Abstract

Background

Previous reports indicated that blockade of AT1 receptor stimulation attenuated adipocyte dysfunction. However, the effects of AT2 receptor stimulation on adipose tissue were not yet clear. In the present study, we examined the adipose tissue dysfunction in atherosclerotic apolipoprotein E knockout (ApoEKO) mice with AT2 receptor deficiency.

Methods

Male ApoEKO and AT2 receptor/ApoE knockout (AT2/ApoEKO) mice at 6 weeks of age were treated with a normal diet or a high-cholesterol diet (HCD: 1.25% cholesterol). Markers for adipocyte differentiation and inflammation in adipose tissue were assayed with real-time reverse-transcription-PCR and western blot.

Results

Compared with ApoEKO mice, AT2/ApoEKO mice with a normal diet showed only a decrease in expression of adiponectin and CCAAT/enhancer binding protein δ (C/EBPδ) in epididymal adipose tissue without changes in body weight, adipose tissue weight, and adipocyte number even at 6 months of age. After HCD for 4 weeks, the weight of both epididymal and retroperitoneal adipose tissue in AT2/ApoEKO mice was greater than that in ApoEKO mice without a change in body weight. Plasma concentrations of cholesterol and fatty acids were higher in AT2/ApoEKO mice than in ApoEKO mice. In adipose tissue of AT2/ApoEKO mice, the adipocyte number was decreased and the expression of peroxisome proliferator–activated receptor γ (PPARγ), C/EBPα, and aP2 was lower than that in ApoEKO mice, in association with an increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.

Conclusions

These results suggest that AT2 receptor stimulation in adipose tissue is involved in the improvement of adipocyte differentiation and adipose tissue dysfunction in atherosclerotic model.

Recent clinical trials showed that an AT1 receptor blocker (ARB) or angiotensin converting enzyme (ACE) inhibitor decreased the clinical onset of diabetes, and organ damage in diabetic patients, indicating that the blockade of renin–angiotensin system improves insulin resistance.1–4 We have previously reported that the treatment with an ARB or an ACE inhibitor improved oral glucose tolerance test and increased glucose uptake into skeletal muscles in type 2 diabetic KK-Ay mice.5,6 The enhancement of insulin resistance by angiotensin II and its improvement by ARBs and ACE inhibitors was also observed in other animal models.7–9

Our recent report indicated that adipocyte size was reduced and adipocyte differentiation was increased in adipose tissue by knocking out of AT1a receptor in atherosclerotic apolipoprotein E knockout (ApoEKO) mice, or by an ARB in diabetic KK-Ay mice.10 These results suggest a possibility that renin–angiotensin system regulates adipose tissue function mainly through the AT1 receptor stimulation. Recent studies indicate that adipose tissue acts as an endocrine organ that produces and secretes signal mediators, such as leptin, adiponectin, tumor necrosis factor-α, monocyte chemoattractant protein-1, and plasminogen activator inhibitor-1 (refs. 11,12). Adipose tissue also produces angiotensinogen, suggesting that the local renin–angiotensin system in adipose tissue regulates adipocyte function.

In contrast, AT2 receptor stimulation often counteracts against AT1 receptor stimulation in cardiovascular and brain damage.13–15 We have reported that the effect of AT2 receptor stimulation is involved in the cardiovascular actions of ARBs.16,17 However, involvement of the AT2 receptor stimulation in adipose tissue dysfunction is still an enigma. In the present study, we examined the role of AT2 receptor in adipose tissue changes including adipocyte differentiation markers in atherosclerotic model. For this purpose, we used ApoEKO mice for an atherosclerosis model, which showed enlarged adipocytes and lowered adipocyte differentiation markers,10 and developed AT2 receptor/ApoE double knockout (AT2/ApoEKO) mice by mating ApoEKO mice with AT2 receptor knockout mice with the same background.16 Using these mice, we analyzed the effects of AT2 receptor deficiency on adipose tissue changes in atherosclerotic model.

Methods

Animals and treatment. Adult male ApoEKO and AT2 receptor/ApoE double knockout (AT2/ApoEKO) mice were used in this study. To generate AT2/ApoEKO mice (backcrossed >10 times, based on C57BL/6J strain), ApoEKO mice (B6.129P2-Apoetm1Unc; apoe−/−: in as backcrossed 10 times, based on C57BL/6J strain; The Jackson Laboratory, Bar Harbor, ME) and AT2KO mice (based on C57BL/6J strain, male agtr2−/y chromosome, female agtr2−/−; since the AT2 receptor is located on the X-chromosome)16 were bred to yield mice heterozygous at the ApoE locus (apoe+/− for both male and female), and heterozygous (female: apoe+/− with agtr2+/−) and hemizygous (male: apoe+/− with agtr2−/y) at the AT2 receptor loci. These mice were crossed and intercrossed to yield AT2/ApoEKO mice.16 The animals were housed in a room with a 12-h light/dark cycle and temperature maintained at 24 °C. The mice were given a normal standard diet (MF; Oriental Yeast, Tokyo, Japan; abbreviated to ND) up to 6 months of age, or a high-cholesterol diet (HCD: 1.25% cholesterol and 10% coconut oil in MF) for 4 weeks from 8 weeks of age and water ad libitum. Some animals were administered valsartan (1 mg/kg/day: provided by Novartis Pharma, Basel, Switzerland) intraperitoneally using an osmotic mini-pump for 2 weeks before sampling. The Animal Studies Committee of Ehime University approved the following experimental protocol.

Measurement of blood pressure and plasma concentrations of adiponectin and lipids. Blood pressure was measured by the indirect tail-cuff method with a blood pressure monitor (MK-1030; Muromachi Kikai, Tokyo, Japan). Plasma concentration of total adiponectin was assayed using a mouse/rat ELISA kit (Otsuka Pharmaceutical, Tokyo, Japan). Plasma concentrations of cholesterol, free fatty acids (FFAs), and triglyceride were measured using colorimetric measurement method kits (Wako Pure Chemical Industries, Osaka, Japan).

Morphological analysis. Epididymal white adipose tissue was taken, fixed with 10% formalin neutral buffer solution, and paraffin-embedded sections were prepared.10 Adipocyte number under three microscopic fields was measured after aldehyde-fuchsin staining of each section, and expressed as the cell number per mm2.

Real-time reverse-transcription-PCR. Total RNA was extracted from epididymal adipose tissue using TRI reagent and a filter cartridge, according to the manufacturer's protocol (Ambion, Tokyo, Japan). Quantitative real-time reverse-transcription-PCR was performed using Premix Ex Taq (Takara Bio, Shiga, Japan) with primers as previously described.10 The level of target gene expression was normalized against the glyceraldehyde-3-phosphate dehydrogenase expression in each sample.

Detection of insulin receptor substrate-1 and nicotinamide adenine dinucleotide phosphate oxidase activity. Protein samples were prepared from epididymal adipose tissue of AT2/ApoEKO and ApoEKO mice. Western blot was performed as previously described.6,17 Anti-insulin receptor substrate-1 (IRS-1) antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-β-tubulin antibody was purchased from Sigma-Aldrich (St Louis, MO). Densitometric analysis was performed using Densitograph Imaging Software (ATTO, Tokyo, Japan). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was measured using protein samples of epididymal adipose tissue according to the method as previously described.18

Statistical analysis. All values are expressed as mean ± s.e.m. The effects of the different treatments on all data were evaluated with factorial analysis of variance. When a significant effect was found, the results were further compared with Bonferroni's multiple range tests. A difference with P < 0.05 was considered significant.

Results

Adipose tissue weight and adipocyte number in ApoEKO and AT2/ApoEKO mice with ND

As previously reported, adipocyte size of epididymal adipose tissue was larger in ApoEKO mice than that in wild-type (C57BL/6J) mice treated with normal diet (ND), whereas adipose tissue weight in ApoEKO mice was not different from that in wild-type mice.10 In the present study, mRNA content for AT2 receptor was decreased ~60% in epididymal adipose tissue of ApoEKO mice in an age-dependent manner at least up to 6 months of age (8.8 ± 3.0, 7.2 ± 2.8, 3.1 ± 0.7*; relative amount to glyceraldehyde-3-phosphate dehydrogenase × 10−5 at 2, 4, and 6 months, respectively, *P < 0.05), whereas AT1 receptor mRNA was not altered (0.015 ± 0.002, 0.017 ± 0.003, 0.012 ± 0.001; relative amount to glyceraldehyde-3-phosphate dehydrogenase at 2, 4, and 6 months, respectively). In AT2/ApoEKO mice with ND, adipose tissue weight and the adipocyte number in epididymal adipose tissue at 6 months of age were not significantly different from those in ApoEKO mice (Figure 1 and Table 1). In AT2/ApoEKO mice, however, the expression of adiponectin and CCAAT/enhancer binding protein δ (C/EBPδ), a marker of adiopcyte differentiation, was lower in adipose tissue than that in ApoEKO mice at 2 months of age, whereas the expression of other transcription factors, such as peroxisome proliferator–activated receptor γ (PPARγ) and C/EBPα, was not significantly different (Figure 2). In addition, plasma cholesterol concentration in AT2/ApoEKO mice with ND was not different from ApoEKO mice (Figure 1b), whereas plasma cholesterol in ApoEKO mice was approximately sevenfold higher than wild-type mice at 6 months of age, as previously reported.10 Systolic blood pressures at sampling were not significantly different among experimental groups (97.2 ± 5.4 mm Hg for ApoEKO+ND, 97.8 ± 6.1 mm Hg for AT2/ApoEKO+ND, 99.3 ± 3.9 mm Hg for ApoEKO+HCD, and 98.1 ± 3.0 mm Hg for T2/ApoEKO+HCD group).

Table 1

Body weight and adipose tissue mass in AT2/ApoEKO and ApoEKO mice

graphic 
graphic 

Adipocyte number in adipose tissue and plasma cholesterol level in AT2/ApoEKO and ApoEKO mice with normal standard diet for 6 months. Epididymal adipose tissue was weighed and fixed with 10% formalin neutral buffer solution. Paraffin-embedded sections were prepared and stained with aldehyde-fuchsin. Adipocyte number was measured under three microscopic fields after staining of each section and expressed as the cell number per mm2. Plasma cholesterol was determined with a colorimetric kit as described in Methods. (a) Representative photos and number of adipocyte. (b) Plasma cholesterol concentration. Values are mean ± s.e.m. of seven to nine mice for each group. ApoEKO, apolipoprotein E knockout; AT2/ApoEKO, AT2 receptor/ApoE double knockout.

Expression of adipocyte differentiation factors in adipose tissue of ApoEKO and AT2/ApoEKO mice with normal standard diet for 6 months. Epididymal adipose tissue was taken and total RNA was prepared as described in Methods. Levels of mRNA were quantified by real-time reverse-transcription-PCR as described in Methods. (a) Adiponectin and C/EBPδ. *P < 0.05 vs. ApoEKO mice. (b) PPARγ, C/EBPα, and aP2. Values are mean ± s.e.m. of seven to nine mice for each group. ApoEKO, apolipoprotein E knockout; AT2/ApoEKO, AT2 receptor/ApoE double knockout; C/EBPδ, CCAAT/enhancer binding protein δ; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPARγ, peroxisome proliferator–activated receptor γ.

Changes in adipose tissue in ApoEKO and AT2/ApoEKO mice after HCD

To exaggerate the atherosclerosis in this model, we treated AT2/ApoEKO and ApoEKO mice with HCD for 4 weeks. After HCD for 4 weeks, plasma concentrations of cholesterol and FFAs were higher in AT2/ApoEKO mice than in ApoEKO mice. Plasma triglyceride concentration was not significantly different in these mice (Figure 3). Weight of both epididymal and retroperitoneal adipose tissue was significantly higher in AT2/ApoEKO mice without significant change in body weight (Table 1). Moreover, adipocyte number was reduced in AT2/ApoEKO mice than in ApoEKO mice after HCD (Figure 4).

Plasma concentration of cholesterol, free fatty acids (FFAs) and triglyceride in AT2/ApoEKO and ApoEKO mice with HCD for 4 weeks. Blood samples were taken after HCD for 4 weeks. Plasma concentration of lipids was measured using colorimetric method kits as described in Methods. *P < 0.05 vs. ApoEKO mice. Values are mean ± s.e.m. of seven to nine mice for each group. ApoEKO, apolipoprotein E knockout; AT2/ApoEKO, AT2 receptor/ApoE double knockout; HCD, high-cholesterol diet.

Adipocyte number in adipose tissue of AT2/ApoEKO and ApoEKO mice with HCD. Epididymal adipose tissue was taken after HCD for 4 weeks. Paraffin-embedded sections were prepared and stained with aldehyde-fuchsin. Adipocyte number was measured under three microscopic fields after staining of each section and expressed as the cell number per mm2. Representative photos are shown in the left panels. Values are mean ± s.e.m. of seven to nine mice for each group. *P < 0.05 vs. ApoEKO mice. ApoEKO, apolipoprotein E knockout; AT2/ApoEKO, AT2 receptor/ApoE double knockout; HCD, high-cholesterol diet.

Adipocyte differentiation factors in AT2/ApoEKO mice treated with HCD

Expression of PPARγ, C/EBPα, and aP2 in adipose tissue of AT2/ApoEKO mice with HCD was lower than that in ApoEKO mice (Figure 5). The adiponectin mRNA content in adipose tissue was not significantly different in AT2/ApoEKO mice with HCD (Figure 5). However, plasma adiponectin concentration was lower in AT2/ApoEKO mice (AT2/ApoEKO vs. ApoEKO, 12.0 ± 0.6 and 15.8 ± 1.2 µg/ml, respectively, P < 0.05). On the other hand, mRNA content of monocyte chemoattractant protein-1 and tumor necrosis factor-α was not different between AT2/ApoEKO and ApoEKO mice (data not shown).

Expression of adipocyte differentiation factors in adipose tissue of AT2/ApoEKO and ApoEKO mice with HCD. Epididymal adipose tissue was taken after HCD for 4 weeks. Total RNA was prepared and the level of mRNA was quantified by real-time reverse-transcription-PCR as described in Methods. (a) Adiponectin and C/EBPδ. (b) PPARγ, C/EBPα, and aP2. *P < 0.05 vs. ApoEKO mice. Values are mean ± s.e.m. of seven to nine mice for each group. ApoEKO, apolipoprotein E knockout; AT2/ApoEKO, AT2 receptor/ApoE double knockout; C/EBPδ, CCAAT/enhancer binding protein δ; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCD, high-cholesterol diet; PPARγ, peroxisome proliferator–activated receptor γ.

NADPH oxidase activity and protein content of IRS-1 in adipose tissue of AT2/ApoEKO mice

IRS-1 is a critical intracellular factor of insulin receptor-mediated signaling. As shown in Figure 6, protein level of total IRS-1 was decreased in adipose tissue of AT2/ApoEKO mice after HCD. On the other hand, tissue NADPH oxidase activity, a critical enzyme for superoxide production, in adipose tissue was higher in AT2/ApoEKO mice than that in ApoEKO mice (17.7 ± 1.6 vs. 12.6 ± 1.0 pmol/µg protein/min for AT2/ApoEKO and ApoEKO mice, respectively).

Protein level of insulin receptor substrate (IRS)-1 in adipose tissue of AT2/ApoEKO and ApoEKO mice with HCD. Epididymal adipose tissue was taken after HCD for 4 weeks. Protein sample was prepared, and IRS-1 was determined by western blot as described in Methods. (a) Representative photo of IRS-1 and β-tubulin. (b) Densitometric analysis of IRS-1. Density was expressed as the ratio to the density of β-tubulin. Values are mean ± s.e.m. of seven to nine mice for each group. *P < 0.05 vs. ApoEKO mice. ApoEKO, apolipoprotein E knockout; AT2/ApoEKO, AT2 receptor/ApoE double knockout; HCD, high-cholesterol diet.

Effect of AT1 receptor antagonism on plasma lipids and adipose tissue of AT2/ApoEKO and ApoEKO mice with HCD

Treatment of mice with an ARB, valsartan, for 2 weeks decreased plasma levels of cholesterol and FFAs in ApoEKO mice (Figure 7a). In addition, valsartan increased adipocyte number and expression of adiponectin and PPARγ in adipose tissue of ApoEKO mice (Figure 7b). The effects of valsartan were not apparent in AT2/ApoEKO mice (Figure 7a,b).

Effects of valsartan (Val) on plasma lipids and adipose tissue of AT2/ApoEKO and ApoEKO mice with HCD. Blood and tissue samples were taken after HCD for 4 weeks. Plasma lipids, adiopcyte number and mRNA levels were measured as in Figures 3–5. (a) Concentration of plasma lipids. (b) Adipocyte number (left) and expression of adiponectin and PPARγ. Values are mean ± s.e.m. of five mice for each group. *P < 0.05 vs. ApoEKO mice without valsartan. ApoEKO, apolipoprotein E knockout; AT2/ApoEKO, AT2 receptor/ApoE double knockout; FFA, free fatty acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCD, high-cholesterol diet; PPARγ, peroxisome proliferator–activated receptor γ.

Discussion

We studied the role of AT2 receptors on adipose tissue in atherosclerotic ApoEKO mice. Without HCD treatment, the effects of AT2 receptor deficiency on adipose tissue were not remarkable. However, after treatment with HCD, a lack of AT2 receptor, i.e., AT2/ApoEKO mice, decreased adipocyte number and increased adipose tissue weight in atherosclerotic ApoEKO mice. Expression of markers for adipocyte differentiation was lowered in AT2/ApoEKO mice after HCD. These results suggest that a lack of AT2 receptor stimulation leads to adipose tissue changes probably through the regulation of adipocyte differentiation in atherosclerotic mice under condition of lipid loading.

We have previously reported that AT1 receptor blockade ameliorated the glucose intolerance and adipose tissue changes in atherosclerotic ApoEKO mice and diabetic KK-Ay mice.10,19,20 As the expression of AT2 receptor in ApoEKO mice was decreased in the adipose tissue age dependently, it is possible that the change in AT2 receptor stimulation influences adipose tissue function. In the present study, adipose tissue mass was increased and adipocyte number was decreased by AT2 receptor deficiency in atherosclerotic ApoEKO mice after HCD (Table 1). Inflammatory cytokines like tumor necrosis factor-α can be secreted from adipose tissue and involved in insulin resistance.12 In our study, however, the mRNA levels of inflammatory factors, monocyte chemoattractant protein-1 and tumor necrosis factor-α, in adipose tissue were not significantly changed in AT2/ApoEKO mice (data not shown). This suggests that AT2 receptor stimulation may not be involved in regulation of inflammatory response in adipose tissue. On the other hand, previous reports indicated that oxidative stress induces insulin resistance.6,8 In the present study, the activity of NADPH oxidase, a key enzyme for superoxide anion formation, was higher in adipose tissue of AT2/ApoEKO mice. These results suggest that oxidative stress might be involved in the adipose tissue dysfunction in AT2/ApoEKO mice. The protein level of IRS-1 was lower in AT2/ApoEKO mice than that in ApoEKO mice. However, we have not actually measured insulin sensitivity in this study. The effect of AT2 receptor stimulation on insulin sensitivity in adipose tissue of atherosclerotic mice remains to be clarified.

Adipocyte differentiation is inversely associated with adipose tissue enlargement (Figure 4) (ref. 21). The expression of adipocyte differentiation factor, C/EBPδ, was decreased in AT2/ApoEKO mice treated with ND, whereas the expression of C/EBPα and aP2 was lowered in AT2/ApoEKO mice after HCD (Figures 2 and 5), suggesting that the change in C/EBPδ probably occurs prior to that in C/EBPα.22 These results suggest that AT2 receptor stimulation is involved in the regulation of adipocyte differentiation, and that these effects of AT2 receptor stimulation may be important, especially in pathological conditions such as hypercholesterolemia or metabolic syndrome.

Adiponectin is an important signaling molecule to improve insulin sensitivity mediated by PPARγ agonists.21 It is reported that an ACE inhibitor or ARB increased plasma adiponectin concentration.23,25 Our previous results indicated that the expression of adiponectin and adipocyte differentiation factors was increased in AT1a receptor/ApoEKO mice or with AT1a-receptor blocker.10 In the present study, the expression of adiponectin mRNA in adipose tissue of AT2/ApoEKO mice was lowered with ND (Figure 2a). After HCD, adiponectin mRNA level in adipose tissue was not significantly different between ApoEKO and AT2/ApoEKO mice. However, plasma adiponectin concentration was lower in AT2/ApoEKO mice than in ApoEKO mice. The discrepancy between adiponectin mRNA and protein in the HCD group is not yet clear. However, one possibility is that the plasma concentration of adiponectin is also regulated by the degradation rate and lack of AT2 receptor stimulation affects degradation of adiponectin protein. The reason why adiponectin mRNA is different in ND but not in HCD is also not yet clarified. One possible explanation is that the interaction between angiotensin II and other factors might cause such difference with HCD, as adiponectin production is regulated not only by angiotensin II but also by other factors, such as nutritional condition or hypoxia.26,27 In addition, when comparing adipocyte number and adiponectin mRNA expression in ND (Figures 1 and 2) to HCD (Figures 4 and 5), ApoEKO mice showed more adiponectin and adipocyte number in HCD than those in ND. One of possible explanations for such a change might be that the adiponectin expression and adipocyte number are influenced by aging. As the sampling was performed at 10 weeks of age for HCD group and at 6 months of age for ND group, the age-dependent changes might affect the basal level of adiponectin expression and adipocyte number. PPARγ is also important for insulin resistance and adipocyte differentiation.23,28 Our previous paper indicated that the reduced expression of PPARγ in adipose tissue in ApoEKO mice was recovered in AT1a/ApoEKO mice.10 In contrast, in the present study, expression of PPARγ in adipose tissue was decreased in AT2/ApoEKO mice with HCD (Figure 5). AT1 receptor antagonism by valsartan decreased plasma cholesterol and FFAs and increased adipocyte number and expression of adiponectin and PPARγ in adipose tissue (Figure 7). The effects of valsartan were not apparent in AT2/ApoEKO mice, suggesting that the contribution of AT1 receptor stimulation was weak in AT2/ApoEKO mice. These results indicate that AT2 receptor stimulation and AT1a receptor stimulation counter-regulate expression of adiponectin and PPARγ in adipose tissue and regulate adipocyte differentiation.

Although adipose tissue is important as a source of plasma angiotensinogen,29,30 expression of angiotensinogen in adipose tissue was not significantly affected by knocking out of the AT2 receptor in our study (data not shown). In AT2/ApoEKO mice with HCD, plasma concentrations of cholesterol and FFAs were increased (Figure 3). The mechanism of action of AT2 receptor stimulation on plasma level of cholesterol and fatty acids is not yet clear. However, it is possible that AT2 receptor is involved in cholesterol and fatty acid metabolism in the liver and other organs. AT2 receptor stimulation may act as a modulator of AT1 receptor stimulation in this metabolism under condition of cholesterol load. In the adipose tissue, protein level of IRS-1 was lower in AT2/ApoEKO mice than that of ApoEKO mice (Figure 6). As insulin sensitivity is regulated for the most by the degree of tyrosine phosphorylation of insulin receptor and IRS substrates,31 our result does not directly indicate the attenuation of insulin sensitivity. Role of AT2 receptor stimulation in insulin signaling of adipose tissue remains to be studied.

We have previously reported that the lack of AT1 a receptor reduced adipose tissue weight and adipocyte size, and recovered the expression of adipocyte differentiation markers in ApoEKO mice.10 Together with the results in the present study, it is suggested that AT1 receptor plays a major role in the regulation of adipose tissue function, and AT2 receptor stimulation acts as modulator of AT1 receptor stimulation. Furthermore, to examine whether the decrease in AT2 receptor expression is involved in adipose tissue change in ApoEKO mice, it would be better to study the effect of recruitment of AT2 receptor in ApoEKO mice. For this purpose, we could plan to perform the experiment with mice crossing ApoEKO mice to AT2 receptor transgenic mice in the future.

Taken together, our results suggest that AT2 receptor stimulation in adipose tissue accelerates adipocyte differentiation and insulin signaling, thereby reduces adipose tissue mass in atherosclerosis with hypercholesterolemia. The effects of AT2 receptor stimulation might be at least partially mediated by attenuating oxidative stress. The results in the present study suggest that AT2 receptor agonists could be useful for treating metabolic syndrome. We also believe that AT1 receptor antagonists that raise angiotensin II levels and indirectly stimulate AT2 receptors will be more metabolically helpful than ACE inhibitors that decrease angiotensin II levels and reduce AT2 stimulation.

Acknowledgments

This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan.

Disclosure

The authors declared no conflict of interest.

References

1.
Elliott
WJ
,
Meyer
PM
.
Incident diabetes in clinical trials of antihypertensive drugs: a network meta-analysis
.
Lancet
 
2007
;
369
:
201
207
.
2.
Dzau
V
.
The cardiovascular continuum and renin-angiotensin-aldosterone system blockade
.
J Hypertens Suppl
 
2005
;
23
:
S9
17
.
3.
Weir
MR
.
Effects of renin-angiotensin system inhibition on end-organ protection: can we do better?
Clin Ther
 
2007
;
29
:
1803
1824
.
4.
Kintscher
U
,
Foryst-Ludwig
A
,
Unger
T
.
Inhibiting angiotensin type 1 receptors as a target for diabetes
.
Expert Opin Ther Targets
 
2008
;
12
:
1257
1263
.
5.
Shiuchi
T
,
Cui
TX
,
Wu
L
,
Nakagami
H
,
Takeda-Matsubara
Y
,
Iwai
M
,
Horiuchi
M
.
ACE inhibitor improves insulin resistance in diabetic mouse via bradykinin and NO
.
Hypertension
 
2002
;
40
:
329
334
.
6.
Shiuchi
T
,
Iwai
M
,
Li
HS
,
Wu
L
,
Min
LJ
,
Li
JM
,
Okumura
M
,
Cui
TX
,
Horiuchi
M
.
Angiotensin II type-1 receptor blocker valsartan enhances insulin sensitivity in skeletal muscles of diabetic mice
.
Hypertension
 
2004
;
43
:
1003
1010
.
7.
Shao
J
,
Iwashita
N
,
Ikeda
F
,
Ogihara
T
,
Uchida
T
,
Shimizu
T
,
Uchino
H
,
Hirose
T
,
Kawamori
R
,
Watada
H
.
Beneficial effects of candesartan, an angiotensin II type 1 receptor blocker, on beta-cell function and morphology in db/db mice
.
Biochem Biophys Res Commun
 
2006
;
344
:
1224
1233
.
8.
Wei
Y
,
Sowers
JR
,
Clark
SE
,
Li
W
,
Ferrario
CM
,
Stump
CS
.
Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-kappaB activation via NADPH oxidase
.
Am J Physiol Endocrinol Metab
 
2008
;
294
:
E345
E351
.
9.
Henriksen
EJ
.
Improvement of insulin sensitivity by antagonism of the renin-angiotensin system
.
Am J Physiol Regul Integr Comp Physiol
 
2007
;
293
:
R974
R980
.
10.
Tomono
Y
,
Iwai
M
,
Inaba
S
,
Mogi
M
,
Horiuchi
M
.
Blockade of AT1 receptor improves adipocyte differentiation in atherosclerotic and diabetic models
.
Am J Hypertens
 
2008
;
21
:
206
212
.
11.
Sharma
AM
,
Staels
B
.
Review: Peroxisome proliferator-activated receptor gamma and adipose tissue—understanding obesity-related changes in regulation of lipid and glucose metabolism
.
J Clin Endocrinol Metab
 
2007
;
92
:
386
395
.
12.
Ahima
RS
.
Adipose tissue as an endocrine organ
.
Obesity (Silver Spring)
 
2006
;
14
(
Suppl 5
):
S242
S249
.
13.
Steckelings
UM
,
Kaschina
E
,
Unger
T
.
The AT2 receptor—a matter of love and hate
.
Peptides
 
2005
;
26
:
1401
1409
.
14.
Stoll
M
,
Unger
T
.
Angiotensin and its AT2 receptor: new insights into an old system
.
Regul Pept
 
2001
;
99
:
175
182
.
15.
de Gasparo
M
,
Catt
KJ
,
Inagami
T
,
Wright
JW
,
Unger
T
.
International union of pharmacology. XXIII. The angiotensin II receptors
.
Pharmacol Rev
 
2000
;
52
:
415
472
.
16.
Iwai
M
,
Chen
R
,
Li
Z
,
Shiuchi
T
,
Suzuki
J
,
Ide
A
,
Tsuda
M
,
Okumura
M
,
Min
LJ
,
Mogi
M
,
Horiuchi
M
.
Deletion of angiotensin II type 2 receptor exaggerated atherosclerosis in apolipoprotein E-null mice
.
Circulation
 
2005
;
112
:
1636
1643
.
17.
Wu
L
,
Iwai
M
,
Nakagami
H
,
Li
Z
,
Chen
R
,
Suzuki
J
,
Akishita
M
,
de Gasparo
M
,
Horiuchi
M
.
Roles of angiotensin II type 2 receptor stimulation associated with selective angiotensin II type 1 receptor blockade with valsartan in the improvement of inflammation-induced vascular injury
.
Circulation
 
2001
;
104
:
2716
2721
.
18.
Tsuda
M
,
Iwai
M
,
Li
JM
,
Li
HS
,
Min
LJ
,
Ide
A
,
Okumura
M
,
Suzuki
J
,
Mogi
M
,
Suzuki
H
,
Horiuchi
M
.
Inhibitory effects of AT1 receptor blocker, olmesartan, and estrogen on atherosclerosis via anti-oxidative stress
.
Hypertension
 
2005
;
45
:
545
551
.
19.
Iwai
M
,
Li
HS
,
Chen
R
,
Shiuchi
T
,
Wu
L
,
Min
LJ
,
Li
JM
,
Tsuda
M
,
Suzuki
J
,
Tomono
Y
,
Tomochika
H
,
Mogi
M
,
Horiuchi
M
.
Calcium channel blocker azelnidipine reduces glucose intolerance in diabetic mice via different mechanism than angiotensin receptor blocker olmesartan
.
J Pharmacol Exp Ther
 
2006
;
319
:
1081
1087
.
20.
Iwai
M
,
Chen
R
,
Imura
Y
,
Horiuchi
M
.
TAK-536, a new AT1 receptor blocker, improves glucose intolerance and adipocyte differentiation
.
Am J Hypertens
 
2007
;
20
:
579
586
.
21.
Kadowaki
T
,
Yamauchi
T
,
Kubota
N
.
The physiological and pathophysiological role of adiponectin and adiponectin receptors in the peripheral tissues and CNS
.
FEBS Lett
 
2008
;
582
:
74
80
.
22.
MacDougald
OA
,
Cornelius
P
,
Liu
R
,
Lane
MD
.
Insulin regulates transcription of the CCAAT/enhancer binding protein (C/EBP) alpha, beta, and delta genes in fully-differentiated 3T3-L1 adipocytes
.
J Biol Chem
 
1995
;
270
:
647
654
.
23.
Zorad
S
,
Dou
JT
,
Benicky
J
,
Hutanu
D
,
Tybitanclova
K
,
Zhou
J
,
Saavedra
JM
.
Long-term angiotensin II AT1 receptor inhibition produces adipose tissue hypotrophy accompanied by increased expression of adiponectin and PPARgamma
.
Eur J Pharmacol
 
2006
;
552
:
112
122
.
24.
Shimamoto
K
,
Miura
T
.
Clinical pathology and treatment of renin-angiotensin system 4. Renin-angiotensin system and insulin resistance
.
Intern Med
 
2007
;
46
:
1303
1304
.
25.
Furuhashi
M
,
Ura
N
,
Higashiura
K
,
Murakami
H
,
Tanaka
M
,
Moniwa
N
,
Yoshida
D
,
Shimamoto
K
.
Blockade of the renin-angiotensin system increases adiponectin concentrations in patients with essential hypertension
.
Hypertension
 
2003
;
42
:
76
81
.
26.
Hosogai
N
,
Fukuhara
A
,
Oshima
K
,
Miyata
Y
,
Tanaka
S
,
Segawa
K
,
Furukawa
S
,
Tochino
Y
,
Komuro
R
,
Matsuda
M
,
Shimomura
I
.
Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation
.
Diabetes
 
2007
;
56
:
901
911
.
27.
Turyn
J
,
Korczynska
J
,
Presler
M
,
Stelmanska
E
,
Goyke
E
,
Swierczynski
J
.
Up-regulation of rat adipose tissue adiponectin gene expression by long-term but not by short-term food restriction
.
Mol Cell Biochem
 
2008
;
312
:
185
191
.
28.
Bouskila
M
,
Pajvani
UB
,
Scherer
PE
.
Adiponectin: a relevant player in PPARgamma-agonist-mediated improvements in hepatic insulin sensitivity?
Int J Obes (Lond)
 
2005
;
29
(
Suppl 1
):
S17
S23
.
29.
Massiéra
F
,
Bloch-Faure
M
,
Ceiler
D
,
Murakami
K
,
Fukamizu
A
,
Gasc
JM
,
Quignard-Boulange
A
,
Negrel
R
,
Ailhaud
G
,
Seydoux
J
,
Meneton
P
,
Teboul
M
.
Adipose angiotensinogen is involved in adipose tissue growth and blood pressure regulation
.
FASEB J
 
2001
;
15
:
2727
2729
.
30.
Massiera
F
,
Seydoux
J
,
Geloen
A
,
Quignard-Boulange
A
,
Turban
S
,
Saint-Marc
P
,
Fukamizu
A
,
Negrel
R
,
Ailhaud
G
,
Teboul
M
.
Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotor activity
.
Endocrinology
 
2001
;
142
:
5220
5225
.
31.
Mahadev
K
,
Motoshima
H
,
Wu
X
,
Ruddy
JM
,
Arnold
RS
,
Cheng
G
,
Lambeth
JD
,
Goldstein
BJ
.
The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction
.
Mol Cell Biol
 
2004
;
24
:
1844
1854
.