057-1063[Abstract/Free Full Text]
17. Berg, A. H., Combs, T. P., Du, X., Brownlee, M., and Scherer, P. E. (2001) Nat. Med. 7, 947-953[CrossRef][Medline] [Order article via Infotrieve]
18. Combs, T. P., Berg, A. H., Obici, S., Scherer, P. E., and Rossetti, L. (2001) J. Clin. Invest. 108, 1875-1881[Abstract/Free Full Text]
19. Fruebis, J., Tsao, T. S., Javorschi, S., Ebbets-Reed, D., Erickson, M. R., Yen, F. T., Bihain, B. E., and Lodish, H. F. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2005-2010[Abstract/Free Full Text]
20. Yamauchi, T., Kamon, J., Waki, H., Terauchi, Y., Kubota, N., Hara, K., Mori, Y., Ide, T., Murakami, K., Tsuboyama-Kasaoka, N., Ezaki, O., Akanuma, Y., Gavrilova, O., Vinson, C., Reitman, M. L., Kagechika, H., Shudo, K., Yoda, M., Nakano, Y., Tobe, K., Nagai, R., Kimura, S., Tomita, M., Froguel, P., and Kadowaki, T. (2001) Nat. Med. 7, 941-946[CrossRef][Medline] [Order article via Infotrieve]
21. Kubota, N., Terauchi, Y., Yamauchi, T., Kubota, T., Moroi, M., Matsui, J., Eto, K., Yamashita, T., Kamon, J., Satoh, H., Yano, W., Froguel, P., Nagai, R., Kimura, S., Kadowaki, T., and Noda, T. (2002) J. Biol. Chem. 277, 25863-25866[Abstract/Free Full Text]
22. Maeda, N., Shimomura, I., Kishida, K., Nishizawa, H., Matsuda, M., Nagaretani, H., Furuyama, N., Kondo, H., Takahashi, M., Arita, Y., Komuro, R., Ouchi, N., Kihara, S., Tochino, Y., Okutomi, K., Horie, M., Takeda, S., Aoyama, T., Funahashi, T., and Matsuzawa, Y. (2002) Nat. Med. 8, 731-737[CrossRef][Medline] [Order article via Infotrieve]
23. Plump, A. S., Smith, J. D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J. G., Rubin, E. M., and Breslow, J. L. (1992) Cell 81, 343-353
24. Zhang, S. H., Reddick, R. L., Piedrahita, J. A., and Maeda, N. (1992) Science 258, 468-471[Medline] [Order article via Infotrieve]
25. Ogawa, Y., Masuzaki, H., Hosoda, K., Aizawa-Abe, M., Suga, J., Suda, M., Ebihara, K., Iwai, H., Matsuoka, N., Satoh, N., Odaka, H., Kasuga, H., Fujisawa, Y., Inoue, G., Nishimura, H., Yoshimasa, Y., and Nakao, K. (1999) Diabetes 48, 1822-1829[Abstract]
26. Yamauchi, T., Oike, Y., Kamon, J., Waki, H., Komeda, K., Tsuchida, A., Date, Y., Li, M. X., Miki, H., Akanuma, Y., Nagai, R., Kimura, S., Saheki, T., Nakazato, M., Naitoh, T., Yamamura, K., and Kadowaki, T. (2002) Nat. Genet. 30, 221-226[CrossRef][Medline] [Order article via Infotrieve]
27. Kubota, N., Tobe, K., Terauchi, Y., Eto, K., Yamauchi, T., Suzuki, R., Tsubamoto, Y., Komeda, K., Nakano, R., Miki, H., Satoh, S., Sekihara, H., Sciacchitano, S., Lesniak, M., Aizawa, S., Nagai, R., Kimura, S., Akanuma, Y., Taylor, S. I., and Kadowaki, T. (2000) Diabetes 49, 1880-1889[Abstract]
28. Imai, Y., Shindo, T., Maemura, K., Sata, M., Saito, Y., Kurihara, Y., Akishita, M., Osuga, J., Ishibashi, S., Tobe, K., Morita, H., Oh-hashi, Y., Suzuki, T., Maekawa, H., Kangawa, K., Minamino, N., Yazaki, Y., Nagai, R., and Kurihara, H. (2002) Arterioscler. Thromb. Vasc. Biol. 22, 1310-1315[Abstract/Free Full Text]
29. Sakamoto, H., Aikawa, M., Hill, C. C., Weiss, D., Taylor, W. R., Libby, P., and Lee, R. T. (2001) Circulation 104, 109-114[Abstract/Free Full Text]
30. Kersten, S., Desvergne, B., and Wahli, W. (2000) Nature 405, 421-424[CrossRef][Medline] [Order article via Infotrieve]
31. Mukherjee, R., Davies, P. J., Crombie, D. L., Bischoff, E. D., Cesario, R. M., Jow, L., Hamann, L. G., Boehm, M. F., Mondon, C. E., Nadzan, A. M., Paterniti, J. R. Jr., and Heyman, R. A. (1997) Nature 386, 407-410[CrossRef][Medline] [Order article via Infotrieve]
32. Hara, K., Boutin, P, Mori, Y., Tobe, K, Dina, C., Yasuda, K, Yamauchi, T., Otabe, S., Okada, T., Eto, K., Kadowaki, H., Hagura, R., Akanuma, Y., Yazaki, Y., Nagai, R., Taniyama, M., Matsubara, K., Yoda, M., Nakano, Y., Kimura, S., Tomita, M., Kimura, S., Ito, C., Froguel, P., and Kadowaki, T. (2002) Diabetes 51, 536-540[Abstract/Free Full Text]
33. Maeda, N., Takahashi, M., Funahashi, T., Kihara, S., Nishizawa, H., Kishida, K., Nagaretani, H., Matsuda, M., Komuro, R., Ouchi, N., Kuriyama, H., Hotta, K., Nakamura, T., Shimomura, I., and Matsuzawa, Y. (2001) Diabetes 50, 2094-2099[Abstract/Free Full Text]


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