Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways*

Background: The autocrine regulation of Ang(1–7) upon adipogenesis is unknown. Results: The autocrine counteractive interplays between Ang(1–7)-Mas and AngII-AT1 signaling upon adipogenesis are revealed. Conclusion: The Ang(1–7)-Mas activation stimulates adipogenesis and antagonizes the antiadipogenic effect of AngII-AT1 activation. Significance: The angiotensin system in adipose tissue may serve as a potential therapeutic target for obesity and related metabolic disorders. Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1–7). Co-expression of AngII receptors (AT1 and AT2) and Ang(1–7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1–7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1–7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1–7)-Mas antagonizes the antiadipogenic effect of AngII-AT1 by inhibiting the AngII-AT1-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT1-ACE2-Ang(1–7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.

RAS is also a crucial regulator of energy metabolism, implicated in metabolic disorders such as obesity and insulin resistance (7)(8)(9). Adipose tissue, a highly active metabolic and endocrine organ, is a source of components of the RAS (10,11). On the other hand, AngII receptors and Mas receptors are co-expressed on adipocytes, implying the involvement of the local RAS system in regulating adipocyte functions (12,13). Several lines of evidence have supported this emerging hypothesis. In particular, recent investigations have begun to reveal that ACE-AngII-AT 1 signaling and ACE2-Ang(1-7)-Mas signaling regulate various functions of adipocytes in an intriguing counteractive manner (6). For example, it has been demonstrated that AngII-AT 1 inhibits lipolysis and insulin-induced glucose uptake in adipocytes (14,15), whereas Ang(1-7)-Mas does the opposite (16,17). Also, AngII-AT 1 induces oxidative stress in adipocytes (18), whereas Ang(1-7)-Mas interaction suppresses it (17).
Previous studies have established the inhibitory effects of AngII-AT 1 on adipogenesis (19)(20)(21). We hypothesize that ACE2-Ang(1-7)-Mas acts as an autocrine balancer (feedback route) to promote adipogenesis. We aim, for the first time, to reveal the molecular mechanisms underlying Ang(1-7)-Mas regulation upon adipogenesis and its cross-talk with AngII-AT 1 pathways.
The 3T3-L1 mouse preadipose cell line (American Type Culture Collection, Manassas, VA) was cultured in DMEM supplemented with 10% (v/v) bovine calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin in a 5% CO 2 humidified atmosphere at 37°C. Adipocyte differentiation of 3T3-L1 cells follows the previously established protocol (24,25). Specifically, 2 days after the cells became confluent, adipocyte differentiation was induced (defined as day 0) by incubation with DMEM and 10% FBS, 5 g/ml insulin, 1 M dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine. On day 2 of differentiation, DMEM containing 10% FBS and 5 g/ml insulin was used as the culture medium. On day 4, the medium was then changed to DMEM with 10% FBS only, and thereafter, the medium was refreshed every 2 days for the following 2-4 days.
In some experiments, 10 M LY294002, 50 M PD98059, 100 nM PMA, 10 M A779, 10 M PD123319, 10 M ZD7155, 1 M AngII, or various concentrations of Ang(1-7) were added to the culture medium. In some experiments, a 30-min pretreatment of LY294002 or 60-min pretreatment of PD98059 was performed prior to the induction of differentiation. Ang(1-7), AngII, ZD7155, and PD123319 were purchased from Tocris Bioscience, and A779 was purchased from GenWay Biotech Inc. All cell culture media, supplements, and sera were purchased from Invitrogen. All other chemicals and reagents were purchased from Sigma-Aldrich.
Confocal Fluorescence Microscopy-Cells grown on glass coverslips were fixed with 3.7% formaldehyde in PBS for 15 min at room temperature and then washed with PBS three times. After being blocked with 1% bovine albumin (BSA) in PBST (PBS with 0.1% Tween 20) at room temperature for 1 h, the cells were incubated (at 4°C) overnight with goat anti-Mas IgG in PBST with 1% BSA. After washing with PBS three times, the cells were incubated in FITC-labeled anti-goat IgG for 1 h at room temperature in the dark, followed by washing with PBS three times. Fluorescence signals were visualized on a confocal laser-scanning microscope (LSM 510 Meta, Carl Zeiss GmbH).
Western Blot Analysis-The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then scraped in radioimmune precipitation assay lysis buffer (sc-24948, Santa Cruz Biotechnology, Inc.) containing freshly added protease/ phosphatase inhibitor mixture (Cell Signaling Technology). After centrifugation at 4°C, the supernatant of the sample was collected, and its protein content was determined using a protein assay kit (Bio-Rad) based on the Bradford method. Each cell lysate with an equal amount of protein content (as the loading control) was separated on SDS-PAGE by electrophoresis, followed by transfer onto a nitrocellulose membrane. After blocking the membrane for 2-3 h in TBST (Tris-buffered saline-Tween; 10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.4) containing 5% BSA (Sigma), the membrane was incubated with primary antibodies (1:200 -400 dilution) in TBST with 1% BSA overnight. After washing three times with TBST buffer, the membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 -3000 dilution) for 6 -8 h. The protein bands were detected in a G:BOX Chemi XT4 imaging system (Syngene) by using Super-Signal West Pico chemiluminescent substrate (Thermo Scientific).
Oil Red O Staining-Lipid droplets in adipocytes were stained with Oil Red O (Sigma-Aldrich) as described previously (28). Briefly, after being fixed in 10% formalin for 60 min, the cells were washed twice with PBS and stained with filtered Oil Red O solution (1.8 mg/ml Oil Red O and 60% (v/v) isopropyl alcohol in distilled water) for 60 min. The cells were then thoroughly washed with distilled water before being photographed under an optical microscope.
Triglyceride Assay-Cellular triglyceride content was determined by using an adipogenesis assay kit (Biovision Inc.) according to the manufacturer's protocol. Briefly, after a 6 -8day differentiation of 3T3-L1 cells, or a 10 -12-day differentiation of human preadipocytes, cells were washed twice with PBS, scraped, sonicated to homogenize the suspension, and then assayed for total triglyceride. The total protein concentration, which was used as an internal control, in these cells was measured with a protein assay kit from Bio-Rad.
Statistical Analysis-Data were analyzed using Student's t test and expressed as mean Ϯ S.E. p Ͻ 0.05 was considered statistically significant.

Ang(1-7)-Mas Receptor Promotes Adipogenesis and Antagonizes the Antiadipogenic Effect of AngII-AT 1 Receptor in Human
Preadipocytes-Preadipocytes isolated from human adipose tissue are used here as the in vitro cell model for adipogenesis (29,30). It has been known that AngII receptors are expressed in preadipocytes, and the majority of AngII receptors found in human preadipocytes and adipocytes are of the AT 1 subtype (13, 31) (supplemental Fig. S1). Here, we report that Mas receptors are also expressed in human preadipocytes and adipocytes ( Fig. 1A and supplemental Fig. S1). To evaluate the roles of Ang(1-7) and AngII in adipogenesis, human preadipocytes were induced to differentiate in the presence of Ang(1-7) and/or AngII.
Transformation of preadipocytes into mature adipocytes requires the coordinated regulation of various transcriptional factors (30). Among them, PPAR␥ is considered as the most essential one (32). Because adipogenesis is accompanied by lipogenesis, enzymes responsible for lipogenesis (e.g. fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC)) are also regarded as the important markers for adipogenesis (24,33).
After 10 -12 days of hormone-induced differentiation, many of the fibroblast-like spindle-shaped human preadipocytes were differentiated into mature adipocytes, as evidenced by their morphological change and intracellular accumulation of lipid droplets (Fig. 1B). Lipid accumulation, an indicator of the extent of adipogenesis, can be assessed visually by Oil Red O staining of lipid droplets and quantitatively by direct measurement of intracellular triglyceride (TG) content. As shown in Fig. 1, C and D, exogenous application of Ang(1-7) during differentiation considerably increased Oil Red O staining and TG content, and the number of lipid-laden adipocytes was also increased by Ang(1-7) treatment ( Fig. 1E and supplemental Fig. S2A). Consistently, the addition of Ang(1-7) enhanced the expression of adipogenic transcriptional factor PPAR␥, lipogenesis markers (FAS and ACC), and adipocyte protein-2 (aP2;
It has been reported that AngII suppresses adipogenesis of human preadipocytes via the AT 1 receptor (19,20). As expected, the addition of AngII during differentiation suppressed adipogenesis, as evidenced by reduced Oil Red O staining, TG content, the number of adipocytes, and the expression of adipogenesis markers (PPAR␥, FAS, ACC, and aP2) (Fig. 1). Such AngII-induced suppression is mediated by AT 1 receptors because it can be rectified by a specific AT 1 antagonist (ZD7155) (34) but not by a specific inhibitor of AT 2 (PD123319) (35) (supplemental Fig. S2B).
Interestingly, co-incubation with Ang(1-7) effectively abolished the antiadipogenic effects of AngII (Fig. 1). To determine if Ang(1-7) mediates its stimulatory actions through the Mas receptor, the selective Mas receptor antagonist A779 (36) was applied during differentiation. As shown in Fig. 1, the addition of A779 completely eliminated the stimulatory effects of Ang(1-7) on adipogenesis and its counteraction on AngII. Taken together, we conclude that Ang(1-7), via its interaction with the Mas receptor, not only  stimulates adipogenesis but also counteracts the antiadipogenic effect of AngII.
In order to evaluate the role of AngII in adipogenesis of 3T3-L1 cells, we have to differentiate the effects mediated by the AT 1 or AT 2 receptor. This is achieved by challenging the AT 2 siRNA knockdown 3T3-L1 cells or AT 1 siRNA knockdown cells with AngII. Knockdown of the AT 1 or AT 2 gene (Agtr1 or Agtr2) was confirmed by Western blot analyses (supplemental Fig. S1). As shown in Fig. 3, activation of AT 1 receptors decreased Oil Red O staining, TG content, and expression of all adipogenic markers (PPAR␥, C/EBP␣, FAS, ACC, and aP2), whereas AT 2 activation did the opposite. These results were corroborated in naive 3T3-L1 cells by selective inhibition of AT 1 or AT 2 using their specific antagonists (supplemental Fig.  S4). These experiments establish the inhibitory role of AngII-AT 1 signaling in adipogenesis, which is in agreement with previous reports (19 -21) and our observations in human preadipocytes (Fig. 1). Also similar to the observations from human preadipocytes (Fig. 1), co-incubation of Ang(1-7) essentially

Ang(1-7) and AngII Regulation upon Adipogenesis
reversed the inhibitory effects of AT 1 receptor activation upon adipogenesis. On the other hand, Ang(1-7) did not affect AT 2stimulated adipogenesis ( Fig. 3 and supplemental Fig. S4). Taken together, we show that 3T3-L1 is a good and convenient alternative model to human preadipocytes to study the stimulatory roles of Ang(1-7)-Mas signaling in adipogenesis and its cross-talk with AngII-AT 1 pathways.
Autocrine Interplays between Ang(1-7) and AngII-It is known that production of AngII and its precursor peptide (angiotensinogen) increases during adipogenesis (20,40). AngII peptides, in turn, are cleaved by a membrane-bound ectoenzyme (ACE2) to form Ang(1-7) (4). It is thus conceivable that Ang(1-7) production might increase as well during adipogenesis. Indeed, as shown in Fig. 4, A and B, both Ang(1-7) and AngII released into the culture media were elevated during adipogenesis. Because the receptors for Ang(1-7) and AngII are expressed in preadipocytes and adipocytes (12, 39) (Figs. 1 and 2 and supplemental Fig. S1), it is likely that these locally produced Ang(1-7) and AngII may serve as the autocrine feedback signals upon adipocyte differentiation in an interactive way.
Ace2 gene expression in 3T3-L1 cells was blocked by specific ACE2 siRNA before the initiation of differentiation (verified by Western blot analyses shown in supplemental Fig. S1). As a result, it was found that the release of Ang(1-7) from these cells was considerably lowered throughout the differentiation, whereas the AngII release was increased in the early stage (day 1-2) (Fig. 4, A and B). It was also noted that adipogenesis was suppressed in ACE2 siRNA knockdown cells, as evidenced by the reduced number of lipid-laden adipocytes and decreased expression of adipocyte markers: aP2 and FAS (Fig. 4, C-F). This is presumably due to the decrease of the adipogenic factor Ang(1-7) and increase of the antiadipogenic factor AngII. In support of this, we show that down-regulation of adipogenesis by ACE2 knockdown can be effectively rectified by application of Ang(1-7) or blocking of the AT 1 receptor. These results demonstrate that ACE2 is a critical linker to control the balance between Ang(1-7) and AngII signaling. Fig. 4, G and H, shows that ACE2 expression steadily increased over time during the 3T3-L1 differentiation, promoting adipogenesis by strengthening Ang(1-7) signaling. Intriguingly, exogenous application of Ang(1-7) suppressed the increase of ACE2 expression (Fig. 4G), whereas AngII-AT 1 activation further enhanced ACE expression in the early differentiation phase (day 1). These observations suggest that a delicate balance is established by the interplays between AngII, ACE2, and Ang(1-7).

Involvement of Mas Receptor-
The experiments on human preadipocytes (Fig. 1) demonstrate that the adipogenic and anti-AngII effects of Ang(1-7) are mediated by the Mas receptor. Here, we further analyzed the involvement of the Mas receptor using Mas1 gene knockdown 3T3-L1 cells. The knockdown of Mas receptor expression by MAS1 siRNA was verified by Western blot analyses (supplemental Fig. S1).
In the Mas knockdown cells, the expressions of adipogenic markers (PPAR␥, FAS, ACC, and aP2) were reduced (Fig. 5, A  MAY 31, 2013 • VOLUME 288 • NUMBER 24 and B), and as expected, Ang(1-7) failed to increase the expression of these markers and to abolish the inhibition by AngII. Some studies have reported low affinity interaction of Ang(1-7) with AT 1 and AT 2 receptors in other cell types (41,42). To rule out the possible contributions from AT 1 and AT 2 receptors, we show that Ang(1-7)-stimulated expression of the adipogenic markers was not compromised when AT 1 and AT 2 receptors were simultaneously blocked by their respective antagonists. These results establish that the adipogenic effects of Ang(1-7) are mediated by Mas receptors.

Ang(1-7) and AngII Regulation upon Adipogenesis
Furthermore, we also observed that Mas receptor expression increased over time during the 3T3-L1 differentiation, and application of exogenous Ang(1-7) elevated Mas receptor expression at the early differentiation phase (day 1-2) (Fig. 5C). These experiments demonstrate the positive regulation of Ang(1-7)-Mas signaling to accelerate adipogenesis, particularly in the early stage.
Stimulation of Adipogenesis by Ang(1-7) is MAPK Kinase/ ERK-and PI3K/Akt-dependent-Next, we explored the signaling pathways responsible for the stimulatory effect of Ang(1-7) on adipogenesis of 3T3-L1 cells. It is known that the phosphorylation (activation) of ERK1/2 is required for mitotic clonal expansion in the early stage of adipogenesis (43,44); however, ERK1/2 activity must quickly return to a low level to avoid inactivation (phosphorylation) of the critical adipogenic factor (PPAR␥) (45,46). Indeed, as shown in Fig. 6A, hormonal stimulation of adipogenesis caused an acute increase of ERK1/2 phosphorylation (within 15 min), which was followed by a quick decay to a low level. Interestingly, such decay was exaggerated by the addition of Ang(1-7) (Fig. 6A). As discussed above, it would help to sustain the adipogenic process by preventing ERK1/2-induced inactivation (phosphorylation) of PPAR␥. This is confirmed in Fig. 6B.
PI3K/Akt signaling-triggered PPAR␥ expression is also well known for its critical role in adipogenesis (47,48). Regulation of Ang(1-7) on the PI3K/Akt pathway has been reported in other cell types (49,50). As shown in Fig. 6C, phosphorylation of Akt was acutely induced (within 5 min) upon induction of adipogenic differentiation, and this acute phosphorylation was largely enhanced by the addition of Ang(1-7), although such enhancement is short lived (within 15 min). ERK1/2 and Akt are the substrates of MAPK kinase and PI3K, respectively. Stimulation of MAPK kinase by phorbol 12-myristate 13-acetate (PMA) produced a significant inhibitory effect on adipogenesis, as evidenced by the reduced number of mature adipocytes and expression of adipogenic markers (aP2, FAS, ACC, and PPAR␥) (Fig. 7, A-G). On the contrary, inhibiting MAPK kinase by PD98059 (51) slightly enhanced adipogenesis ( Fig. 7 and supplemental Fig. S5). PD98059 inhibition and PMA stimulation upon adipogenesis have also been reported previously (52)(53)(54). Here, we further show that PMA was able to partially eliminate Ang(1-7)-induced adipogenesis ( Fig. 7 and supplemental Fig. S5). In another line of experiments, it was found that blocking PI3K by LY294002 (55) significantly inhibited adipogenesis and abolished the stimulatory effect of Ang(1-7) (Fig. 7, A-G).
The ERK1/2 inhibition and Akt stimulation upon adipogenesis were further confirmed by knockdown of ERK1/2 (Mapk3 and Mapk1) or Akt1/2 genes before the initiation of 3T3-L1 cell differentiation. The knockdown of the protein expression was verified by Western blot (supplemental Fig. S5). In the ERK1/2 knockdown cells, the expressions of adipogenic markers (aP2 and FAS) were significantly increased, whereas in the Akt knockdown cells, the expressions of these markers were largely suppressed (Fig. 7, H-J). Also, as expected, Ang(1-7) failed to increase the expressions of these markers in the Akt knockdown cells (Fig. 7, H-J).
Taken together, it may be concluded that Ang(1-7) stimulates adipogenesis through two distinct pathways: 1) inhibiting MAPK kinase-ERK1/2 signaling, which leads to PPAR␥ inactivation, and 2) enhancing PI3K/Akt signaling, which leads to PPAR␥ expression. The latter pathway appears to be more potent.
(shown in Fig. 6) suggests the importance of Ang(1-7) signaling in the early stage of differentiation. In support of this view, it was found, based on the number of differentiated adipocytes and expressions of adipogenic markers (aP2 and FAS), that administration of Ang(1-7) even 1 day after the hormonal initiation of differentiation failed to stimulate adipogenesis, whereas incubation with Ang(1-7) only for the first day of differentiation was as potent as its presence for the whole differentiation process (6 days) (Fig. 8).
To further confirm that Ang(1-7) acts mainly on the early stage of differentiation, the Mas receptor was knocked down at different stages of differentiation (at day Ϫ2 (2 days before the initiation of differentiation) or at day 0, day 2, or day 4) (Fig. 8, G-I). The Mas receptor expression was largely suppressed even 1 day after the transfection with MAS1 siRNA, which was verified by Western blot analyses (supplemental Fig. S1). Consistent with our data shown in Fig. 5, Ang(1-7) failed to increase the expression of adipogenic markers (aP2 and FAS) in the 3T3-L1 cells transfected with MAS1 siRNA at day Ϫ2; however, Ang(1-7) was able to significantly stimulate the expressions of these markers in the cells transfected with MAS1 siRNA at day 0, day 2, or day 4 (Fig. 8, G-I).
It is known that, in the early stage of differentiation, preadipocytes undergo several rounds of cell division, known as mitotic clonal expansion, before undertaking terminal differentiation, through which they acquire the characteristics of maturity (33,43). We show in Fig. 8C that induction of differentiation resulted in 2-3-fold increase in cell number within 2 days, but Ang(1-7) treatment had an insignificant effect on cell proliferation. This observation rules out the possibility that the adipogenic effects of Ang(1-7) is due to increased mitotic clonal expansion.

DISCUSSION
Adipocytes, which were previously regarded as the passive storage depots for excess energy (fat), actually play perplexing and integrative roles in regulating metabolism (56). Complicated cross-talks exist between adipocytes and other cells in the metabolic networks (57). For example, the functions of adipocytes (adipogenesis, lipid, and carbohydrate metabolism) are  MAY 31, 2013 • VOLUME 288 • NUMBER 24 highly regulated by insulin secreted by pancreatic beta cells (58), catecholamines secreted by chromaffin cells (59), and glucocorticoid secreted by adrenal cortex (60). On the other hand, adipocytes release a variety of adipokines (e.g. leptin, apelin, and AngII) to control various aspects of metabolism and influence the secretion of other metabolic factors or hormones from other cells (59,61). Intriguingly, the receptors of some adipokines are identified in adipocytes, implying the existence of autocrine regulatory loops on adipocyte functions (39,62,63). For instance, adiponectin-adiponectin receptor (AdipoR1 and AdipoR2) promotes adipogenesis and insulin-induced glucose uptake and inhibits lipolysis (64,65); leptin-leptin receptor (ObR) inhibits adipogenesis and glucose uptake and promotes lipolysis (54,66); and apelin-apelin receptor inhibits adipogenesis and lipolysis (26) and promotes insulin sensitivity and glucose uptake (67).
It is increasingly accepted that formation of new adipocytes (adipogenesis) is a physiological adaptive response to excessive calorie intake in order to maintain an overall healthier adipose tissue because newly differentiated adipocytes are more insulin-sensitive (thus more capable of energy storage) (68,69). Otherwise, hypertrophic adipocytes, which are less insulin-sensitive, will result (70,71). The adverse metabolic consequences of enlarged adipocytes (particularly in visceral fat) have been extensively documented, including insulin resistance-caused type 2 diabetes and hypertriglyceridemia (72,73). The adipogenic potential of preadipocytes from different fat depots differs. Subcutaneous preadipocytes are more sensitive to the adipogenic stimuli (e.g. PPAR␥ agonist) than visceral ones (74,75). Adipogenesis in subcutaneous adipose tissues thus assists in preventing the notorious hypertrophy of visceral adipose (76). Therefore, the adipogenic effect of Ang(1-7) signaling should, albeit counterintuitively, be beneficial. This is supported by previous studies on animal models. For instance, increasing the circulating level of Ang(1-7) in rats improves insulin sensitivity in adipose tissue and decreases the plasma level of triglyceride and visceral fat mass (7,77), whereas knockdown of Mas receptors in mice results in impaired insulin sensitivity and increased visceral fat mass (78). In contrast, the antiadipogenic AngII-AT 1 signaling increases visceral adipose tissue growth and insulin resistance (19,79,80), whereas administration of AT 1 receptor blockers reduces fat mass and improves insulin sensitivity in human and several animal models (81)(82)(83).
Through circulation, the autocrine balance between AngII and Ang(1-7) signaling can be influenced by other angiotensinpeptide producing cells (e.g. vascular endothelial and smooth muscle cells, cardiac myocytes, pancreatic beta cells, hepatocytes, etc.) (2, 6). On the other hand, adipose tissue is a major source of angiotensin peptides (10,84), which play critical roles in regulating, for example, blood vessel contraction (85), angiogenesis (86), glucose uptake in skeletal muscles (9), and hormone secretion (39,87). Such cross-talks may underlie the association between obesity and other metabolic disorders (e.g. diabetes and cardiovascular diseases). Hence, this study not only reveals the intriguing autocrine interplays between AngII and Ang(1-7) signaling upon adipogenesis but also raises the possibility of targeting these molecular pathways to treat metabolic disorders.