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Endocrinology, doi:10.1210/en.2006-1270
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Endocrinology Vol. 148, No. 6 2690-2697
Copyright © 2007 by The Endocrine Society

Apelin, an APJ Receptor Ligand, Regulates Body Adiposity and Favors the Messenger Ribonucleic Acid Expression of Uncoupling Proteins in Mice

Keiko Higuchi, Takayuki Masaki, Koro Gotoh, Seiichi Chiba, Isao Katsuragi, Katsuhiro Tanaka, Tetsuya Kakuma and Hironobu Yoshimatsu

Department of Anatomy, Biology, and Medicine, Internal Medicine 1, Faculty of Medicine, Oita University, Oita 879-5593, Japan

Address all correspondence and requests for reprints to: Hironobu Yoshimatsu, M.D., Ph.D., Department of Anatomy, Biology, and Medicine, Internal Medicine 1, Faculty of Medicine, Oita University, 1-1 Idai-ga-oka, Hasama-cho, Yufu-city, Oita 879-5593, Japan. E-mail: higukei{at}med.oita-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apelin, the endogenous ligand of the APJ receptor, has been identified in a variety of tissues, including stomach, heart, skeletal muscle, and white adipose tissue. We sought to clarify the effects of apelin on body adiposity and the expression of uncoupling proteins (UCPs) in C57BL/6 mice. Treatment with ip apelin at a dose of 0.1 µmol/kg·d for 14 d decreased the weight of white adipose tissue and serum levels of insulin and triglycerides, compared with controls, without influencing food intake. Apelin treatment also decreased body adiposity and serum levels of insulin and triglycerides in obese mice fed a high-fat diet. Apelin increased the serum adiponectin level and decreased that of leptin. Additionally, apelin treatment increased mRNA expression of UCP1, a marker of peripheral energy expenditure, in brown adipose tissue (BAT) and of UCP3, a regulator of fatty acid export, in skeletal muscle. In addition, immunoblot bands and relative densities of UCP1 content in BAT were also higher in the apelin group than controls. Furthermore, apelin treatment increased body temperature and O2 consumption and decreased the respiratory quotient. In conclusion, apelin appears to regulate adiposity and lipid metabolism in both lean and obese mice. In addition, apelin regulates insulin resistance by influencing the circulating adiponectin level, the expression of BAT UCP1, and energy expenditure in mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APELIN WAS FIRST isolated from bovine stomach as an endogenous ligand of the seven-transmembrane G protein-coupled receptor, APJ (1). The expression of both apelin and APJ in rodents has been identified in several tissues, including brain, heart, stomach, skeletal muscle, and the vascular system (2, 3, 4, 5). Apelin has been implicated in a variety of roles, including cardiac function (6, 7), drinking behavior (2, 8, 9, 10), and gastric cell proliferation (11). Recently apelin and its receptors have been found in adipose tissues (12, 13), although their function there is still unclear.

It is well known that adipose tissue is not just the main endogenous source of circulating lipids but is also the site for the production and secretion of several hormones and cytokines. Studies have demonstrated that these adipose tissue-derived signaling molecules play key roles in a complex network that appears to modulate obesity and related metabolic disorders, including insulin resistance (14). As mentioned above, apelin is produced by adipocytes and released into the circulation (13). Apelin expression in adipose tissue is regulated by nutritional status, such as fasting and refeeding (13). In addition, insulin exerts a direct action on adipocyte apelin production and may influence its plasma level in obese animal models (13). These results suggest that apelin has functional roles in regulating energy metabolism as an adipose tissue-derived signaling molecule.

In general, adipose tissue is classified into brown adipose tissue (BAT) and white adipose tissue (WAT). Uncoupling protein (UCP)-1 in BAT plays a role in energy expenditure and nonshivering thermogenesis (15, 16, 17, 18). UCP2 is expressed ubiquitously in peripheral tissues, including WAT (18, 19, 20), and UCP3 is expressed mainly in skeletal muscle (MSL) and adipose tissues (20, 21, 22). Gene expression of these proteins is regulated by several humoral factors and environmental temperature (23, 24, 25, 26). Thus, BAT UCP1 and MSL UCP3 can be considered indicators of energy metabolism (18, 23). We suggest that apelin affects adiposity and energy metabolism by modulating the expression of UCPs.

Thus, we investigated the effects of apelin-13, the most effective form of apelin, on food intake, body weight, adiposity, serum metabolic parameters [such as glucose, free fatty acids (FFAs), triglycerides, and insulin], WAT mRNA expression and serum leptin and adiponectin levels, UCP expression in peripheral tissues, body temperature changes, O2 consumption, and the respiratory quotient. The goal of this study was to confirm the usefulness of apelin to regulate adiposity, lipid metabolism, and energy expenditure in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All animals were treated in accordance with the Oita University Guidelines for the Care and Use of Laboratory Animals.

Mice (C57BL/6; Seac Yoshitomi, Fukuoka, Japan) were housed in a light-, temperature-, and humidity-controlled room (12-h light, 12-h dark cycle, lights on/off at 0700 and 1900 h, respectively, 21 ± 1 C, 55 ± 5% relative humidity). The mice were allowed free access to standard laboratory food (CLEA Japan, Tokyo, Japan) and water.

Materials
Apelin (Peptide Institute, Osaka, Japan) was dissolved in saline to a concentration of 0.1 µM and used at 0, 0.05, or 0.1 µmol/kg·d for ip injection. The dose of apelin was based on our preliminary study. Each solution was prepared freshly on the day it was administered. The pH was adjusted to between 6.8 and 7.4.

Measuring food intake, body weight, and histological examination
Mice were divided into apelin-treated [apelin (AP)] and nontreated [saline (CONT)] groups. First, to evaluate any dose-response effect of apelin on body weight regulation, apelin was administered by ip injection at doses of 0, 0.05, and 0.1 µmol/kg for 7 d. We observed a dose-response effect of apelin and chose to use a dose of 0.1 µmol/kg. In the apelin-treated group, apelin was given at a dose of 0.1 µmol/kg·d by ip injection for 14 d. In the control group, 100 µl of saline were given in the same way. Food intake and body weight were measured at 14:30 each day, and the apelin injection was given at 1500 h. Animals were killed 24 h after the last dose. WAT and interscapular BAT were removed and frozen in liquid nitrogen before being stored at –80 C until mRNA extraction. Epididymal WAT was dissected from the fat located in the upper region of the testis, mesenteric WAT was dissected from the fat located in the lower region of the duodenum, and retroperitoneal WAT was dissected from the fat located at the back of the kidney. The mass of body fat was measured to assess changes in body fat accumulation. The histology of epididymal WAT and mRNA levels of the UCPs, leptin, and adiponectin were assessed in all animals at the end of the 14-d treatment period.

High-fat diet and apelin treatment
Mice were allowed free access to 60% high-fat food (catalog no. D12492; 20% protein, 20% carbohydrate, 60% fat, 5.2 kcal/g; Research Diet, Tokyo, Japan) and water. The high-fat food contained soybean oil (25/773.85 g) and lard (245/773.85 g). Mice were selected and divided into treatment groups as described above. High-fat food was administered for 6 wk (from 8 to 14 wk of age). Apelin was injected ip at a dose of 0.1 µmol/kg·d, and controls received the vehicle for 14 consecutive days (the last 2 wk). Cumulative food intake was measured once daily for each of the 14 d of treatment. Body weight, fat weight, serum glucose, and lipid profiles were measured in all animals at the end of the 14-d treatment period.

Measuring blood sampling
We measured the body weight at 1430 h and took blood for hormones at 1500 h. Blood was collected after a 16-h fast; serum was separated and frozen immediately at –20 C until assayed. Serum levels of glucose, insulin, triglycerides, and FFAs were measured using commercial kits (Wako Chemical, Tokyo, Japan). Serum apelin concentrations were measured using a commercially available kit, using a sandwich enzyme immunoassay (Phoenix Pharmaceuticals, Belmont, CA). The assay had a sensitivity of 0–100 ng/ml.

Intraperitoneal glucose tolerance test
Glucose [2 g/kg body weight in normal (0.9%) saline] was administered ip on d 14 after treatment with apelin. Blood was drawn from a tail vein at 0, 30, 60, and 90 min for measurement of plasma glucose levels. Plasma glucose levels were determined by the glucose oxidation method (Glutest sensor; Sanwa Kagaku, Nagoya, Japan).

Triglycerides in WAT
Epididymal WAT (100 mg) was homogenized in 2 ml of a solution containing 150 mM NaCl, 0.1% Triton X-100, and 10 mM Tris, using a polytron homogenizer (NS-310E; MicroTech Nichion, Chiba, Japan) for 1 min. The triglyceride content of 100 µl of this solution was determined using a commercial kit (Wako Chemical).

Histological analysis
Small pieces of epididymal WAT, BAT, and MSL were dissected, washed in saline, fixed in 10% formalin, and embedded in paraffin. Tissue sections were cut at a thickness of 20 µm and stained with hematoxylin and eosin.

Real-time quantitative RT-PCR
BAT UCP1, WAT UCP2, MSL UCP3, adiponectin, and leptin mRNAs were amplified by PCR and quantified using real-time quantitative PCR as follows. Total cellular RNA was prepared from selected mouse tissues using TRIzol (Lifetech, Tokyo, Japan) according to the manufacturer’s protocol. Total RNA (20 µg) was electrophoresed on 1.2% formaldehyde agarose gels. RNA quality and quantity were assessed using EtBr agarose gel electrophoresis and by measuring the absorbance at 260 nm relative to that at 280 nm. cDNA was synthesized from total RNA (150 ng) in a volume of 20 µl using a ReverTra-Dash reverse transcriptase kit (Toyobo, Tokyo, Japan) with random hexamer primers. The reactions were diluted to 50 µl with sterile distilled water and stored at –20 C. Primers were designed, synthesized, optimized, and obtained as preoptimized kits: adiponectin (catalog no. Mm00456425m1), leptin (catalog no. Mm00434759m1), UCP1 (catalog no. Mm00494069m1), UCP2 (catalog no. Mm00495907m1), and UCP3 (catalog no. Mm00494074m1). Primers for ribosomal RNA for use as an internal control were also obtained as a preoptimized kit (catalog no. Hs99999901). These preoptimized kits were purchased from Applied Biosystems (Foster City, CA). Using an ABI PRISM 7000 sequence detector (Applied Biosystems), PCR amplification was performed in 50-µl volumes containing 100 ng cDNA template in PCR Master Mix (Roche, Nutley, NJ), according to the following protocol: 50 C for 2 min, 95 C for 10 min, and 40 cycles at 95 C for 15 sec and 60 C for 1 min. Samples were analyzed in duplicate. Target mRNA amounts were normalized to ribosomal RNA. In brief, target genes and ribosomal RNA values were calculated from standard curves obtained by amplification of 2-fold serial dilutions of cDNA from the tissue. We verified that the cDNAs and ribosomal RNA were amplified at approximately the same efficiency. Results are expressed as the percent of ribosomal RNA-normalized target mRNA in experimental groups vs. control groups. The results were analyzed using sequence detection software (Applied Biosystems), as outlined in PerkinElmer’s user bulletin no. 2 (PerkinElmer, Wellesley, MA).

Western blotting
Western blotting was performed as previously described (27). The frozen tissue preparations were homogenized with sodium dodecyl sulfate sample buffer, centrifuged, and boiled. The total protein concentration of tissue was quantified by the Bradford method (28). After determining the total protein concentration, an equal amount of total protein was loaded on 8% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis and was then transferred electrophoretically onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Richmond, CA). The membranes were blocked with 5% nonfat milk for 1 h and then incubated overnight with primary antibodies at 4 C and then for 1 h at room temperature with the secondary antibody. UCP1 was detected by enhanced chemiluminescence (Amersham Life Science, Buckinghamshire, UK) and quantitated using National Institutes of Health imaging software.

Immunohistochemistry
Mice were anesthetized with Nembutal and perfused transcardially with isotonic PBS, followed by 4% paraformaldehyde in 0.1 M phosphate buffer after the apelin or vehicle injection. BAT and MSL were removed and postfixed. Slices (5 µm) were cut from the BAT and MSL using a vibrotome. Tissues were washed three times in PBS and incubated for 1 h in 0.3% H2O2 to quench endogenous peroxidase activity. Slices were then transferred without rinsing to the primary antibody solution, consisting of 0.005 g/ml polyclonal rabbit antiserum with specificity for UCP1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After 24-h incubation on ice, the slices were washed three times in PBS and processed using the ABC method (Vector Laboratories, Burlingame, CA). Slices were transferred to a biotinylated antirabbit antibody solution for 1 h, washed, transferred to avidin-biotinylated peroxidase for 1 h, washed, and then developed with diaminobenzidine substrate for 10 min. Slices were then washed, mounted on slides, and coverslipped with Permount.

Body temperature
The core body temperature of male mice 60 min after food deprivation was measured at room temperature at 1300 h, using a rodent telethermometer (EIK, Tokyo, Japan), equipped with a rectal probe, which was inserted 1 cm into the rectum. Body temperature was also assessed by rectal temperature at 0 and 60 min after injection of apelin (0.1 µmol/kg) or the vehicle.

Indirect calorimetry
In vivo indirect calorimetry was performed using the Oxymax system (no. 05142; Columbus Instruments, Columbus, OH). Constant airflow (0.6 l/min) was drawn through the chamber and monitored by a flowmeter. To calculate oxygen consumption (VO2), carbon dioxide production, and respiratory quotient (RQ; ratio of carbon dioxide production to VO2), gas concentrations were monitored at the inlet and outlet of the scaled chambers. The 12-wk-old animals were randomly placed into the experimental chambers at 25 ± 1 C, 55 ± 5% humidity, with free access to food and water. Mice were individually housed in Plexiglas cages, through which air of known O2 concentration was passed at a constant flow rate. After a 24-h acclimation period, exhaust air was sampled in the fed state to determine O2 and CO2 levels. After confirming the stability of VO2 and RQ in both groups, apelin or vehicle was administered. Calorimetry was performed after a single injection of apelin (0.1 µmol/kg) at 1530 h. Samples were collected 6 h after treatment approximately every 5 min. For each time point, the samples for each group were averaged.

Statistical analyses
All data are expressed as the mean ± SEM. We used ANOVA with a post hoc Bonferroni test to analyze differences in multiple comparisons (StatView 5.0; SAS Institute, Cary, NC), or a Mann-Whitney U test, when appropriate. In the dose-dependency study, we used a simple regression test and Pearson’s coefficient test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of apelin treatment on food intake and body weight
Figure 1AGo shows average food intakes. No significant difference in average food intake was observed between apelin-treated [apelin (AP)] and nontreated [saline (CONT)] animals (CONT vs. AP; P > 0.1). In contrast, body weight gain was attenuated in the apelin group, compared with the control group, as shown in Fig. 1BGo (CONT vs. AP; 0.61 ± 0.24 vs. –0.13 ± 0.21 g, P < 0.05).


Figure 1
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  		FIG. 1. Effects of apelin on average food intake (A), body weight (B), weight of epididymal, mesenteric, and retroperitoneal WAT (C), triglyceride content of epididymal WAT (D), and histological analysis of epididymal WAT (E) in mice in the CONT and AP groups. Each value and vertical bar represents the mean ± SEM (*, P < 0.05, **, P < 0.01 vs. control, n = 6).

 
Effect of apelin treatment on WAT weight, triglyceride content of WAT, and WAT morphology
Apelin treatment decreased the WAT weight, compared with the saline-treated group (CONT vs. AP: 0.32 ± 0.03 vs. 0.22 ± 0.02 g, P < 0.01 for epididymal WAT; CONT vs. AP: 0.057 ± 0.003 vs. 0.023 ± 0.002 g, P < 0.01 for mesenteric WAT; CONT vs. AP: 0.17 ± 0.01 vs. 0.067 ± 0.01 g, P < 0.01 for retroperitoneal WAT; Fig. 1CGo). In addition, the triglyceride content of the epididymal WAT tissue decreased in the apelin group, compared with the controls (P < 0.05; Fig. 1DGo). Figure 1EGo shows the morphology of epididymal WAT; the cell size in the epididymal WAT in the apelin group decreased, compared with the controls.

Time course of concentration changes in apelin after treatment
Figure 2AGo shows the time course of changes in serum apelin levels after apelin treatment. Apelin treatment increased the serum apelin level 1 h after treatment (CONT vs. AP: 17.93 ± 4.78 vs. 64.48 ± 21.30 ng/ml, P < 0.05; Fig. 2AGo).


Figure 2
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  		FIG. 2. Time course of changes in serum apelin levels after apelin treatment in mice in the CONT and AP groups (A) and the dose-dependent effect of apelin on body weight in mice (B) (P < 0.001, r = –0.82). Each value and vertical bar represents the mean ± SEM (*, P < 0.05 vs. control, n = 4).

 
Dose-dependent effect of apelin on body weight
Figure 2BGo shows the dose-dependent effect of apelin on body weight (apelin dose: 0, 0.05, 0.1 µmol/kg, P < 0.001, r = –0.82; Fig. 2BGo).

Effect of apelin treatment on serum glucose, insulin, FFAs, and triglycerides
No significant difference was observed in basal serum glucose or FFA levels between the apelin group and controls (both P > 0.1; Fig. 3Go, A–D). The serum insulin and triglyceride levels were reduced in the apelin group, compared with the controls (both P < 0.05; Fig. 3Go, B and E).


Figure 3
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  		FIG. 3. Effects of apelin on serum glucose (A), serum insulin (B), glucose level changes during an ip glucose tolerance test (C), serum FFA (D), and serum triglyceride (E) in mice in the CONT and AP groups. Each value and vertical bar represents the mean ± SEM (*, P < 0.05, **, P < 0.01 vs. control, n = 4).

 
Intraperitoneal glucose tolerance test
During the ip glucose tolerance test, apelin-treated mice clearly showed reduced levels of blood glucose at 30 min after treatment, compared with controls (Fig. 3CGo; P < 0.01).

Effect of apelin treatment on food intake, body weight, WAT weight, serum glucose, insulin, FFAs, and triglycerides in diet-induced obese mice
There was no significant difference in daily food consumption or body weight change between apelin-treated (HFAP) and nontreated (HF) diet-induced obese mice (HF vs. HFAP: P > 0.1; Fig. 4Go, A and B). Epididymal adiposity was attenuated in the apelin group, compared with controls (HF vs. HFAP: 1.71 ± 0.12 vs. 1.25 ± 0.07 g, P < 0.01 for epididymal WAT; Fig. 4CGo). In addition, serum insulin, FFA, and triglyceride levels were all decreased in the apelin group, compared with the controls (HF vs. HFAP: 1.9 ± 0.1 vs. 1.2 ± 0.1 ng/ml, P < 0.05 for insulin; HF vs. HFAP; 1.4 ± 0.09 vs. 1.2 ± 0.03 meq/liter, P < 0.05 for FFA; HF vs. HFAP: 42 ± 4.0 vs. 26 ± 2.9 mg/dl, P < 0.05 for triglyceride; P < 0.05 for each; Fig. 4Go, E, F, and G). There was no significant difference in serum glucose level between the apelin group and controls (HF vs. HFAP: 268 ± 22 vs. 273 ± 13 mg/dl, P > 0.1 for glucose; Fig. 4DGo).


Figure 4
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  		FIG. 4. Effects of apelin on average food intake (A), body weight (B), weight of epididymal WAT (C), serum glucose (D), serum insulin (E), serum FFA (F), and serum triglyceride (G) in mice in the HF and HFAP groups. Each value and vertical bar represents the mean ± SEM (*, P < 0.05, **, P < 0.01 vs. control, n = 3).

 
Effect of apelin treatment on the expression of leptin mRNA in WAT and its serum levels
Figure 5AGo shows the mRNA levels of leptin in epididymal WAT. Leptin mRNA did not change significantly in the apelin group, compared with the controls (CONT vs. AP: 100 ± 7.0 vs. 115 ± 10.2%, P > 0.1; Fig. 5AGo). Figure 5BGo shows the serum leptin level, which was lower in the apelin group, compared with controls (CONT vs. AP: 1.65 ± 0.16 vs. 1.29 ± 0.15 ng/ml, P < 0.01; Fig. 5BGo).


Figure 5
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  		FIG. 5. Effects of apelin on the expression of leptin mRNA in epididymal WAT (A), serum leptin (B), expression of adiponectin mRNA in epididymal WAT (C), and serum adiponectin (D) in mice in the CONT and AP groups. Each value and vertical bar represents the mean ± SEM (*, P < 0.05, **, P < 0.01 vs. control, n = 8).

 
Effect of apelin treatment on the expression of adiponectin mRNA in WAT and its serum levels
Figure 5CGo shows the mRNA levels of adiponectin in epididymal WAT. The expression of adiponectin mRNA did not significantly change in the apelin group, compared with the controls (CONT vs. AP: 100 ± 11.4 vs. 84.5 ± 10.3%, P > 0.1; Fig. 5CGo). Figure 5DGo shows the serum adiponectin level, which increased in the apelin group, compared with the controls (CONT vs. AP: 9.9 ± 2.9 vs. 14.1 ± 5.9 µg/ml, P < 0.05; Fig. 5DGo).

Effects of apelin on BAT UCP1 proteins, as assessed by immunohistochemistry and Western blotting, and BAT UCP1, WAT UCP2, and MSL UCP3 mRNA expression
Figure 6AGo shows that immunostained UCP1-positive cells were relatively higher in the apelin group than in the control group. Figure 6BGo shows the immunoblot bands and relative densities of UCP1 in BAT. UCP1 content was higher in the apelin group than in the controls (CONT vs. AP: 100 ± 9.5 vs. 472.2 ± 122.4%, P < 0.05; Fig. 6BGo). Figure 6CGo shows the change in BAT UCP1 mRNA levels after treatment with apelin. BAT UCP1 mRNA expression increased by 519% after treatment with apelin, compared with the controls (P < 0.05; Fig. 6CGo). No significant change occurred in WAT UCP2 mRNA expression in the apelin group, compared with controls (P > 0.1; Fig. 6DGo). Figure 6EGo shows that MSL UCP3 mRNA expression was increased by 623% after treatment with apelin, compared with saline-treated mice (P < 0.05, Fig. 6EGo).


Figure 6
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  		FIG. 6. Effects of apelin on BAT UCP1 immunostaining (A), immunoblot bands and relative densities of UCP1 in BAT (B), BAT UCP1 mRNA (C), WAT UCP2 mRNA (D), and MSL UCP3 mRNA (E) within the CONT and AP groups. Each value and vertical bar represents the mean ± SEM (*, P < 0.05 vs. control, n = 4).

 
Effects of apelin on body temperature
As shown in Fig. 7AGo, apelin treatment increased rectal temperature by 0.75 C, compared with controls 60 min after the injection (P < 0.05, Fig. 7AGo).


Figure 7
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  		FIG. 7. A, Effects of apelin on body temperature. CONT-PRE, Pretreatment of saline; CONT-POST, 1 h after saline treatment; AP-PRE, pretreatment of apelin; AP-POST, 1 h after apelin treatment. B and C, Effects of apelin on oxygen consumption (B), and the respiratory quotient (C) in mice. CONT-PRE, Pretreatment of saline; CONT-POST, 6 h after saline treatment; AP-PRE, pretreatment of apelin; AP-POST, 6 h after apelin treatment. Each value and vertical bar represents the mean ± SEM (*, P < 0.05 vs. control, n = 4).

 
Effects of apelin treatment on oxygen consumption and respiratory quotient
Figure 7Go, B and C, shows the change in oxygen consumption (Fig. 7BGo) and the RQ (Fig. 7CGo). Acute treatment with apelin increased the oxygen consumption (P < 0.05; Fig. 7BGo) and decreased the RQ (P < 0.05; Fig. 7CGo), both of which were assessed pretreatment and 6 h after treatment, compared with the controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrated that apelin attenuated body adiposity, without affecting food intake both in normal and diet-induced obese C57BL/6 mice. In addition, apelin treatment dose-dependently decreased body weight gain. The possible involvement of apelin in regulating adiposity has been suggested by the distribution of APJ receptors in WAT, suggesting a direct action of apelin on WAT lipid metabolism (3, 5, 12) both in normal and diet-induced obese C57BL/6 mice.

In this study, apelin treatment decreased serum insulin levels both in normal and die-induced obese mice. In addition, apelin-treated mice showed reduced levels of blood glucose after treatment, compared with controls during the ip glucose tolerance test. From these observations, it is suggested that apelin treatment increased in insulin sensitivity in vivo. It is possible that the lowered insulin and triglyceride levels simply resulted from the reduction in adiposity. In this study, the apelin-induced reduction in adiposity may have caused the decreased leptin level and increased adiponectin level. Adiponectin stimulates glucose use and fatty-acid oxidation by activating AMP kinase (29, 30, 31). Accelerated fatty acid oxidation also leads to the attenuation of triglyceride synthesis and consequently prevents tissue triglyceride accumulation (17).

Next, we considered how apelin reduces body adiposity. A previous study showed that central administration of apelin reduces food intake (32). However, our results demonstrated that the peripheral administration of apelin did not affect food intake. It appears that the peripheral administration of apelin, at least at the doses we used, is insufficient to regulate food intake.

Given this, the effect of apelin on energy metabolism may be an important factor in the apelin-induced reduction in adiposity. UCP is an inner mitochondrial membrane transporter of FFAs that dissipates the proton gradient by releasing stored energy as heat (16, 17). UCP1 in BAT plays a crucial role in regulating energy expenditure and thermogenesis in rodents and the neonates of larger mammalian species, including humans (15, 16, 17, 18). Our study demonstrated that apelin treatment increased BAT UCP1 mRNA and protein expression, reflecting an apelin-induced increase in energy expenditure. It is not clear whether apelin affects BAT UCP1 expression directly because APJ receptors have not been identified in BAT.

It is also possible that other factors mediate the accelerating effect of apelin on UCP1. First, activation of adiponectin by apelin, as seen here, might increase UCP1 expression because we previously observed up-regulation of UCP1 by adiponectin (33). Second, the possible involvement of the central nervous system cannot be excluded because BAT UCP1 is regulated by the sympathetic nervous system and upper brain structures (34, 35, 36). More specifically, a neuroanatomical study using a trans-synaptic retrograde tracer identified the paraventricular nucleus in the hypothalamus as a major origin of sympathetic nerves that innervate BAT (37). Indeed, stimulation of the paraventricular nucleus can accelerate sympathetic nerve activity (38, 39).

Our data (Higuchi, K., T. Masaki, and H. Yoshimatsu, unpublished observations) revealed that the apelin concentration in the hypothalamus is increased after ip apelin injection. This suggests that peripherally administered apelin can cross the blood-brain barrier and may have central effects on body weight and adiposity. However, intralateral ventrical-administered apelin at a dose of 1 nmol/kg·d (comparable with the concentration in the hypothalamus) had no effect on body adiposity. These observations indicate that central apelin, at least at the doses we used, did not have an effect on body adiposity, whereas peripheral apelin effectively reduced body weight and adiposity.

In this study, we confirmed the functional role of the apelin-induced increase in UCP1 mRNA and protein expression in BAT using body temperature measurements and calorimetry. In fact, apelin treatment increased body temperature and O2 consumption, indicating an apelin-induced acceleration of energy expenditure, probably mediated by BAT UCP1. In addition, apelin treatment decreased the respiratory quotient. This suggests that apelin treatment decreases the use of carbohydrates and increases the use of fat, which may also contribute to the reduction in body fat.

In addition to the change in BAT UCP1, in this study, apelin treatment increased muscle UCP3 mRNA expression. The functional meaning of this is unclear. Recently it was suggested that UCP3 contributes to the export of fatty acids from the mitochondrial matrix rather than the regulation of energy expenditure via thermogenesis (40). The export of fatty acid from the mitochondrial matrix by UCP3 may prevent the accumulation of fatty acids in mitochondria and help to maintain muscular fat oxidative capacity. The apelin-induced up-regulation of muscle UCP3 might also contribute to regulating fatty acid mobilization and use.

The present study has some limitations. First, apelin has a potent diuretic action (41) that could affect body weight. Second, we did not determine the endogenous/exogenous apelin level or the bioactivity of apelin. Third, the involvement of apelin in human obesity remains unresolved. Further studies are necessary to address in more detail the signaling mechanisms by which apelin affects BAT UCP1, body temperature, oxygen consumption, and body weight.

In summary, apelin may regulate the adiposity of WAT, lipid metabolism, the expression of BAT UCP1, and energy expenditure in mice.


   Footnotes
 
First Published Online March 8, 2007

Abbreviations: BAT, Brown adipose tissue; FFA, free fatty acid; MSL, skeletal muscle; RQ, respiratory quotient; UCP, uncoupling protein; VO2, oxygen consumption; WAT, white adipose tissue.

Disclosure Statement: We have nothing to disclose.

Received September 15, 2006.

Accepted for publication February 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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