Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis

Brown adipose tissue dissipates energy in the form of heat. Recent studies have shown that adult humans possess both classical brown and beige adipocytes (brown-like adipocytes in white adipose tissue, WAT), and stimulating brown and beige adipocyte formation can be a new avenue to treat obesity. Angiotensin II (AngII) is a peptide hormone that plays important roles in energy metabolism via its angiotensin type 1 or type 2 receptors (AT1R and AT2R). Adipose tissue is a major source of AngII and expresses both types of its receptors, implying the autocrine and paracrine role of AngII in regulating adipose functions and self-remodeling. Here, based on the in vitro studies on primary cultures of mouse white adipocytes, we report that, AT2R activation, either by AngII or AT2R agonist (C21), induces white adipocyte browning, by increasing PPARγ expression, at least in part, via ERK1/2, PI3kinase/Akt and AMPK signaling pathways. It is also found that AngII–AT2R enhances brown adipogenesis. In the in vivo studies on mice, administration of AT1R antagonist (ZD7155) or AT2R agonist (C21) leads to the increase of WAT browning, body temperature and serum adiponectin, as well as the decrease of WAT mass and the serum levels of TNFα, triglycerides and free fatty acids. In addition, AT2R-induced browning effect is also observed in human white adipocytes, as evidenced by the increased UCP1 expression and oxygen consumption. Finally, we provide evidence that AT2R plays important roles in hormone T3-induced white adipose browning. This study, for the first time, reveals the browning and brown adipogenic effects of AT2R and suggests a potential therapeutic target to combat obesity and related metabolic disorders.


INTRODUCTION
The excess white adipose tissue (WAT) that characterizes obesity is a major risk factor for the development of many diseases, such as diabetes, coronary heart disease, hypertension, stoke and some types of cancers (for example, colorectal cancer). 1,2 In contrast to the notorious WAT that stores energy as lipids, brown adipose tissue (BAT) dissipates energy directly as heat by uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) production through the action of brown adipocyte-specific uncoupling protein 1 (UCP1). 3 BAT also improves insulin sensitivity and regulates glucose homeostasis. 4 Recent studies have shown that metabolically active BAT, which was thought to disappear after infancy, is actually present in adult humans. 5 Although BAT is rare (present as small clusters in supraclavicular and paraspinal regions) in adult humans and even less in obese people, brownlike adipose cells (called beige cells) have been identified in certain WAT depots (for example, subcutaneous WAT). 6,7 Emerging evidence suggests that beige adipocytes in WAT promote body energy consumption, and confer beneficial effects on obesity and insulin resistance. 6,8 The development of beige cells in WAT (a process known as WAT browning) occurs in response to prolonged cold exposure (or β-adrenergic stimulation), and some hormones (for example, thyroid hormone T3, fibroblast growth factor 21). 8 Thus, the recent discovery of inducible beige cells in WAT have inspired the possibility to treat obesity and related metabolic disorders through stimulating ameliorative selfremodeling of WAT.
WAT is not just a passive depot for lipid storage, but also the largest endocrine organ secreting a variety of signaling molecules called adipokines, which play critical roles in regulating energy metabolism in adipose tissue and whole body. 9,10 Recent studies demonstrated that adipokines also modulate WAT browning. For examples, TNFα suppresses UCP1 expression in white adipocytes via extracellular signal-related kinase (ERK) activation 11 whereas both apelin and adiponectin enhance WAT browning. 12,13 Angiotensin II (AngII) is an adipokine well-known for its crucial roles in regulating energy homeostasis (for example, by influencing food intake, insulin secretion, glucose uptake), and its involvements with the development of obesity and type2 diabetes. 14 AngII, the major bioactive component of the reninangiotensin system (RAS), is produced from angiotensinogen by the actions of renin and angiotensin-converting enzyme (ACE), and acts through angiotensin type 1 and type 2 receptors (AT1R and AT2R). Studies have revealed that all components of RAS (including AT1R and AT2R) are present in adipose tissue, [15][16][17] implying the involvement of local RAS in regulating adipose functions and self-remodeling. This hypothesis has been supported by a number of investigations on RAS regulations of adipocyte differentiation, lipid metabolism, as well as expression and release of adipokines. 15,17,18 Most of AngII effects reported in adipocytes are negative and mediated by AT1R, such as, reduction of glucose uptake, induced oxidative stress, increased release of pro-inflammatory cytokines, and stimulation of adipocyte hypertrophy. 17,19 In contrast, except for its roles in promoting adipocyte differentiation and insulin sensitivity via PPARγ 1 School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore; 2 Duke-NUS Graduate Medical School, Singapore, Singapore and activation, 20,21 AT2R's effects in adipocyte are much less elucidated. In particular, the implication of AT2R signaling on adipose browning is unknown.
It has been shown that chronic treatment with AT2R agonist (C21) reduces the size of hypertrophic adipocytes and adipose tissue mass in rodents fed with high-fat diet, 20,21 suggesting the favorable effects of AT2R activation for obesity. Because the plasma level of AngII is elevated in obesity, 22 we speculate that AT2R activation serves to counteract the often detrimental actions by AT1R. Here, we reveal the currently-elusive AT2R signaling implicated in WAT browning, brown adipogenesis, and thyroid hormone T3-induced white adipocyte browning.

MATERIALS AND METHODS
Primary pre-adipocyte culture and differentiation The primary white and brown pre-adipocytes were isolated from mouse inguinal WAT and interscapular BAT as described before. 13 Briefly, after collagenase (0.2%) digestion, filtration and centrifugation (1500 r.p.m., 5 min), the stromal vascular fraction/pre-adipose cells were re-suspended in basal growth medium (Dulbecco's modified Eagle's medium, DMEM, supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin), and cultured at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air. For adipocyte differentiation, fully confluent preadipose cells (defined as day 0) were treated for 2 days with induction medium (growth medium supplemented with 0.5 μg ml − 1 insulin, 0.5 mM isobutylmethylxanthine (IBMX), 100 nM dexamethasone, 1 μM rosiglitazone). After 48 h, the cells were incubated for another 2 days with growth medium containing insulin, and thereafter, the medium was replenished every other day for the next 4-6 days before conducting the experiments (days 8-10).

Animal studies
All experimental protocols were approved by the SingHealth Research Facilities Institutional Animal Care and Use Committee (Singapore). Mice (C57BL/6J, 8-10 weeks old, male), housed in light-and temperaturecontrolled facility (12-h light/12-h dark cycle, 22°C), were allowed free access to standard laboratory food and water. Mice were divided into ZD7155, M024/C21 or saline (as control) -treated groups. Based on the previous studies, mice were subjected to intraperitoneal injection of ZD7155 (1 mg kg − 1 day) or M024/C21 (0.1 mg kg − 1 per day) dissolved in 100 μl of phosphate buffer saline (PBS) for 14 days. 20,24 Body surface temperatures (which correlates to body core temperature 25 and oxygen consumption rate) 26 were monitored at room temperature (RT) by taking the thermographic images of mice using infrared thermal imaging camera (Flir T420; the thermal sensitivity, o0.045°C and the spectral range, 7.5-13 μm) and analyzing with a FLIR-Tools software (FLIR System Inc, Wilsonville, OR, USA).

Assessments on serological parameters
Serum adiponectin, TNFα and insulin levels in mice were analyzed by a mouse adiponectin ELISA kit (ThermoFisher Scientific), a mouse TNFα ELISA kit (Sigma-Aldrich) and a mouse insulin ELISA kit (ThermoFisher Scientific), AT2 receptor stimulates adipose tissue browning A Than et al respectively, according to manufacturer's protocols. Serum levels of free fatty acids, triglycerides and glucose were measured by standard assay kits from Biovision. Serum cholesterol level was determined by a cholesterol quantitation kit (Sigma-Aldrich).
Oxygen consumption rate assay Oxygen consumption was analyzed using oxygen consumption rate assay kit from Cayman Chemical (MitoXpress-Xtra HS method) according to manufacturer's instructions. Briefly, adipocytes grown in 96-well plates were treated with 10 μl MitoXpress-Xtra (a phosphorescent oxygensensitive probe whose signal increases overtime when oxygen in solution is depleting) immediately before measuring the fluorescence intensity (Ex/ Em: 380 nm/650 nm, 0.5 min interval) using a time-resolved fluorescence plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). Measurement was performed at 37°C under a sealed environment by overlaying with 100 μl of HS Mineral Oil to limit the exchange of oxygen.
Oxygen-consuming glucose oxidase and a potent mitochondrial uncoupling agent FCCP (4-trifluoromethoxy-carbonyl cyanide phenylhydrazone) was used as a reference.

Statistical analyses
Data were expressed as mean ± s.e.m. from at least 3-4 independently performed experiments. Statistical differences between two means were assessed using unpaired Student's t-test. One-way analysis of variance (followed by Tukey's multiple comparisons test) was used to compare differences among multiple groups. A P-value o0.05 was considered statistically significant.

RESULTS
AngII induces the browning characteristics in mouse white adipocytes via AT2R It has been known that AngII stimulates sympathetic neurotransmission to adipose tissue, while catecholamine released from sympathetic nerves are considered major stimulating factors for browning of white adipose tissues. 29 As both AngII and AngII receptors (AT1R and AT2R) are expressed in white adipocytes (Figure 1a), 15,16 we sought to investigate whether AngII has a direct role in browning of white adipocytes. As shown in Figures 1b and c, mouse white adipocytes have negligible amount of uncoupling protein 1 (UCP1, brown adipocyte-specific protein and thermogenic marker), which is consistent with our previous finding and other studies. 13,30 Nevertheless, UCP1 level was significantly increased in AngII-treated white adipocytes. Expression of beige adipocyte-specific marker, CITED1, 7 was also upregulated with AngII treatment. Blockage of AT1R (by ZD7155, a selective non-peptide AT1R antagonist) largely enhanced AngII-induced UCP1 and CITED1 expressions, and such enhancement was effectively prevented by co-application of PD123319 (a selective non-peptide AT2R antagonist). These observations suggest the stimulatory role of AT2R and inhibitory role of AT1R on white adipose browning in response to paracrine or endocrine AngII stimulation. Furthermore, blockage of AT1R alone, but not AT2R, increased UCP1 and CITED1 expressions in white adipocytes (Figures 1b and c,  Supplementary Figure S1). It implies that under basal condition the autocrine browning stimulation of AT2R by the endogenously produced AngII is suppressed by autocrine activation of AT1R.
Consistently, expressions of PPARγ and PRDM16 (major transcription factors known to promote expressions of genes required for adipose browning and thermogenesis) 31 were significantly increased by AngII-AT2R interaction, and such upregulations could be rectified by co-treatment with AT2R antagonist (Figures 1b and c). Also, brown adipocyte-like features, namely multilocular appearance (multiple small lipid droplets, LDs) and dark-brown color due to high mitochondrial contents, 30 were frequently observed in AngII+ZD7155 treated cells (allowing selective stimulation of AT2R), while the unilocular appearance (large central LD) was mostly found in the control and AngII +PD123319 treated cells (Figure 1d). Figure 1e shows the increased proportion of multilocular adipose cells (adipocytes with the largest LD smaller than 10-15 μm) after AngII+ZD7155 treatment. We further found that the AngII+ZD7155 treated adipocytes with multilocular appearance were usually UCP1expressing cells (as evidenced by the immunostaining of UCP1), whereas the multilocular ones found in control and AngII +PD123319 treated groups were not UCP1 expressing (Figure 1f, Supplementary Figure S2). In addition, AngII+ZD7155 treatment (AT2R stimulation) was associated with increased oxygen consumption rate (OCR) which reflects the mitochondrial respiratory activity and metabolic rate in adipocytes (Figures 1g and h). Simultaneous blockage of both AT1R and AT2R (AngII+ ZD7155 +PD123319) effectively blocked OCR increase and induction of brown-like phenotype. All these suggest that AngII promotes the browning of white adipocytes by stimulating the expressions of PPARγ and PRDM16 via AT2R.
Consistently with the experiments shown in Figures 1b and c using specific antagonists, activation of AT2R by AngII in AT1R knockdown cells increased both UCP1 and CITED1 expressions whereas AT1R activation in AT2R knockdown cells failed to do so (Figure 2b, Supplementary Figure S2). In addition, the expressions of UCP1 and CITED1 in the AT1R knockdown cells were elevated, confirming our view that AT1R suppresses the endogenous AngII-AT2R browning pathway. This is consistent with a recent study showing the upregulation of thermogenic gene expression in WAT of AT1R knockout mice. 34 Finally, we observed that M024/C21 (also known as C21; a selective non-peptide AT2R agonist), [35][36][37] but not CGP42112 (a selective AT2R ligand), stimulated the expressions of  Data represent mean ± s.e.m. *P o0.05, **P o0.01, ***P o0.001 versus control; # Po0.05, ## P o0.01, ### P o0.001 between indicated pairs. UCP1, CITED1, PPARγ and PRDM16; and these M024/C21-induced upregulations could be effectively abolished by co-incubation with AT2R antagonist-PD123319 (Figure 2c). Taken together of these multiple lines of evidence, we conclude that AngII stimulates browning by directly activating AT2R but at basal state this endogenous autocrine stimulation is suppressed by AT1R.
AngII-AT2R-induced white adipocyte browning is dependent on ERK1/2, Akt and AMPK signaling pathways We and others previously revealed that AngII-AT1R interaction triggered MEK/ERK pathway in white adipocytes. 38,39 In contrast, a number of studies in different cell types (for example, neurons) showed that AT2R activation decreases ERK1/2 activity via activation of phosphatases, such as MAP kinase phosphatase-1 (MKP-1). 35 Studies have also showed that AngII regulates Akt and AMPK signaling pathways in adipocytes and other cell types. 40,41 Here, we found that selective activation of AT2R by co-treatment with AngII and AT1R antagonist reduced ERK1/2 phosphorylation (in 5 min) but increased Akt and AMPK phosphorylation (in 15 and 30 min, respectively) in white adipocytes (Figures 3a-c). Similarly, M024/C21 treatment reduced ERK1/2 phosphorylation and increased Akt phosphorylation in white adipocytes, and such changes were effectively prevented by co-application of AT2R antagonist PD123319 (Supplementary Figure S4). It has also been demonstrated in adipocytes and other cell types that inhibition of ERK signaling (by either chemical inhibitor or knockdown of ERK) is accompanied by improved AMPK and Akt signaling. 40,42,43 Hence, one may conclude that AngII-AT2R interaction rapidly thwarts ERK signaling which subsequently promotes the downstream Akt (and AMPK) pathway in white adipocytes.
Consistently, AngII-AT2R-induced expressions of PPARγ, PRDM16, UCP1 and CITED1 were concealed in both Akt and AMPK knockdown cells (but not in ERK1/2 knockdown cells) (Figures 3d and e). AngII-AT2R-induced expressions of UCP1 and CITED1 were also significantly abolished by the pharmacologic inhibition of Akt and AMPK signaling, but not of MEK/ERK signaling (Supplementary Figure S4). Recent studies have showed that both Akt and AMPK signaling are important for inducing brown-like phenotypes in WAT, 13,44 whereas ERK signaling suppresses expression of genes controlling thermogenesis and browning of WAT in mice. 11,45 Taken together, it may be concluded that AngII-AT2R-induced white adipose browning is, at least in part, dependent on ERK, Akt and AMPK signaling pathways.
AngII-AT2R enhances brown adipogenesis Angiotensinogen (the sole precursor of AngII) is expressed in interscapular brown adipose tissue (classical BAT). 46 Here, we found that both AT1R and AT2R are expressed in brown adipocytes isolated from interscapular BAT (Figure 4a). We conceive that, in addition to stimulation of white adipocyte browning, AngII-AT2R signaling may also play regulatory roles in brown adipogenesis (brown pre-adipocytes of interscapular BAT differentiate into mature brown adipocytes).
As shown in Figures 4b and c, exogenous application of AngII, ZD7155 or AngII+ZD7155 (since the induction of brown preadipocyte differentiation at day 0) significantly increased the   Figure 4d). Blockage of AT2R (using PD123319) mostly removed the stimulatory effects of AngII and/or ZD7155 on brown adipogensis. On the other hand, AngII, ZD7155 and PD123319 (treatment at day 8) exerted no significant effect on the expressions of PPARγ, UCP1 (a key thermogenic protein), CIDE-A, aP2 and FAS in mature brown adipocytes (Figure 4e). Therefore, AT2R activation (induced by either exogenously applied AngII or endogenously produced AngII) has stimulatory role in brown adipogenesis, but doesn't significantly affect the basal activities of mature brown adipocytes.

AT2R activation promotes WAT browning and reduces WAT mass in vivo
To further investigate the role of AT2R in WAT browning in vivo, we gave mice intraperitoneal injection of ZD7155 (specific AT1R antagonist that permits selective AT2R activation by endogenously produced AngII) or M024/C21 (specific AT2R agonist that directly activates AT2R). No significant difference in body weight (Figure 5a) or food intake (data not shown) was observed between the control group (saline injection) and ZD7155 or M024/C21 treated group. Interestingly, WAT mass (both inguinal and epididymal WAT) was obviously reduced in both ZD7155 and M024/C21 treated mice, whereas the weight of interscapular BAT was similar to that of the control (Figures 5b and c). In addition, similar to the mice treated with CL316, 243 (a specific β3-adrenergic receptor agonist; as a positive control; Supplementary Figure S5), the body surface temperature was elevated in both ZD7155 and M024/C21 treated mice (Figures 5d and e), indicating increased body metabolic rate and heat production. Consistently, both ZD7155 and M024/C21 treatments similarly promoted the browning of WAT, as evidenced by the increased expressions of PPARγ, UCP1 and CITED1  20,47 Increased surface temperature around the interscapular area (which reflects BAT-derived thermogenesis) 26 in ZD7155 or M024/C21 treated mice (Figures 5d and e) is also consistent with our results of increased brown adipogenesis induced by AT2R activation (Figure 4). Furthermore, mice receiving either ZD7155 or M024/C21 showed a significant increase of serum adiponectin concentration, and reduction of serum levels of TNFα, triglycerides, and free fatty acid (Figures 5j-m). On the other hand, there was no significant difference in the serum levels of cholesterol (Figure 5n), glucose (Figure 5o), and insulin (Figure 5p) between the groups. In sum, the in vivo experiments further confirm the stimulatory roles of AT2R activation on WAT browning, which is accompanied with whole body energy metabolism (as indicated by the reduction of WAT mass and serum levels of triglycerides and free fatty acids) and thermogenesis (as indicated by the raised body temperature).
AngII-AT2R increases UCP1 expression and basal metabolic activity in human white adipocytes To test the human relevance of our discovery in mouse white adipocytes, we also conducted the experiments using human white adipocytes. We first confirmed the presence of both AT1R and AT2R in human white adipocytes (Figure 6a), in agreement with our previous work and other studies. 18,48,49 Similarly, AT2R activation (AngII+ZD7155) increased number of multilocular adipose cells (adipocytes with the largest LD smaller than 10-15 μm ; Figures 6b and c) and brown-like phenotypes in human white adipocytes, namely the common appearance of UCP1-positive dark-brown multilocular beige adipocytes Top panels, representative immunoblots; bottom panels, statistics (mean ± s.e.m., n = 3) of the optical density ratio between pERK and ERK, pAkt and Akt, or pAMPK and AMPK. (d and e) Mouse white adipose cells (day 4) were transfected with control siRNA, ERK1/2-siRNAs, Akt-siRNA or AMPK-siRNAs for 2-3 days before treated without ( − ) (control) or with (+) AngII and ZD7155 for 4 days. The representative immunoblots and the statistics (mean ± s.e.m., n = 4; normalized to actin densities) are shown accordingly. *P o0.05, **P o0.01, ***P o0.001 versus control; # Po 0.05 and ## P o0.01 between indicated pairs. AT2 receptor stimulates adipose tissue browning A Than et al (Figures 6b and d). As expected, the expressions of UCP1, CITED1, PPARγ and PRDM16 proteins were also boosted in the AT2Ractivated white adipocytes (AngII+ ZD7155), but not in AT1Ractivated cells (AngII+PD123319; Figures 6e and f). UCP1 promotes mitochondrial respiration and heat generation by decreasing the proton gradient generated in oxidative phosphorylation, hence the mitochondrial membrane potential. 50 In line with the increase of UCP1 expression, AT2R activation lowered the mitochondrial membrane potential as evidenced by the decreased fluorescence intensity of MitoTracker Red staining (Figures 6g and h) and increased the basal activity of mitochondrial respiration as indicated by the increased oxygen consumption rate (Figure 6i). Taken together with the results shown in Figures 1-5, one may conclude that AngII-AT2R signaling promotes white adipose browning in both human and rodents.
AT2R is upregulated by thyroid hormone T3 and plays a significant role in T3-induced white adipose browning All components of angiotensin system (including ACE, ACE2, AT1R and AT2R) are present in white adipocytes and closely linked to the development of obesity and related metabolic disorders. 16,17 As white adipocytes are inducible into brown-like adipocytes in response to various activators, especially cold exposure and thyroid hormone, we have investigated whether adipose angiotensin system (RAS) acts the mediator.
As shown in Figure 7a, selective activation of either AT2R (by AngII while blocking AT1R), β3-adrenergic receptor (by CL316,243) or thyroid nuclear receptors (by thyroid hormone T3) significantly upregulated UCP1 expression in human white adipocytes. Interestingly, T3 treatment stimulated the expression of AT2R (Figures 7a and  e) but not the other RAS components (AT1R, ACE, ACE2, TACE and AGT; Figure 7a and Supplementary Figure S7), implying that AT2R has a critical role in T3's browning effects. This notion is corroborated by the observations that T3-induced increase of browning markers (UCP1, CITED1 and PRDM16) was partially reversed by blocking AT2R (Figure 7b). In contrast, β3-adrenergic receptor agonist had no significant effects on the expression of angiotensin system including AT2R (Figure 7a and Supplementary Figure S7).
Activation of AT2R, on the other hand, reduced the expression of thyroid hormone receptor β1 (TRβ1, the specific TR isoform responsible for the upregulation of UCP1 in brown adipocytes), but without significant effect on the expression TRα1 (major TR isoform present in adipocytes) and β adrenergic receptors (Figures 7c and d). The observation of AT2R-suppressed TRβ1 expression may explain why there was no synergistic effect on adipose browning when AT2R and TR were simultaneously activated (Figure 7b). Herein, we reveal the intricate crosstalks between AT1R, AT2R and TR in white adipocyte browning.

DISCUSSION
With the recent identification of WAT remodeling into brown-like adipose tissue (that is, beige or brite adipocytes), there is enormous interest in discovering how to enhance white adipose browning as a therapeutic strategy for treating obesity. WAT Browning not only suppresses obesity through increased energy expenditure, but also reduces adverse effects of excess WAT such as insulin resistance, cardiovascular complications, etc. 6,8 Recent evidences suggest that adipokines secreted by adipose tissue act as the autocrine and paracrine signals to regulate its own browning. [11][12][13] In this study, we provide in vitro and in vivo evidence that AngII, which is an adipokine secreted by adipocytes or endothelial cells in adipose tissue and an endocrine hormone supplying to adipose via circulation, promotes white adipose browning and brown adipogensis via activation of AT2R. Figure 8 summaries our major findings, some of which are also supported by ours and others' previous studies.
Adipocytes, a major contributor to plasma AngII level, express both AngII receptors (AT1R and AT2R) with the majority of AT1R subtype. 15 Normally AT1R functions to suppress adipogenesis, lipolysis and glucose uptake in adipocytes. But in obesity, elevated AngII level together with overexpression of adipose AT1R is one of the main mechanisms that links obesity and metabolic AT2 receptor stimulates adipose tissue browning A Than et al disorders (for example, insulin resistance). 17,22 For example, AT1Rexerted suppression of adipogenesis 18,38 causes excessive lipid accumulation; thus converting adipocytes into hypertrophic and insulin-resistant state in obese rodents. 19,51 In obesity, AngII-AT1R increases adipose production of several pro-inflammatory cytokines, particularly TNFα, 17,52 which in turn suppress white adipocyte browning 11 and trigger apoptosis of brown adipocytes. 53 Reduced BAT mass (hence energy expenditure) further exacerbates the growth of WAT for excess energy storage. 5,6 On the other hand, AT2R activation is able to enhance both white 18,21 and brown adipogenesis (Figure 4). AT2R stimulates white adipocyte browning through ERK1/2, Akt and AMPK signaling pathways (Figure 3), leading to increased expressions of transcriptional factors PPARγ and PRDM16, and  . AT2R also promotes adipose production of anti-inflammatory adipokines, 54 including adiponectin ( Figure 5j) which is known to be beneficial to WAT browning. 12 As a result of increased number of brown and brownlike adipocytes, body energy expenditure increases (Figure 5d), and WAT depots are reduced (Figures 5b and c). Taken together, AT2R offers multiple beneficial effects to combat obesity. This notion is consistent with some previous studies. Specifically, it has been shown in mice that chronic AT2R activation using C21 causes an increase of white adipocyte differentiation and PPARγ expression, and a decrease in adiposity, adipose tissue inflammation and insulin resistance, 20,21,47 while knockout of AT2R does the opposite. 55,56 And resting metabolic rate, detected by direct calorimetry method, is significantly decreased in AT2R knockout mice. 57 Browning of WAT can be induced by prolonged cold exposure and other stress conditions through the activation of sympathetic nervous system. 12,58 Sympathetic norepinephrine activation of β-adrenergic receptor increases cAMP accumulation, which in turn induces UCP1 expression and brown-like phenotypes (for example, mitochondrial biogenesis) in white adipocytes. 6 cAMP also stimulates expression and release of angiotensinogen (AGT), a precursor peptide of AngII, in human adipose cells. 59 In fact, plasma AngII level increases markedly after cold exposure or under stress conditions (for example, exercise) in human. 60 Plasma AngII level can also be increased by thyroid hormone T3, another well-known activator of adipose browning. 61 Here, we found that T3 also stimulates adipose AT2R expression. It has been shown that hormone estrogen induced UCP1 expression is associated with increased AT2R expression in WAT. 47 Evidently, AT2R is critically involved in the browning process induced by stresses or endocrine hormones.
Previous studies have revealed the autocrine and paracrine regulations by adipokines on self-modeling of adipose tissue including browning. 9,13,62 TNFα, an adipokine known to inhibit white adipocyte browning, suppresses adipose expression of the precursor peptide of AngII (AGT). 63 Apelin, an adipokine known to promote white adipose browning, inhibits AT1R by triggering heterodimerization between APJ receptor and AT1R. 64 It has also been demonstrated that AT1R blockage (and AT2R activation) increases apelin production and decreases TNFα production in white adipocytes. 52,65 All these observations, together with our findings here, suggest the central role of AT2R in autocrine and paracrine regulation of browning. Interestingly, AT2R activation may serve as the protective response to diet-induced obesity because high-fat diet stimulates adipose expression of AT2R. 47 AngII-AT2R signaling can be exploited for obesity treatment. It has been reported that systemic infusion of AngII or AngII analog increases energy expenditure and reduces WAT mass in rodents. 66,67 However, AngII infusion is usually associated with many systemic adverse effects produced by AT1R (for example, hypertension). 66 Therefore, direct activation of AT2R by introducing exogenous selective agonists and making endogenous AngIIinduced basal AT2R activity more prominent by inhibiting AT1R should be better strategies. Here, we show that both specific AT1R antagonist and AT2R agonist lead to brown-like phenotypes in white adipocytes, and reduction of fat mass as well as serum levels of free fatty acids and triglycerides. In summary, this study establishes the central roles of AT2R in white adipose browning and brown adipogenesis and suggests new strategies to combat obesity.