Attenuation of Diet-Induced Weight Gain and Adiposity through Increased Energy Expenditure in Mice Lacking Angiotensin II Type 1a Receptor
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内分泌学杂志 2005年第8期
Departments of Molecular Medicine and Metabolism (R.K., T.S., J.N., M.T., T.T., M.K., T.C., Y.O.) and Clinical and Molecular Endocrinology (R.K., Y.H.), Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone (M.T., T.T., Y.O.), Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062; Department of Medicine, Division of Atherosclerosis and Diabetes, National Cardiovascular Center Hospital (Y.M., Y.Y.), Suita, Osaka 560-0005; Center for Tsukuba Advanced Research Alliance, Aspect of Functional Genomic Biology, Institute of Applied Biochemistry, University of Tsukuba (A.F.), Ten-noudai, Ibaraki 305-8577; and Department of Medical Biochemistry, Ehime University School of Medicine (M.H.), Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan
Address all correspondence and requests for reprints to: Dr. Yoshihiro Ogawa, Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: ogawa.mmm@mri.tmd.ac.jp.
Abstract
Given that angiotensin II (AII) type 1 and 2 receptors (Agtr1 and Agtr2) are expressed in adipose tissue, AII may act directly on adipose tissue. However, regardless of whether AII directly modulates adipose tissue growth and metabolism in vivo and, if so, whether it is mediated via Agtr1 are still matters of debate. To understand the functional role of Agtr1 in adipose tissue growth and metabolism in vivo, we examined the metabolic phenotypes of mice lacking Agtr1a (Agtr1a–/– mice) during a high-fat diet. The Agtr1a–/– mice exhibited the attenuation of diet-induced body weight gain and adiposity, and insulin resistance relative to wild-type littermates (Agtr1a+/+ mice). They also showed increased energy expenditure accompanied by sympathetic activation, as revealed by increased rectal temperature and oxygen consumption, increased expression of uncoupling protein-1 mRNA in brown adipose tissue, and increased urinary catecholamine excretion. The heterozygous Agtr1a-deficient mice (Agtr1a+/– mice) also exhibited metabolic phenotypes similar to those of Agtr1a–/– mice. Using mouse embryonic fibroblasts derived from Agtr1a+/+ and Agtr1a–/– mice, we found no significant difference between genotypes in the ability to differentiate into lipid-laden mature adipocytes. In primary cultures of mouse mature adipocytes, AII increased the expression of mRNAs for some adipocytokines, which was abolished by pharmacological blockade of Agtr1. This study demonstrates that Agtr1a–/– mice exhibit attenuation of diet-induced weight gain and adiposity through increased energy expenditure. The data also suggest that AII does not affect directly adipocyte differentiation, but can modulate adipocytokine production via Agtr1.
Introduction
THE RENIN-ANGIOTENSIN system (RAS) plays an important role in the regulation of body fluid homeostasis and blood pressure control. Angiotensin II (AII) is a potent hypertensive octapeptide that induces blood pressure elevation primarily through the activation of AII type 1 receptor (Agtr1) (1). Clinically, Agtr1 blockers (ARBs) have been used widely as antihypertensive drugs. Evidence has accumulated suggesting that activation of the RAS is a common feature in patients with the metabolic syndrome (2), a condition of clustering of independent risks of atherosclerosis, including abdominal obesity, insulin resistance (or impaired glucose metabolism), atherogenic dyslipidemia, and blood pressure elevation (3, 4). One of the keys to effectively preventing cardiovascular diseases, therefore, is to suppress RAS activation at an early stage of the metabolic syndrome. Several animal studies (5, 6) and recent clinical trials (7, 8) have suggested the antiatherogenic and antidiabetic effects of some ARBs beyond a reduction in blood pressure, which has been called the pleiotropic effect.
Adipose tissue is an important endocrine organ that secretes many biologically active substances, such as leptin, adiponectin, and IL-6, which are collectively termed adipocytokines (9). The dysregulation of adipocytokine functions seen in abdominal obesity may be involved in the pathogenesis of the metabolic syndrome. Angiotensinogen (Agt), the precursor of AII, is produced primarily by the liver. It also occurs in adipose tissue, where it is up-regulated during the development of obesity (10, 11, 12). A previous study demonstrated that Agt-deficient mice (Agt–/– mice) exhibit not only hypotension, but also impairment of diet-induced weight gain accompanied by increased locomotor activity (13). Furthermore, Massiéra et al. (14) showed that transgenic overexpression of Agt in adipose tissue leads to its secretion into the circulation and rescues hypotension and leanness of Agt–/– mice. These observations suggest that adipose-derived Agt, thus AII, is involved in blood pressure regulation and adipose tissue growth. Given that Agtr1 and Agtr2, another subtype of AII receptor, are both expressed in adipose tissue (15, 16), it is tempting to speculate that local as well as systemic AII may affect adipose tissue growth and metabolism. In this regard, Sharma et al. (17) demonstrated that AII inhibits directly human adipocyte differentiation via Agtr1in vitro and hypothesized that blockade of the RAS by either angiotensin-converting enzyme inhibitors or ARBs promotes the formation of fat cells and thus counteracts lipid accumulation in nonadipose tissue (skeletal muscle, liver, and pancreas, etc.) or lipotoxicity, thereby preventing diabetes (18). In contrast, a recent study showed that AII directly increases leptin release from isolated mature adipocytes in vitro, whereas systemic infusion of AII counteracts it through sympathetic activation in vivo (19), suggesting a differential effect of local vs. systemic AII in the adipose metabolism. Whether AII directly modulates adipose tissue growth and metabolism in vivo is still a matter of debate, and, if so, whether it is mediated through the activation of Agtr1 has not been tested rigorously.
It has been recognized that only rodents have duplicated Agtr1 genes (Agtr1a and Agtr1b) (20), whereas other mammals have a single Agtr1 gene (1). A large number of studies with Agtr1a–/– mice have revealed the physiological and pathophysiological roles of Agtr1 in the regulation of cardiovascular homeostasis in vivo (21, 22, 23, 24) and provided new insight into its clinical implication in human cardiovascular medicine. To explore the functional role of Agtr1 in adipose tissue growth and metabolism in vivo, we studied the metabolic phenotypes of Agtr1a–/– mice during a high-fat diet. Using mouse embryonic fibroblasts (MEFs) and primary cultures of mouse mature adipocytes, we also examined the role of Agtr1 in adipocyte differentiation and adipose gene expression in vitro.
Materials and Methods
Animals
The generation of male Agtr1a–/– mice on the C57BL/6J background was reported previously (21). Four-week-old male C57BL/6J mice were purchased from Oriental Yeast, Co., Ltd. (Tokyo, Japan). The animals were housed in individual cages in a temperature-, humidity-, and light-controlled room (12-h light, 12-h dark cycle) and were allowed free access to water and standard chow (362 kcal/100 g; 5.4% energy as fat; Oriental MF, Oriental Yeast, Co., Ltd.) unless otherwise noted. In the high-fat feeding experiments, Agtr1a–/– mice were given free access to water and either the standard chow or high-fat diet (556 kcal/100 g; 60% energy as fat). All animal experiments were conducted according to the guidelines of Tokyo Medical and Dental University committee on animal research.
Blood and urinary parameter analyses
Inferior vena cava puncture was performed at the time mice were killed in the fed state. Blood was also sampled from the tail vein of mice when glucose and insulin tolerance tests (GTT and ITT, respectively) were performed. Blood glucose was measured by the blood glucose test meter (Glutest PRO R, Sanwa-Kagaku, Nagoya, Japan). Enzymatic assay kits were used for the determination of serum triglyceride and free fatty acid (Wako Pure Chemical, Osaka, Japan). Serum insulin concentrations were measured by a commercially available ELISA kit (Morinaga, Tokyo, Japan). Serum creatinine and blood urea nitrogen were measured by the standard method. For urinary parameter analysis, 6-wk-old mice were placed in metabolic cages and given free access to food and water. Daily urinary catecholamine excretion was determined by reverse phase HPLC (25).
GTT and ITT
GTT was performed in 13-wk-old mice after a 6-h fast. Blood glucose concentrations were measured at 0 min before and 30, 60, and 120 min after ip injection of glucose (1 g/kg body weight). For ITT, insulin (0.7 U/kg body weight in 0.1% BSA; Humulin R-Insulin, Eli Lilly & Co., Indianapolis, IN) was injected ip after a 1-h fast. Blood glucose concentrations were measured 0 min before and 30 and 60 min after the injection.
Oxygen consumption and rectal temperature analysis
Oxygen consumption was measured with an O2/CO2 metabolism-measuring system (model MK-5000, Muromachikikai, Tokyo, Japan) (26). Each mouse was placed in a sealed chamber (560-ml volume) with an air flow of 0.60 liters/min for 22 h at 23 C. Air was taken every 3 min, and the consumed oxygen concentration was converted to milliliters per minute by multiplying it by the flow. Oxygen consumption was normalized by kilogram0.75 body weight. Respiratory quotient, the ratio of carbon dioxide production to oxygen consumption was also measured. Rectal temperature was measured using an electron thermistor equipped with rectal probe (Model TD-300, Shibaura Electronics, Saitama, Japan).
Blood pressure analysis
Systolic blood pressure was measured by the indirect tail-cuff method (BP-98A, Softron, Tokyo, Japan) as previously described (27). At least six readings were taken for each measurement.
Histological analysis
The epididymal white adipose tissue (WAT) was fixed with neutral-buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and studied under x200 magnification to compare adipocyte size. The adipocyte diameter and area were measured using Win Roof software (Mitani Co., Ltd., Chiba, Japan).
Quantitative real-time PCR
Total RNA was extracted from mouse epididymal and mesenteric WATs, interscapular brown adipose tissue (BAT), and mature adipocytes and stromal vascular fraction (SVF) obtained from mouse epididymal WAT using the acid guanidinium-phenol-chloroform method. Total RNA (5 μg) was reverse transcribed in a 20-μl reaction containing random primers and SuperScript II enzyme (Invitrogen Life Technologies, Inc., Carlsbad, CA). Quantitative real-time PCR was performed with an ABI PRISM 7000 Sequence Detection System using TaqMan or SYBR Green PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA). The primers used were as follows: adiponectin: sense, 5'-ATGGCAGAGATGGCACTCCT-3'; antisense, 5'-CCTTCAGCTCCTGTCATTCCA-3'; and oligonucleotide probe, 5'-AGGTCTTCTTGGTCCTAAGGGTGAGACA-3'; monocyte chemoattractant protein-1 (MCP-1): sense, 5'-CCACTCACCTGCTGCTACTCAT-3'; antisense, 5'-TGGTGATCCTCTTGTAGCTCTCC-3'; and oligonucleotide probe, 5'-CACCAGCAAGATGATCCCAATGAGTAGGC-3'; IL-6: sense, 5'-ACAACCACGGCCTTCCCTACTT-3'; and antisense: 5'-CACGATTTCCCAGAGAACATGTG-3'; plasminogen activator inhibitor-1 (PAI-1): sense, 5'-GGCAGATCCAAGATGCTATGG-3'; and antisense, 5'-CAAAGATGGCATCCGCAGTA-3'; uncoupling protein-1 (UCP-1): sense, 5'-CGCTGGACACTGCCAAAGT-3'; and antisense, 5'-GGTGGTGATGGTCCCTACCA-3'; Agtr1a: sense, 5'-TTTCCAGATCAAGTGCATTTTGA-3'; and antisense, 5'-AGAGTTAAGGGCCATTTTGCTTT-3'; and 36B4: sense, 5'-GGCCCTGCACTCTCGCTTTC-3'; and antisense, 5'-TGCCAGGACGCGCTTGT-3'. The sense and antisense primers and specific oligonucleotide probe for leptin were obtained from Applied Biosystems. Levels of mRNA were normalized to those of 18S rRNA (primers obtained from Applied Biosystems) or 36B4 mRNA. Leptin, adiponectin, and MCP-1 mRNA levels were normalized with 18S rRNA, whereas IL-6, PAI-1, UCP-1, and AT1a mRNA levels were normalized with 36B4 mRNA.
Cell culture experiments
The lipid-laden mature adipocytes and SVF were prepared from the epididymal WAT from 8-wk-old Agtr1a+/+ and Agtr1a–/– mice as previously described (28). They were subjected to total RNA extraction for Agtr1a mRNA analysis. The mature adipocytes were also treated for 24 h with AII (100 nM) in the presence or absence of valsartan (10 μM; Novartis Pharmaceuticals, East Hanover, NJ), an ARB.
MEFs were prepared from Agtr1a+/+ and Agtr1a–/– mice as previously described (29). They were maintained in DMEM containing 10% fetal bovine serum (FBS), grown to 2 d after confluence (designated d 0), and then subjected to differentiation by DMEM containing 10% FBS, 5 μg/ml insulin, 0.25 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methlyxanthine until d 2. They were fed DMEM supplemented with 10% FBS/5 μg/ml insulin for 2 d, then fed every other day with DMEM containing 10% FBS in the presence or absence of AII (100 nM). Cells at d 8 were also stained with Oil-Red O. Lipid accumulation was quantified by a model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA).
Statistical analysis
Data were expressed as the mean ± SE. Statistical analysis was performed using ANOVA, followed by Scheffé’s test. P < 0.05 was considered statistically significant.
Results
High-fat diet experiments
Reduced adiposity in Agtr1a–/– mice on a high-fat diet.
We examined the effect of high-fat diet on the metabolic phenotypes of Agtr1a–/– mice. On a standard diet, body weight gain was indistinguishable between wild-type Agtr1a+/+ and Agtr1a–/– mice throughout the experiments (Fig. 1A). The Agtr1a+/+ mice fed a high-fat diet weighed 44% more than those fed a standard diet for 8 wk (39.7 ± 1.0 vs. 27.6 ± 0.6 g; P < 0.05). There was no significant difference in body weight between Agtr1a+/+ and Agtr1a–/– mice fed a standard diet (27.6 ± 0.6 vs. 29.9 ± 0.7 g). By contrast, Agtr1a–/– mice were protected from high-fat diet-induced obesity. At the end of the experiments, the body weight of Agtr1a–/– mice was 31.7 ± 1.1 g, which was approximately 80% that of Agtr1a+/+ mice (P < 0.05).
FIG. 1. Attenuation of diet-induced weight gain and adiposity in Agtr1a–/– mice. A, Growth curve of Agtr1a+/+ and Agtr1a–/– mice on either a standard diet (SD) or a high-fat diet (HFD). Agtr1a+/+ (n = 6; ) and Agtr1a–/– (n = 7; ) mice on SD are shown. Agtr1a+/+ (n = 16; ) and Agtr1a–/– (n = 14; ) mice on HFD are shown. *, P < 0.05. B, Abdominal dissection of Agtr1a+/+ and Agtr1a–/– mice on either SD or HFD. C, Weight of the epididymal (Epi), mesenteric (Mes), and sc (Sub) WAT; interscapular BAT; and liver in Agtr1a+/+ and Agtr1a–/– mice on either SD or HFD. D, Hematoxylin and eosin staining of epididymal WAT from Agtr1a+/+ and Agtr1a–/– mice on either SD or HFD. Original magnification, x200. Scale bars, 50 μm. E, Adipocyte diameter and area in epididymal WAT. Lane 1, Agtr1a+/+/SD (n = 6); lane 2, Agtr1a+/+/HFD (n = 16); lane 3, Agtr1a–/–/SD (n = 7); lane 4, Agtr1a–/–/HFD (n = 14). *, P < 0.01 vs. Agtr1a+/+/SD; #, P < 0.05 vs. Agtr1a–/–/SD; , P < 0.05 between the indicated groups.
Gross inspection of abdominal dissection revealed a reduced mass of adipose tissue in Agtr1a–/– mice compared with Agtr1a+/+ mice (Fig. 1B). The weights of the epididymal, mesenteric, and sc fat depots were increased significantly in Agtr1a+/+ mice fed a high-fat diet relative to those in Agtr1a+/+ mice fed a standard diet (P < 0.05; Fig. 1C). The weights of both epididymal and sc fat depots were increased significantly in Agtr1a–/– mice fed a high-fat diet relative to those fed a standard diet (P < 0.05), which were markedly reduced compared with those of Agtr1a+/+ mice fed a high-fat diet. The weight of interscapular BAT was also increased significantly in Agtr1a+/+ mice fed a high-fat diet compared with those of Agtr1a+/+ mice fed a standard diet (P < 0.05), whereas no significant difference was noted between Agtr1a–/– mice fed a high-fat diet and those fed a standard diet. In this study no significant differences in liver weight were noted between genotypes on either a standard or a high-fat diet (Fig. 1C). Histological examination revealed no appreciable difference in the size of epididymal mature adipocytes between Agtr1a+/+ and Agtr1a–/– mice fed a standard diet. However, the size of mature adipocytes in Agtr1a–/– mice was significantly smaller than that in Agtr1a+/+ mice on a high-fat diet (P < 0.05; Fig. 1, D and E).
Increased energy expenditure in Agtr1a–/– mice on a high-fat diet.
Agtr1a–/– mice ate more daily than Agtr1a+/+ mice on a calorie basis (P < 0.05) when fed a standard diet (Fig. 2A). However, Agtr1a–/– mice did not differ from Agtr1a+/+ mice in daily food intake on a high-fat diet. In this study, rectal temperature was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice on either a standard or a high-fat diet (P < 0.05; Fig. 2B). Oxygen consumption, an indirect measurement of metabolism, was increased in Agtr1a–/– mice compared with Agtr1a+/+ mice in both the light and dark phases on a high-fat diet (P < 0.05; Fig. 2C). There was no significant difference in respiratory quotient between Agtr1a+/+ and Agtr1a–/– mice on a high-fat diet (data not shown).
FIG. 2. Increased energy expenditure in Agtr1a–/– mice. A, Daily food intake on a calorie basis during a high fat diet (HFD) from 7–12 wk of age. B, Rectal temperature at 14 wk of age. C, Oxygen consumption after 8 wk of an HFD. Upper panel, Daily profile of oxygen consumption. Agtr1a+/+ () and Agtr1a–/– () mice on an HFD are shown (n = 10 in each group). Lower panel, Quantification of oxygen consumption. Lane 1, Agtr1a+/+/standard diet (SD; n = 5); lane 2, Agtr1a+/+/HFD (n = 10); lane 3, Agtr1a–/–/SD (n = 6); lane 4, Agtr1a–/–/HFD (n = 10). *, P < 0.05 vs. Agtr1a+/+/SD; , P < 0.05 between the indicated groups.
Blood and urinary parameters and blood pressure in Agtr1a–/– mice on a high-fat diet.
We next examined blood glucose and lipid metabolism in Agtr1a+/+ and Agtr1a–/– mice on a high-fat diet. Blood glucose and serum insulin concentrations were increased in Agtr1a+/+ mice fed a high-fat diet compared with those fed a standard diet (P < 0.05; Fig. 3A). However, these levels were suppressed in Agtr1a–/– mice during a high-fat diet (P < 0.05). There was no significant difference in serum triglyceride and free fatty acids between Agtr1a+/+ and Agtr1a–/– mice on a high-fat diet (Fig. 3A).
FIG. 3. Glucose and lipid metabolism in Agtr1a+/+ and Agtr1a–/– mice. A, Nonfasting blood glucose, serum insulin, free fatty acid, and triglyceride concentrations at 14 wk of age. Lane 1, Agtr1a+/+/standard diet (SD; n = 6); lane 2, Agtr1a+/+/high-fat diet (HFD; n = 16); lane 3, Agtr1a–/–/SD (n = 7); lane 4, Agtr1a–/–/HFD (n = 14). *, P < 0.05 vs. Agtr1a+/+/SD; #, P < 0.05 vs. Agtr1a–/–/SD; , P < 0.05 between the indicated groups. B, The ip GTT after a 6-h fast at 13 wk of age. C, The ip ITT after a 1-h fast at 14 wk of age. Agtr1a+/+ () and Agtr1a–/– () mice on SD, and Agtr1a+/+ () and Agtr1a–/– () mice on HFD are shown (n = 6 in each group). , P < 0.05 between Agtr1a+/+/HFD and Agtr1a–/–/HFD.
The ip GTT revealed increased glucose disposal in Agtr1a–/– mice compared with Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 3B). Furthermore, the ip ITT demonstrated that the hypoglycemic response is exaggerated in Agtr1a–/– mice compared with Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 3C). These observations indicate that Agtr1a–/– mice are protected from high-fat diet-induced impairment of glucose tolerance and insulin resistance.
The tail cuff systolic blood pressure of Agtr1a–/– mice was significantly lower than that of Agtr1a+/+ mice (P < 0.05; Table 1). However, no significant difference in urine volume was noted between Agtr1a+/+ and Agtr1a–/– mice. Serum creatinine levels tended to be increased in Agtr1a–/– mice compared with Agtr1a+/+ mice. Urinary norepinephrine excretion was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice (P < 0.05), whereas urinary epinephrine excretion tended to be increased in Agtr1a–/– mice compared with Agtr1a+/+ mice (Table 1).
TABLE 1. Blood pressure and urinary parameters at 6 wk of age
Systolic blood pressure tended to be increased in Agtr1a+/+ mice fed a high-fat diet for 16 wk compared with those fed a standard diet (Table 2). Blood pressure was significantly lower in Agtr1a–/– mice than in Agtr1a+/+ mice on a standard diet as reported previously (P < 0.05). No significant difference in blood pressure was noted between Agtr1a–/– mice fed a high-fat diet and those fed a standard diet. Urinary norepinephrine excretion was increased significantly in Agtr1a+/+ mice fed a high-fat diet for 16 wk compared with those fed a standard diet (P < 0.05; Table 2). There was no significant difference between Agtr1a–/– mice fed a high-fat diet and those a fed standard diet. Urinary norepinephrine excretion was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice on either a standard or a high-fat diet (P < 0.05). Urinary epinephrine excretion was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice on a standard diet (P < 0.05). During a high-fat diet, urinary epinephrine excretion tended to be increased in Agtr1a–/– mice compared with Agtr1a+/+ mice. These observations indicate the sympathetic activation in Agtr1a–/– mice compared with Agtr1a+/+ mice.
TABLE 2. Blood pressure and urinary parameters after 16 wk of a high-fat diet
Quantitative real-time PCR analysis.
By quantitative real-time PCR analysis, we examined the expression of mRNAs for leptin, adiponectin, MCP-1, IL-6, and PAI-1 in epididymal and mesenteric WAT from Agtr1a+/+ and Agtr1a–/– mice during a high-fat diet. The expression of leptin mRNA was increased significantly in both epididymal and mesenteric WAT from Agtr1a+/+ mice fed a high-fat diet compared with those fed a standard diet (P < 0.05; Fig. 4A). In this study, leptin mRNA expression was also up-regulated in the epididymal WAT from Agtr1a–/– mice fed a high-fat diet compared with those fed a standard diet (P < 0.05). No significant difference in leptin mRNA expression in epididymal WAT was noted between genotypes. In mesenteric WAT, the expression of leptin mRNA was markedly reduced in Agtr1a–/– mice compared with Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 4A). Adiponectin mRNA expression was decreased in the epididymal WAT from Agtr1a+/+ mice fed a high-fat diet compared with those fed a standard diet (P < 0.05). It tended to be decreased in mesenteric WAT from Agtr1a+/+ mice fed a high-fat diet. No significant difference in adiponectin mRNA expression was noted between genotypes.
FIG. 4. Adipose gene expression in Agtr1a+/+ and Agtr1a–/– mice. A, Expression of mRNAs for leptin, adiponectin, MCP-1, IL-6, and PAI-1 in epididymal (Epi) and mesenteric (Mes) WAT from Agtr1a+/+ and Agtr1a–/– mice. B, Expression of UCP-1 mRNA in BAT from Agtr1a+/+ and Agtr1a–/– mice. Lane 1, Agtr1a+/+/standard diet (SD; n = 5); lane 2, Agtr1a+/+/high-fat diet (HFD; n = 10); lane 3, Agtr1a–/–/SD (n = 6); lane 4, Agtr1a–/–/HFD (n = 10). *, P < 0.05 vs. Agtr1a+/+/SD; #, P < 0.05 vs. Agtr1a–/–/SD; , P < 0.05 between the indicated groups.
During a high-fat diet, MCP-1 mRNA expression was increased significantly in epididymal and mesenteric WAT from Agtr1a+/+ mice (P < 0.05), but was reduced significantly in those from Agtr1a–/– mice (P < 0.05; Fig. 4A). During a high-fat diet, IL-6 mRNA expression was also increased significantly in epididymal WAT from Agtr1a+/+ mice (P < 0.05) and was reduced significantly in Agtr1a–/– mice (P < 0.05; Fig. 4A). There was no significant difference in IL-6 mRNA levels in mesenteric WAT from Agtr1a+/+ and Agtr1a–/– mice. In this study, there was no significant difference in PAI-1 mRNA expression in epididymal WAT from Agtr1a+/+ and Agtr1a–/– mice. In mesenteric WAT, PAI-1 mRNA expression was significantly increased in Agtr1a+/+ mice fed a high-fat diet (P < 0.05), whereas it was markedly reduced in Agtr1a–/– mice (P < 0.05).
In this study, we found no significant difference in UCP-1 mRNA expression in interscapular BAT between Agtr1a+/+ mice fed a standard diet and those fed a high-fat diet (Fig. 4B). The expression of UCP-1 mRNA in BAT was increased significantly in Agtr1a–/– mice fed a high-fat diet compared with those fed a standard diet (P < 0.05). Consequently, UCP-1 mRNA level in BAT from Agtr1a–/– mice was approximately 1.4-fold higher than that in Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 4B).
Metabolic phenotypes of heterozygous Agtr1a+/– mice.
We also examined the metabolic phenotypes of heterozygous Agtr1a+/– mice on a high-fat diet. There was no significant difference in body weight between Agtr1a+/+ and Agtr1a+/– mice on a standard diet at 14 wk of age (29.6 ± 0.6 vs. 29.3 ± 0.7 g). The body weight of Agtr1a+/– mice fed a high-fat diet was also significantly increased compared with those fed standard diet (P < 0.05). However, the body weight of Agtr1a+/– was reduced significantly compared with those of Agtr1a+/+ mice during a high-fat diet at 14 wk of age (32.4 ± 0.6 vs. 40.4 ± 0.7 g; P < 0.05). We also observed a reduced mass of adipose tissue in Agtr1a+/– mice compared with Agtr1a+/+ mice during a high-fat diet (data not shown).
Agtr1a+/– mice did not differ from Agtr1a+/+ mice in daily food intake on a high-fat diet (data not shown). In this study, rectal temperature was increased in Agtr1a+/– mice compared with Agtr1a+/+ mice on a high-fat diet (P < 0.05; data not shown). Oxygen consumption was increased significantly in Agtr1a+/– mice compared with Agtr1a+/+ mice in both the light and dark phases on a high-fat diet (P < 0.05; data not shown).
The ip GTT revealed increased glucose disposal in Agtr1a+/– mice compared with Agtr1a+/+ mice during a high-fat diet for 16 wk (P < 0.05; data not shown).
Blood pressure was reduced significantly in Agtr1a+/– mice compared with Agtr1a+/+ mice on a standard diet as reported previously (P < 0.05; data not shown). However, no significant differences in urine volume or serum creatinine were noted between Agtr1a+/+ and Agtr1a+/– mice. Urinary norepinephrine excretion was increased significantly in Agtr1a+/– mice compared with Agtr1a+/+ mice (P < 0.05; data not shown). These observations indicate that heterozygous Agtr1a+/– mice show reduced weight gain and adiposity during a high-fat diet.
Cell culture experiments
Functional significance of Agtr1 in adipocyte differentiation.
Agtr1a mRNA was expressed in MEFs, the level of which was unchanged during the course of adipocyte differentiation (our unpublished observations). We examined the functional significance of Agtr1a in adipocyte differentiation using MEFs obtained from Agtr1a+/+ and Agtr1a–/– mice. In this study, treatment with AII at a dose of 100 nM did not affect adipocyte differentiation of MEFs derived from Agtr1a+/+ mice, as revealed by Oil-Red O staining (Fig. 5A). Furthermore, there was no significant difference in adipocyte differentiation and lipid accumulation between AII- and vehicle-treated MEFs derived from Agtr1a–/– mice (Fig. 5A).
FIG. 5. Functional significance of Agtr1 in adipocyte differentiation and adipose gene expression in vitro. A, Left, Adipocyte differentiation and lipid accumulation revealed by Oil-Red O staining in AII- and vehicle-treated MEFs obtained from Agtr1a+/+ and Agtr1a–/– mice. Right, Quantification of lipid accumulation. diff., Differentiation. n = 6 in each group. B, Expression of Agtr1a mRNA in mature adipocytes and SVF obtained from epididymal WAT from Agtr1a+/+ mice. M, Mature adipocytes. n = 4 in each group. C, Effects of AII and/or ARB on adipose gene expression in mature adipocytes obtained from epididymal WAT from Agtr1a+/+ mice. Val, Valsartan. *, P < 0.05 vs. control; #, P < 0.05 vs. AII-treated group. n = 6 in each group.
Expression of Agtr1a mRNA and its functional significance in mature adipocytes.
We confirmed that Agtr1a mRNA is expressed in both mature adipocytes and SVF obtained from Agtr1a+/+ mice (Fig. 5B). Using primary cultures of mature adipocytes obtained from epididymal WAT, we also examined the effect of AII on adipose gene expression. We found no significant change in leptin and adiponectin mRNA levels between AII- and vehicle-treated mature adipocytes. By contrast, treatment with AII significantly increased MCP-1, IL-6, and PAI-1 mRNA expression in mature adipocytes (P < 0.05; Fig. 5C). Increased expression of MCP-1, IL-6, and PAI-1 mRNAs was abolished by coadministration of valsartan (10 μM; P < 0.05; Fig. 5C).
Discussion
The RAS plays a critical role in the regulation of body fluid homeostasis and blood pressure control. Recent observations, however, have suggested that it also participates in the regulation of glucose and lipid metabolism (16). Agtr1a–/– mice have facilitated understanding of the functional role of Agtr1 in the regulation of cardiovascular homeostasis in vivo (21, 22, 23, 24). This study was designed to elucidate the functional role of Agtr1 in adipose tissue growth and metabolism using Agtr1a–/– mice.
We have demonstrated for the first time that Agtr1a–/– mice exhibit the attenuation of diet-induced weight gain and adiposity accompanied by increased sympathetic activity and energy expenditure compared with Agtr1a+/+ mice. In this study, Agtr1a+/+ and Agtr1a–/– mice do not differ significantly in food intake on a high-fat diet. Therefore, the attenuation of diet-induced weight gain and adiposity in Agtr1a–/– mice may be due to increased energy expenditure. The Agtr1a–/– mice are also hypotensive and show attenuation of impaired glucose tolerance and insulin resistance compared with Agtr1a+/+ mice on a high-fat diet. In contrast, there is no appreciable difference in lipid metabolism between Agtr1a+/+ and Agtr1a–/– mice. These observations indicate that Agtr1a–/– mice are protected from some components of the metabolic syndrome during a high-fat diet, suggesting that they are the unique experimental model system to investigate the role of Agtr1 in this pathological condition.
The metabolic phenotypes of Agtr1a–/– mice revealed in this study are similar to, although less severe than, those of Agt–/– mice as described previously (13). Furthermore, heterozygous Agtr1a+/– mice exhibit similar phenotypes to those of Agtr1a–/– mice on a high-fat diet. These observations suggest that Agtr1a is important for AII regulation of adipose tissue growth and metabolism in vivo. In this context, Tsuchida et al. (30, 31) previously demonstrated that targeted disruption of genes encoding both Agtr1a and Agtr1b simultaneously in mice produces specific abnormalities in renal morphology and function that are quantitatively similar to those found in Agt–/– mice, whereas mice lacking either Agtr1a or Agtr1b show milder renal abnormalities. These observations suggest that major biological functions of endogenous AII revealed by the abnormal renal phenotypes of Agt–/– mice are mediated by both Agtr1a and Agtr1b. Recently, we examined the effect of valsartan on the metabolic phenotypes of Agtr1a+/+ and Agtr1a–/– mice fed a standard diet and found that urinary catecholamine excretion is significantly reduced in valsartan-treated Agtr1a–/– mice compared with vehicle-treated groups (our unpublished observations). It has been reported that Agtr1b is expressed in a variety of tissues, such as the adrenal gland and sympathetic nerve terminals (32, 33, 34), suggesting that sympathetic activation in Agtr1a–/– mice is due at least in part to Agtr1b. In contrast, a previous study showed that AII increases lipogenesis in 3T3-L1 and human mature adipocytes, possibly through the activation of Agtr2 in vitro (35). In this context, Yvan-Charvet et al. (36) recently demonstrated that deletion of Agtr2 protects from diet-induced obesity and insulin resistance. We also examined the effect of a high-fat diet on the metabolic phenotypes of Agtr2y–/– mice and found that body weight and epididymal adipose tissue weight were reduced significantly in Agtr2y/– mice compared with Agtr2y/+ mice on a high-fat diet (our unpublished observations). These observations, taken together, suggest that Agtr2 is important for the regulation of energy homeostasis. Because plasma AII concentrations are elevated in Agtr1a–/– mice compared with Agtr1a+/+ mice (24), it is possible that the effect of AII via Agtr1b and Agtr2 contributes to the metabolic phenotypes of Agtr1a–/– mice. There are several reports that systemic or central administration of AII in rats decreases body weight and adipose tissue weight, which is attributable to both reduced food intake and increased peripheral metabolism (37, 38, 39). Reductions in body weight and adipose tissue growth in rats treated with AII and in Agtr1a–/– mice may be related to the activation of Agtr1b and Agtr2. Additional studies are needed to elucidate the possible roles of Agtr1b and Agtr2 in the metabolic phenotypes of Agtr1a–/– mice.
The tissue(s) or organ(s) responsible for the metabolic phenotypes of Agtr1a–/– mice is currently unclear. Massiéra et al. (14) demonstrated that transgenic overexpression of Agt in adipose tissue from Agt–/– mice, in which Agt is present in the systemic circulation at 20–30% the levels in Agt+/+ mice, rescues hypotension and leanness in Agt–/– mice. These observations suggest that the adipose-derived Agt, thus AII, is involved in blood pressure regulation and adipose tissue growth in the absence of systemic AII. It is unlikely that adipose-derived or peripherally produced AII is transported directly into the brain through the blood-brain barrier (40), where it may modulate the sympathetic nervous system and adipose tissue growth. In this regard, there are a couple of reports suggesting that circulating AII increases peripheral sympathetic nerve activity via Agtr1 (41, 42). Improved insulin resistance in Agtr1a–/– mice on a high-fat diet may be secondary to the attenuation of diet-induced weight gain and adiposity in these animals. Glucose metabolism might also be increased in skeletal muscle from Agtr1a–/– mice, because treatment with valsartan enhances insulin sensitivity in skeletal muscle from diabetic mice (6).
It is important to know whether AII can directly regulate adipose tissue growth and metabolism. In this study using MEFs derived from Agtr1a+/+ and Agtr1a–/– mice, we found no significant difference between genotypes in the ability to differentiate into lipid-laden mature adipocytes. We also confirmed no significant difference in lipid accumulation between AII- and vehicle-treated groups in the 3T3-L1 preadipocyte cell line (our unpublished observations). These findings indicate that AII does not directly affect adipocyte differentiation through the activation of Agtr1a in vitro. We, therefore, speculate that basal adipose tissue growth is not related to the direct action of AII on adipose tissue. However, part of the metabolic phenotypes of Agtr1a–/– mice fed a high-fat diet might be due to the direct action of AII on adipose tissue, because AII is capable of regulating the expression of some of the adipocytokines. In this context, Sharma et al. (17) previously reported that AII inhibits adipocyte differentiation from human preadipocytes via Agtr1 in vitro, whereas blockade of this receptor enhances adipogenesis, and they hypothesized that blockade of the formation of AII produced by large insulin-resistant adipocytes promotes preadipocyte recruitment and increases the number of small adipocytes, resulting in improved insulin sensitivity (18). There might be a species difference with regard to the role of AII in adipocyte differentiation.
We also found that AII increases the expression of mRNAs for some adipocytokines (MCP-1, IL-6, and PAI-1) in primary cultures of mature mouse adipocytes, which was abolished by pharmacological blockade of Agtr1. These observations indicate that AII is capable of directly regulating the production of some adipocytokines via Agtr1. It was reported that AII directly stimulates the release of PAI-1 and IL-6 from cultured human adipocytes via Agtr1 in vitro (43, 44). In this study, there was no significant effect of AII on leptin and adiponectin mRNA expression. Recently, Cassis et al. (19) reported that AII directly increases leptin release from isolated mature adipocytes in vitro, whereas systemic infusion of AII counteracts it through sympathetic activation in vivo, suggesting a differential effect of local vs. systemic AII in adipose metabolism. Furthermore, Furuhashi et al. (45) showed that blockade of the RAS by temocapril or candesartan increases adiponectin concentrations in patients with essential hypertension. However, Fasshauer et al. (46) showed that AII does not affect adiponectin gene expression in 3T3-L1 adipocytes. Adipose tissue is composed of various cell types: mature adipocytes and SVF, including preadipocytes, endothelial cells, lymphocytes, and macrophages. Because Agtr1 is expressed in both mature adipocytes and SVF, it is tempting to speculate about a regulatory role for Agtr1 in the paracrine interaction between adipocytes and other cell types within adipose tissue in vivo.
In conclusion, this study demonstrates that Agtr1a–/– mice exhibit attenuation of diet-induced weight gain and adiposity through increased energy expenditure. The data from this study also suggest that AII does not directly affect adipocyte differentiation, but can modulate adipocytokine production via Agtr1.
Acknowledgments
We thank Dr. Mutsuo Taiji for advice on oxygen consumption measurement, Ms. Rie Kodani for technical assistance, and Ms. Ai Togo for secretarial assistance. We also acknowledge Novartis Pharma Co. for valsartan.
References
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Address all correspondence and requests for reprints to: Dr. Yoshihiro Ogawa, Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: ogawa.mmm@mri.tmd.ac.jp.
Abstract
Given that angiotensin II (AII) type 1 and 2 receptors (Agtr1 and Agtr2) are expressed in adipose tissue, AII may act directly on adipose tissue. However, regardless of whether AII directly modulates adipose tissue growth and metabolism in vivo and, if so, whether it is mediated via Agtr1 are still matters of debate. To understand the functional role of Agtr1 in adipose tissue growth and metabolism in vivo, we examined the metabolic phenotypes of mice lacking Agtr1a (Agtr1a–/– mice) during a high-fat diet. The Agtr1a–/– mice exhibited the attenuation of diet-induced body weight gain and adiposity, and insulin resistance relative to wild-type littermates (Agtr1a+/+ mice). They also showed increased energy expenditure accompanied by sympathetic activation, as revealed by increased rectal temperature and oxygen consumption, increased expression of uncoupling protein-1 mRNA in brown adipose tissue, and increased urinary catecholamine excretion. The heterozygous Agtr1a-deficient mice (Agtr1a+/– mice) also exhibited metabolic phenotypes similar to those of Agtr1a–/– mice. Using mouse embryonic fibroblasts derived from Agtr1a+/+ and Agtr1a–/– mice, we found no significant difference between genotypes in the ability to differentiate into lipid-laden mature adipocytes. In primary cultures of mouse mature adipocytes, AII increased the expression of mRNAs for some adipocytokines, which was abolished by pharmacological blockade of Agtr1. This study demonstrates that Agtr1a–/– mice exhibit attenuation of diet-induced weight gain and adiposity through increased energy expenditure. The data also suggest that AII does not affect directly adipocyte differentiation, but can modulate adipocytokine production via Agtr1.
Introduction
THE RENIN-ANGIOTENSIN system (RAS) plays an important role in the regulation of body fluid homeostasis and blood pressure control. Angiotensin II (AII) is a potent hypertensive octapeptide that induces blood pressure elevation primarily through the activation of AII type 1 receptor (Agtr1) (1). Clinically, Agtr1 blockers (ARBs) have been used widely as antihypertensive drugs. Evidence has accumulated suggesting that activation of the RAS is a common feature in patients with the metabolic syndrome (2), a condition of clustering of independent risks of atherosclerosis, including abdominal obesity, insulin resistance (or impaired glucose metabolism), atherogenic dyslipidemia, and blood pressure elevation (3, 4). One of the keys to effectively preventing cardiovascular diseases, therefore, is to suppress RAS activation at an early stage of the metabolic syndrome. Several animal studies (5, 6) and recent clinical trials (7, 8) have suggested the antiatherogenic and antidiabetic effects of some ARBs beyond a reduction in blood pressure, which has been called the pleiotropic effect.
Adipose tissue is an important endocrine organ that secretes many biologically active substances, such as leptin, adiponectin, and IL-6, which are collectively termed adipocytokines (9). The dysregulation of adipocytokine functions seen in abdominal obesity may be involved in the pathogenesis of the metabolic syndrome. Angiotensinogen (Agt), the precursor of AII, is produced primarily by the liver. It also occurs in adipose tissue, where it is up-regulated during the development of obesity (10, 11, 12). A previous study demonstrated that Agt-deficient mice (Agt–/– mice) exhibit not only hypotension, but also impairment of diet-induced weight gain accompanied by increased locomotor activity (13). Furthermore, Massiéra et al. (14) showed that transgenic overexpression of Agt in adipose tissue leads to its secretion into the circulation and rescues hypotension and leanness of Agt–/– mice. These observations suggest that adipose-derived Agt, thus AII, is involved in blood pressure regulation and adipose tissue growth. Given that Agtr1 and Agtr2, another subtype of AII receptor, are both expressed in adipose tissue (15, 16), it is tempting to speculate that local as well as systemic AII may affect adipose tissue growth and metabolism. In this regard, Sharma et al. (17) demonstrated that AII inhibits directly human adipocyte differentiation via Agtr1in vitro and hypothesized that blockade of the RAS by either angiotensin-converting enzyme inhibitors or ARBs promotes the formation of fat cells and thus counteracts lipid accumulation in nonadipose tissue (skeletal muscle, liver, and pancreas, etc.) or lipotoxicity, thereby preventing diabetes (18). In contrast, a recent study showed that AII directly increases leptin release from isolated mature adipocytes in vitro, whereas systemic infusion of AII counteracts it through sympathetic activation in vivo (19), suggesting a differential effect of local vs. systemic AII in the adipose metabolism. Whether AII directly modulates adipose tissue growth and metabolism in vivo is still a matter of debate, and, if so, whether it is mediated through the activation of Agtr1 has not been tested rigorously.
It has been recognized that only rodents have duplicated Agtr1 genes (Agtr1a and Agtr1b) (20), whereas other mammals have a single Agtr1 gene (1). A large number of studies with Agtr1a–/– mice have revealed the physiological and pathophysiological roles of Agtr1 in the regulation of cardiovascular homeostasis in vivo (21, 22, 23, 24) and provided new insight into its clinical implication in human cardiovascular medicine. To explore the functional role of Agtr1 in adipose tissue growth and metabolism in vivo, we studied the metabolic phenotypes of Agtr1a–/– mice during a high-fat diet. Using mouse embryonic fibroblasts (MEFs) and primary cultures of mouse mature adipocytes, we also examined the role of Agtr1 in adipocyte differentiation and adipose gene expression in vitro.
Materials and Methods
Animals
The generation of male Agtr1a–/– mice on the C57BL/6J background was reported previously (21). Four-week-old male C57BL/6J mice were purchased from Oriental Yeast, Co., Ltd. (Tokyo, Japan). The animals were housed in individual cages in a temperature-, humidity-, and light-controlled room (12-h light, 12-h dark cycle) and were allowed free access to water and standard chow (362 kcal/100 g; 5.4% energy as fat; Oriental MF, Oriental Yeast, Co., Ltd.) unless otherwise noted. In the high-fat feeding experiments, Agtr1a–/– mice were given free access to water and either the standard chow or high-fat diet (556 kcal/100 g; 60% energy as fat). All animal experiments were conducted according to the guidelines of Tokyo Medical and Dental University committee on animal research.
Blood and urinary parameter analyses
Inferior vena cava puncture was performed at the time mice were killed in the fed state. Blood was also sampled from the tail vein of mice when glucose and insulin tolerance tests (GTT and ITT, respectively) were performed. Blood glucose was measured by the blood glucose test meter (Glutest PRO R, Sanwa-Kagaku, Nagoya, Japan). Enzymatic assay kits were used for the determination of serum triglyceride and free fatty acid (Wako Pure Chemical, Osaka, Japan). Serum insulin concentrations were measured by a commercially available ELISA kit (Morinaga, Tokyo, Japan). Serum creatinine and blood urea nitrogen were measured by the standard method. For urinary parameter analysis, 6-wk-old mice were placed in metabolic cages and given free access to food and water. Daily urinary catecholamine excretion was determined by reverse phase HPLC (25).
GTT and ITT
GTT was performed in 13-wk-old mice after a 6-h fast. Blood glucose concentrations were measured at 0 min before and 30, 60, and 120 min after ip injection of glucose (1 g/kg body weight). For ITT, insulin (0.7 U/kg body weight in 0.1% BSA; Humulin R-Insulin, Eli Lilly & Co., Indianapolis, IN) was injected ip after a 1-h fast. Blood glucose concentrations were measured 0 min before and 30 and 60 min after the injection.
Oxygen consumption and rectal temperature analysis
Oxygen consumption was measured with an O2/CO2 metabolism-measuring system (model MK-5000, Muromachikikai, Tokyo, Japan) (26). Each mouse was placed in a sealed chamber (560-ml volume) with an air flow of 0.60 liters/min for 22 h at 23 C. Air was taken every 3 min, and the consumed oxygen concentration was converted to milliliters per minute by multiplying it by the flow. Oxygen consumption was normalized by kilogram0.75 body weight. Respiratory quotient, the ratio of carbon dioxide production to oxygen consumption was also measured. Rectal temperature was measured using an electron thermistor equipped with rectal probe (Model TD-300, Shibaura Electronics, Saitama, Japan).
Blood pressure analysis
Systolic blood pressure was measured by the indirect tail-cuff method (BP-98A, Softron, Tokyo, Japan) as previously described (27). At least six readings were taken for each measurement.
Histological analysis
The epididymal white adipose tissue (WAT) was fixed with neutral-buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and studied under x200 magnification to compare adipocyte size. The adipocyte diameter and area were measured using Win Roof software (Mitani Co., Ltd., Chiba, Japan).
Quantitative real-time PCR
Total RNA was extracted from mouse epididymal and mesenteric WATs, interscapular brown adipose tissue (BAT), and mature adipocytes and stromal vascular fraction (SVF) obtained from mouse epididymal WAT using the acid guanidinium-phenol-chloroform method. Total RNA (5 μg) was reverse transcribed in a 20-μl reaction containing random primers and SuperScript II enzyme (Invitrogen Life Technologies, Inc., Carlsbad, CA). Quantitative real-time PCR was performed with an ABI PRISM 7000 Sequence Detection System using TaqMan or SYBR Green PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA). The primers used were as follows: adiponectin: sense, 5'-ATGGCAGAGATGGCACTCCT-3'; antisense, 5'-CCTTCAGCTCCTGTCATTCCA-3'; and oligonucleotide probe, 5'-AGGTCTTCTTGGTCCTAAGGGTGAGACA-3'; monocyte chemoattractant protein-1 (MCP-1): sense, 5'-CCACTCACCTGCTGCTACTCAT-3'; antisense, 5'-TGGTGATCCTCTTGTAGCTCTCC-3'; and oligonucleotide probe, 5'-CACCAGCAAGATGATCCCAATGAGTAGGC-3'; IL-6: sense, 5'-ACAACCACGGCCTTCCCTACTT-3'; and antisense: 5'-CACGATTTCCCAGAGAACATGTG-3'; plasminogen activator inhibitor-1 (PAI-1): sense, 5'-GGCAGATCCAAGATGCTATGG-3'; and antisense, 5'-CAAAGATGGCATCCGCAGTA-3'; uncoupling protein-1 (UCP-1): sense, 5'-CGCTGGACACTGCCAAAGT-3'; and antisense, 5'-GGTGGTGATGGTCCCTACCA-3'; Agtr1a: sense, 5'-TTTCCAGATCAAGTGCATTTTGA-3'; and antisense, 5'-AGAGTTAAGGGCCATTTTGCTTT-3'; and 36B4: sense, 5'-GGCCCTGCACTCTCGCTTTC-3'; and antisense, 5'-TGCCAGGACGCGCTTGT-3'. The sense and antisense primers and specific oligonucleotide probe for leptin were obtained from Applied Biosystems. Levels of mRNA were normalized to those of 18S rRNA (primers obtained from Applied Biosystems) or 36B4 mRNA. Leptin, adiponectin, and MCP-1 mRNA levels were normalized with 18S rRNA, whereas IL-6, PAI-1, UCP-1, and AT1a mRNA levels were normalized with 36B4 mRNA.
Cell culture experiments
The lipid-laden mature adipocytes and SVF were prepared from the epididymal WAT from 8-wk-old Agtr1a+/+ and Agtr1a–/– mice as previously described (28). They were subjected to total RNA extraction for Agtr1a mRNA analysis. The mature adipocytes were also treated for 24 h with AII (100 nM) in the presence or absence of valsartan (10 μM; Novartis Pharmaceuticals, East Hanover, NJ), an ARB.
MEFs were prepared from Agtr1a+/+ and Agtr1a–/– mice as previously described (29). They were maintained in DMEM containing 10% fetal bovine serum (FBS), grown to 2 d after confluence (designated d 0), and then subjected to differentiation by DMEM containing 10% FBS, 5 μg/ml insulin, 0.25 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methlyxanthine until d 2. They were fed DMEM supplemented with 10% FBS/5 μg/ml insulin for 2 d, then fed every other day with DMEM containing 10% FBS in the presence or absence of AII (100 nM). Cells at d 8 were also stained with Oil-Red O. Lipid accumulation was quantified by a model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA).
Statistical analysis
Data were expressed as the mean ± SE. Statistical analysis was performed using ANOVA, followed by Scheffé’s test. P < 0.05 was considered statistically significant.
Results
High-fat diet experiments
Reduced adiposity in Agtr1a–/– mice on a high-fat diet.
We examined the effect of high-fat diet on the metabolic phenotypes of Agtr1a–/– mice. On a standard diet, body weight gain was indistinguishable between wild-type Agtr1a+/+ and Agtr1a–/– mice throughout the experiments (Fig. 1A). The Agtr1a+/+ mice fed a high-fat diet weighed 44% more than those fed a standard diet for 8 wk (39.7 ± 1.0 vs. 27.6 ± 0.6 g; P < 0.05). There was no significant difference in body weight between Agtr1a+/+ and Agtr1a–/– mice fed a standard diet (27.6 ± 0.6 vs. 29.9 ± 0.7 g). By contrast, Agtr1a–/– mice were protected from high-fat diet-induced obesity. At the end of the experiments, the body weight of Agtr1a–/– mice was 31.7 ± 1.1 g, which was approximately 80% that of Agtr1a+/+ mice (P < 0.05).
FIG. 1. Attenuation of diet-induced weight gain and adiposity in Agtr1a–/– mice. A, Growth curve of Agtr1a+/+ and Agtr1a–/– mice on either a standard diet (SD) or a high-fat diet (HFD). Agtr1a+/+ (n = 6; ) and Agtr1a–/– (n = 7; ) mice on SD are shown. Agtr1a+/+ (n = 16; ) and Agtr1a–/– (n = 14; ) mice on HFD are shown. *, P < 0.05. B, Abdominal dissection of Agtr1a+/+ and Agtr1a–/– mice on either SD or HFD. C, Weight of the epididymal (Epi), mesenteric (Mes), and sc (Sub) WAT; interscapular BAT; and liver in Agtr1a+/+ and Agtr1a–/– mice on either SD or HFD. D, Hematoxylin and eosin staining of epididymal WAT from Agtr1a+/+ and Agtr1a–/– mice on either SD or HFD. Original magnification, x200. Scale bars, 50 μm. E, Adipocyte diameter and area in epididymal WAT. Lane 1, Agtr1a+/+/SD (n = 6); lane 2, Agtr1a+/+/HFD (n = 16); lane 3, Agtr1a–/–/SD (n = 7); lane 4, Agtr1a–/–/HFD (n = 14). *, P < 0.01 vs. Agtr1a+/+/SD; #, P < 0.05 vs. Agtr1a–/–/SD; , P < 0.05 between the indicated groups.
Gross inspection of abdominal dissection revealed a reduced mass of adipose tissue in Agtr1a–/– mice compared with Agtr1a+/+ mice (Fig. 1B). The weights of the epididymal, mesenteric, and sc fat depots were increased significantly in Agtr1a+/+ mice fed a high-fat diet relative to those in Agtr1a+/+ mice fed a standard diet (P < 0.05; Fig. 1C). The weights of both epididymal and sc fat depots were increased significantly in Agtr1a–/– mice fed a high-fat diet relative to those fed a standard diet (P < 0.05), which were markedly reduced compared with those of Agtr1a+/+ mice fed a high-fat diet. The weight of interscapular BAT was also increased significantly in Agtr1a+/+ mice fed a high-fat diet compared with those of Agtr1a+/+ mice fed a standard diet (P < 0.05), whereas no significant difference was noted between Agtr1a–/– mice fed a high-fat diet and those fed a standard diet. In this study no significant differences in liver weight were noted between genotypes on either a standard or a high-fat diet (Fig. 1C). Histological examination revealed no appreciable difference in the size of epididymal mature adipocytes between Agtr1a+/+ and Agtr1a–/– mice fed a standard diet. However, the size of mature adipocytes in Agtr1a–/– mice was significantly smaller than that in Agtr1a+/+ mice on a high-fat diet (P < 0.05; Fig. 1, D and E).
Increased energy expenditure in Agtr1a–/– mice on a high-fat diet.
Agtr1a–/– mice ate more daily than Agtr1a+/+ mice on a calorie basis (P < 0.05) when fed a standard diet (Fig. 2A). However, Agtr1a–/– mice did not differ from Agtr1a+/+ mice in daily food intake on a high-fat diet. In this study, rectal temperature was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice on either a standard or a high-fat diet (P < 0.05; Fig. 2B). Oxygen consumption, an indirect measurement of metabolism, was increased in Agtr1a–/– mice compared with Agtr1a+/+ mice in both the light and dark phases on a high-fat diet (P < 0.05; Fig. 2C). There was no significant difference in respiratory quotient between Agtr1a+/+ and Agtr1a–/– mice on a high-fat diet (data not shown).
FIG. 2. Increased energy expenditure in Agtr1a–/– mice. A, Daily food intake on a calorie basis during a high fat diet (HFD) from 7–12 wk of age. B, Rectal temperature at 14 wk of age. C, Oxygen consumption after 8 wk of an HFD. Upper panel, Daily profile of oxygen consumption. Agtr1a+/+ () and Agtr1a–/– () mice on an HFD are shown (n = 10 in each group). Lower panel, Quantification of oxygen consumption. Lane 1, Agtr1a+/+/standard diet (SD; n = 5); lane 2, Agtr1a+/+/HFD (n = 10); lane 3, Agtr1a–/–/SD (n = 6); lane 4, Agtr1a–/–/HFD (n = 10). *, P < 0.05 vs. Agtr1a+/+/SD; , P < 0.05 between the indicated groups.
Blood and urinary parameters and blood pressure in Agtr1a–/– mice on a high-fat diet.
We next examined blood glucose and lipid metabolism in Agtr1a+/+ and Agtr1a–/– mice on a high-fat diet. Blood glucose and serum insulin concentrations were increased in Agtr1a+/+ mice fed a high-fat diet compared with those fed a standard diet (P < 0.05; Fig. 3A). However, these levels were suppressed in Agtr1a–/– mice during a high-fat diet (P < 0.05). There was no significant difference in serum triglyceride and free fatty acids between Agtr1a+/+ and Agtr1a–/– mice on a high-fat diet (Fig. 3A).
FIG. 3. Glucose and lipid metabolism in Agtr1a+/+ and Agtr1a–/– mice. A, Nonfasting blood glucose, serum insulin, free fatty acid, and triglyceride concentrations at 14 wk of age. Lane 1, Agtr1a+/+/standard diet (SD; n = 6); lane 2, Agtr1a+/+/high-fat diet (HFD; n = 16); lane 3, Agtr1a–/–/SD (n = 7); lane 4, Agtr1a–/–/HFD (n = 14). *, P < 0.05 vs. Agtr1a+/+/SD; #, P < 0.05 vs. Agtr1a–/–/SD; , P < 0.05 between the indicated groups. B, The ip GTT after a 6-h fast at 13 wk of age. C, The ip ITT after a 1-h fast at 14 wk of age. Agtr1a+/+ () and Agtr1a–/– () mice on SD, and Agtr1a+/+ () and Agtr1a–/– () mice on HFD are shown (n = 6 in each group). , P < 0.05 between Agtr1a+/+/HFD and Agtr1a–/–/HFD.
The ip GTT revealed increased glucose disposal in Agtr1a–/– mice compared with Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 3B). Furthermore, the ip ITT demonstrated that the hypoglycemic response is exaggerated in Agtr1a–/– mice compared with Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 3C). These observations indicate that Agtr1a–/– mice are protected from high-fat diet-induced impairment of glucose tolerance and insulin resistance.
The tail cuff systolic blood pressure of Agtr1a–/– mice was significantly lower than that of Agtr1a+/+ mice (P < 0.05; Table 1). However, no significant difference in urine volume was noted between Agtr1a+/+ and Agtr1a–/– mice. Serum creatinine levels tended to be increased in Agtr1a–/– mice compared with Agtr1a+/+ mice. Urinary norepinephrine excretion was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice (P < 0.05), whereas urinary epinephrine excretion tended to be increased in Agtr1a–/– mice compared with Agtr1a+/+ mice (Table 1).
TABLE 1. Blood pressure and urinary parameters at 6 wk of age
Systolic blood pressure tended to be increased in Agtr1a+/+ mice fed a high-fat diet for 16 wk compared with those fed a standard diet (Table 2). Blood pressure was significantly lower in Agtr1a–/– mice than in Agtr1a+/+ mice on a standard diet as reported previously (P < 0.05). No significant difference in blood pressure was noted between Agtr1a–/– mice fed a high-fat diet and those fed a standard diet. Urinary norepinephrine excretion was increased significantly in Agtr1a+/+ mice fed a high-fat diet for 16 wk compared with those fed a standard diet (P < 0.05; Table 2). There was no significant difference between Agtr1a–/– mice fed a high-fat diet and those a fed standard diet. Urinary norepinephrine excretion was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice on either a standard or a high-fat diet (P < 0.05). Urinary epinephrine excretion was increased significantly in Agtr1a–/– mice compared with Agtr1a+/+ mice on a standard diet (P < 0.05). During a high-fat diet, urinary epinephrine excretion tended to be increased in Agtr1a–/– mice compared with Agtr1a+/+ mice. These observations indicate the sympathetic activation in Agtr1a–/– mice compared with Agtr1a+/+ mice.
TABLE 2. Blood pressure and urinary parameters after 16 wk of a high-fat diet
Quantitative real-time PCR analysis.
By quantitative real-time PCR analysis, we examined the expression of mRNAs for leptin, adiponectin, MCP-1, IL-6, and PAI-1 in epididymal and mesenteric WAT from Agtr1a+/+ and Agtr1a–/– mice during a high-fat diet. The expression of leptin mRNA was increased significantly in both epididymal and mesenteric WAT from Agtr1a+/+ mice fed a high-fat diet compared with those fed a standard diet (P < 0.05; Fig. 4A). In this study, leptin mRNA expression was also up-regulated in the epididymal WAT from Agtr1a–/– mice fed a high-fat diet compared with those fed a standard diet (P < 0.05). No significant difference in leptin mRNA expression in epididymal WAT was noted between genotypes. In mesenteric WAT, the expression of leptin mRNA was markedly reduced in Agtr1a–/– mice compared with Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 4A). Adiponectin mRNA expression was decreased in the epididymal WAT from Agtr1a+/+ mice fed a high-fat diet compared with those fed a standard diet (P < 0.05). It tended to be decreased in mesenteric WAT from Agtr1a+/+ mice fed a high-fat diet. No significant difference in adiponectin mRNA expression was noted between genotypes.
FIG. 4. Adipose gene expression in Agtr1a+/+ and Agtr1a–/– mice. A, Expression of mRNAs for leptin, adiponectin, MCP-1, IL-6, and PAI-1 in epididymal (Epi) and mesenteric (Mes) WAT from Agtr1a+/+ and Agtr1a–/– mice. B, Expression of UCP-1 mRNA in BAT from Agtr1a+/+ and Agtr1a–/– mice. Lane 1, Agtr1a+/+/standard diet (SD; n = 5); lane 2, Agtr1a+/+/high-fat diet (HFD; n = 10); lane 3, Agtr1a–/–/SD (n = 6); lane 4, Agtr1a–/–/HFD (n = 10). *, P < 0.05 vs. Agtr1a+/+/SD; #, P < 0.05 vs. Agtr1a–/–/SD; , P < 0.05 between the indicated groups.
During a high-fat diet, MCP-1 mRNA expression was increased significantly in epididymal and mesenteric WAT from Agtr1a+/+ mice (P < 0.05), but was reduced significantly in those from Agtr1a–/– mice (P < 0.05; Fig. 4A). During a high-fat diet, IL-6 mRNA expression was also increased significantly in epididymal WAT from Agtr1a+/+ mice (P < 0.05) and was reduced significantly in Agtr1a–/– mice (P < 0.05; Fig. 4A). There was no significant difference in IL-6 mRNA levels in mesenteric WAT from Agtr1a+/+ and Agtr1a–/– mice. In this study, there was no significant difference in PAI-1 mRNA expression in epididymal WAT from Agtr1a+/+ and Agtr1a–/– mice. In mesenteric WAT, PAI-1 mRNA expression was significantly increased in Agtr1a+/+ mice fed a high-fat diet (P < 0.05), whereas it was markedly reduced in Agtr1a–/– mice (P < 0.05).
In this study, we found no significant difference in UCP-1 mRNA expression in interscapular BAT between Agtr1a+/+ mice fed a standard diet and those fed a high-fat diet (Fig. 4B). The expression of UCP-1 mRNA in BAT was increased significantly in Agtr1a–/– mice fed a high-fat diet compared with those fed a standard diet (P < 0.05). Consequently, UCP-1 mRNA level in BAT from Agtr1a–/– mice was approximately 1.4-fold higher than that in Agtr1a+/+ mice during a high-fat diet (P < 0.05; Fig. 4B).
Metabolic phenotypes of heterozygous Agtr1a+/– mice.
We also examined the metabolic phenotypes of heterozygous Agtr1a+/– mice on a high-fat diet. There was no significant difference in body weight between Agtr1a+/+ and Agtr1a+/– mice on a standard diet at 14 wk of age (29.6 ± 0.6 vs. 29.3 ± 0.7 g). The body weight of Agtr1a+/– mice fed a high-fat diet was also significantly increased compared with those fed standard diet (P < 0.05). However, the body weight of Agtr1a+/– was reduced significantly compared with those of Agtr1a+/+ mice during a high-fat diet at 14 wk of age (32.4 ± 0.6 vs. 40.4 ± 0.7 g; P < 0.05). We also observed a reduced mass of adipose tissue in Agtr1a+/– mice compared with Agtr1a+/+ mice during a high-fat diet (data not shown).
Agtr1a+/– mice did not differ from Agtr1a+/+ mice in daily food intake on a high-fat diet (data not shown). In this study, rectal temperature was increased in Agtr1a+/– mice compared with Agtr1a+/+ mice on a high-fat diet (P < 0.05; data not shown). Oxygen consumption was increased significantly in Agtr1a+/– mice compared with Agtr1a+/+ mice in both the light and dark phases on a high-fat diet (P < 0.05; data not shown).
The ip GTT revealed increased glucose disposal in Agtr1a+/– mice compared with Agtr1a+/+ mice during a high-fat diet for 16 wk (P < 0.05; data not shown).
Blood pressure was reduced significantly in Agtr1a+/– mice compared with Agtr1a+/+ mice on a standard diet as reported previously (P < 0.05; data not shown). However, no significant differences in urine volume or serum creatinine were noted between Agtr1a+/+ and Agtr1a+/– mice. Urinary norepinephrine excretion was increased significantly in Agtr1a+/– mice compared with Agtr1a+/+ mice (P < 0.05; data not shown). These observations indicate that heterozygous Agtr1a+/– mice show reduced weight gain and adiposity during a high-fat diet.
Cell culture experiments
Functional significance of Agtr1 in adipocyte differentiation.
Agtr1a mRNA was expressed in MEFs, the level of which was unchanged during the course of adipocyte differentiation (our unpublished observations). We examined the functional significance of Agtr1a in adipocyte differentiation using MEFs obtained from Agtr1a+/+ and Agtr1a–/– mice. In this study, treatment with AII at a dose of 100 nM did not affect adipocyte differentiation of MEFs derived from Agtr1a+/+ mice, as revealed by Oil-Red O staining (Fig. 5A). Furthermore, there was no significant difference in adipocyte differentiation and lipid accumulation between AII- and vehicle-treated MEFs derived from Agtr1a–/– mice (Fig. 5A).
FIG. 5. Functional significance of Agtr1 in adipocyte differentiation and adipose gene expression in vitro. A, Left, Adipocyte differentiation and lipid accumulation revealed by Oil-Red O staining in AII- and vehicle-treated MEFs obtained from Agtr1a+/+ and Agtr1a–/– mice. Right, Quantification of lipid accumulation. diff., Differentiation. n = 6 in each group. B, Expression of Agtr1a mRNA in mature adipocytes and SVF obtained from epididymal WAT from Agtr1a+/+ mice. M, Mature adipocytes. n = 4 in each group. C, Effects of AII and/or ARB on adipose gene expression in mature adipocytes obtained from epididymal WAT from Agtr1a+/+ mice. Val, Valsartan. *, P < 0.05 vs. control; #, P < 0.05 vs. AII-treated group. n = 6 in each group.
Expression of Agtr1a mRNA and its functional significance in mature adipocytes.
We confirmed that Agtr1a mRNA is expressed in both mature adipocytes and SVF obtained from Agtr1a+/+ mice (Fig. 5B). Using primary cultures of mature adipocytes obtained from epididymal WAT, we also examined the effect of AII on adipose gene expression. We found no significant change in leptin and adiponectin mRNA levels between AII- and vehicle-treated mature adipocytes. By contrast, treatment with AII significantly increased MCP-1, IL-6, and PAI-1 mRNA expression in mature adipocytes (P < 0.05; Fig. 5C). Increased expression of MCP-1, IL-6, and PAI-1 mRNAs was abolished by coadministration of valsartan (10 μM; P < 0.05; Fig. 5C).
Discussion
The RAS plays a critical role in the regulation of body fluid homeostasis and blood pressure control. Recent observations, however, have suggested that it also participates in the regulation of glucose and lipid metabolism (16). Agtr1a–/– mice have facilitated understanding of the functional role of Agtr1 in the regulation of cardiovascular homeostasis in vivo (21, 22, 23, 24). This study was designed to elucidate the functional role of Agtr1 in adipose tissue growth and metabolism using Agtr1a–/– mice.
We have demonstrated for the first time that Agtr1a–/– mice exhibit the attenuation of diet-induced weight gain and adiposity accompanied by increased sympathetic activity and energy expenditure compared with Agtr1a+/+ mice. In this study, Agtr1a+/+ and Agtr1a–/– mice do not differ significantly in food intake on a high-fat diet. Therefore, the attenuation of diet-induced weight gain and adiposity in Agtr1a–/– mice may be due to increased energy expenditure. The Agtr1a–/– mice are also hypotensive and show attenuation of impaired glucose tolerance and insulin resistance compared with Agtr1a+/+ mice on a high-fat diet. In contrast, there is no appreciable difference in lipid metabolism between Agtr1a+/+ and Agtr1a–/– mice. These observations indicate that Agtr1a–/– mice are protected from some components of the metabolic syndrome during a high-fat diet, suggesting that they are the unique experimental model system to investigate the role of Agtr1 in this pathological condition.
The metabolic phenotypes of Agtr1a–/– mice revealed in this study are similar to, although less severe than, those of Agt–/– mice as described previously (13). Furthermore, heterozygous Agtr1a+/– mice exhibit similar phenotypes to those of Agtr1a–/– mice on a high-fat diet. These observations suggest that Agtr1a is important for AII regulation of adipose tissue growth and metabolism in vivo. In this context, Tsuchida et al. (30, 31) previously demonstrated that targeted disruption of genes encoding both Agtr1a and Agtr1b simultaneously in mice produces specific abnormalities in renal morphology and function that are quantitatively similar to those found in Agt–/– mice, whereas mice lacking either Agtr1a or Agtr1b show milder renal abnormalities. These observations suggest that major biological functions of endogenous AII revealed by the abnormal renal phenotypes of Agt–/– mice are mediated by both Agtr1a and Agtr1b. Recently, we examined the effect of valsartan on the metabolic phenotypes of Agtr1a+/+ and Agtr1a–/– mice fed a standard diet and found that urinary catecholamine excretion is significantly reduced in valsartan-treated Agtr1a–/– mice compared with vehicle-treated groups (our unpublished observations). It has been reported that Agtr1b is expressed in a variety of tissues, such as the adrenal gland and sympathetic nerve terminals (32, 33, 34), suggesting that sympathetic activation in Agtr1a–/– mice is due at least in part to Agtr1b. In contrast, a previous study showed that AII increases lipogenesis in 3T3-L1 and human mature adipocytes, possibly through the activation of Agtr2 in vitro (35). In this context, Yvan-Charvet et al. (36) recently demonstrated that deletion of Agtr2 protects from diet-induced obesity and insulin resistance. We also examined the effect of a high-fat diet on the metabolic phenotypes of Agtr2y–/– mice and found that body weight and epididymal adipose tissue weight were reduced significantly in Agtr2y/– mice compared with Agtr2y/+ mice on a high-fat diet (our unpublished observations). These observations, taken together, suggest that Agtr2 is important for the regulation of energy homeostasis. Because plasma AII concentrations are elevated in Agtr1a–/– mice compared with Agtr1a+/+ mice (24), it is possible that the effect of AII via Agtr1b and Agtr2 contributes to the metabolic phenotypes of Agtr1a–/– mice. There are several reports that systemic or central administration of AII in rats decreases body weight and adipose tissue weight, which is attributable to both reduced food intake and increased peripheral metabolism (37, 38, 39). Reductions in body weight and adipose tissue growth in rats treated with AII and in Agtr1a–/– mice may be related to the activation of Agtr1b and Agtr2. Additional studies are needed to elucidate the possible roles of Agtr1b and Agtr2 in the metabolic phenotypes of Agtr1a–/– mice.
The tissue(s) or organ(s) responsible for the metabolic phenotypes of Agtr1a–/– mice is currently unclear. Massiéra et al. (14) demonstrated that transgenic overexpression of Agt in adipose tissue from Agt–/– mice, in which Agt is present in the systemic circulation at 20–30% the levels in Agt+/+ mice, rescues hypotension and leanness in Agt–/– mice. These observations suggest that the adipose-derived Agt, thus AII, is involved in blood pressure regulation and adipose tissue growth in the absence of systemic AII. It is unlikely that adipose-derived or peripherally produced AII is transported directly into the brain through the blood-brain barrier (40), where it may modulate the sympathetic nervous system and adipose tissue growth. In this regard, there are a couple of reports suggesting that circulating AII increases peripheral sympathetic nerve activity via Agtr1 (41, 42). Improved insulin resistance in Agtr1a–/– mice on a high-fat diet may be secondary to the attenuation of diet-induced weight gain and adiposity in these animals. Glucose metabolism might also be increased in skeletal muscle from Agtr1a–/– mice, because treatment with valsartan enhances insulin sensitivity in skeletal muscle from diabetic mice (6).
It is important to know whether AII can directly regulate adipose tissue growth and metabolism. In this study using MEFs derived from Agtr1a+/+ and Agtr1a–/– mice, we found no significant difference between genotypes in the ability to differentiate into lipid-laden mature adipocytes. We also confirmed no significant difference in lipid accumulation between AII- and vehicle-treated groups in the 3T3-L1 preadipocyte cell line (our unpublished observations). These findings indicate that AII does not directly affect adipocyte differentiation through the activation of Agtr1a in vitro. We, therefore, speculate that basal adipose tissue growth is not related to the direct action of AII on adipose tissue. However, part of the metabolic phenotypes of Agtr1a–/– mice fed a high-fat diet might be due to the direct action of AII on adipose tissue, because AII is capable of regulating the expression of some of the adipocytokines. In this context, Sharma et al. (17) previously reported that AII inhibits adipocyte differentiation from human preadipocytes via Agtr1 in vitro, whereas blockade of this receptor enhances adipogenesis, and they hypothesized that blockade of the formation of AII produced by large insulin-resistant adipocytes promotes preadipocyte recruitment and increases the number of small adipocytes, resulting in improved insulin sensitivity (18). There might be a species difference with regard to the role of AII in adipocyte differentiation.
We also found that AII increases the expression of mRNAs for some adipocytokines (MCP-1, IL-6, and PAI-1) in primary cultures of mature mouse adipocytes, which was abolished by pharmacological blockade of Agtr1. These observations indicate that AII is capable of directly regulating the production of some adipocytokines via Agtr1. It was reported that AII directly stimulates the release of PAI-1 and IL-6 from cultured human adipocytes via Agtr1 in vitro (43, 44). In this study, there was no significant effect of AII on leptin and adiponectin mRNA expression. Recently, Cassis et al. (19) reported that AII directly increases leptin release from isolated mature adipocytes in vitro, whereas systemic infusion of AII counteracts it through sympathetic activation in vivo, suggesting a differential effect of local vs. systemic AII in adipose metabolism. Furthermore, Furuhashi et al. (45) showed that blockade of the RAS by temocapril or candesartan increases adiponectin concentrations in patients with essential hypertension. However, Fasshauer et al. (46) showed that AII does not affect adiponectin gene expression in 3T3-L1 adipocytes. Adipose tissue is composed of various cell types: mature adipocytes and SVF, including preadipocytes, endothelial cells, lymphocytes, and macrophages. Because Agtr1 is expressed in both mature adipocytes and SVF, it is tempting to speculate about a regulatory role for Agtr1 in the paracrine interaction between adipocytes and other cell types within adipose tissue in vivo.
In conclusion, this study demonstrates that Agtr1a–/– mice exhibit attenuation of diet-induced weight gain and adiposity through increased energy expenditure. The data from this study also suggest that AII does not directly affect adipocyte differentiation, but can modulate adipocytokine production via Agtr1.
Acknowledgments
We thank Dr. Mutsuo Taiji for advice on oxygen consumption measurement, Ms. Rie Kodani for technical assistance, and Ms. Ai Togo for secretarial assistance. We also acknowledge Novartis Pharma Co. for valsartan.
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