Regulation and Functions of the Renin-Angiotensin System in White and Brown Adipose Tissue

Abstract

The renin angiotensin system (RAS) is a major regulator of blood pressure, fluid, and electrolyte homeostasis. RAS precursor angiotensinogen (Agt) is cleaved into angiotensin I (Ang I) and II (Ang II) by renin and angiotensin converting enzyme (ACE), respectively. Major effects of Ang II, the main bioactive peptide of this system, is mediated by G protein coupled receptors, Angiotensin Type 1 (AGTR1, AT1R) and Type 2 (AGTR2, AT2R) receptors. Further, the discovery of additional RAS peptides such as Ang 1–7 generated by the action of another enzyme ACE2 identified novel functions of this complex system. In addition to the systemic RAS, several local RAS exist in organs such as the brain, kidney, pancreas, and adipose tissue. The expression and regulation of various components of RAS in adipose tissue prompted extensive research into the role of adipose RAS in metabolic diseases. Indeed, animal studies have shown that adipose-derived Agt contributes to circulating RAS, kidney, and blood pressure regulation. Further, mice overexpressing Agt have high blood pressure and increased adiposity characterized by inflammation, adipocyte hypertrophy, and insulin resistance, which can be reversed at least in part by RAS inhibition. These findings highlight the importance of this system in energy homeostasis, especially in the context of obesity. This overview article discusses the depot-specific functions of adipose RAS, genetic and pharmacological manipulations of RAS, and its applications to adipogenesis, thermogenesis, and overall energy homeostasis.

Introduction

The renin angiotensin system (RAS) is classically known for its role in regulating blood pressure, fluid, and electrolyte homeostasis. The major precursor protein for RAS is angiotensinogen (Agt), which is sequentially cleaved by the enzymes renin and angiotensin-converting enzyme (ACE) into angiotensin I (Ang I) and II (Ang II), respectively. Ang II is considered the main bioactive peptide of this system and its effects are primarily mediated by two G-protein coupled receptors, Angiotensin Type 1 (AT1R, AGTR1) and Type 2 (AT2R, AGTR2) receptors (76). The contemporary understanding of this system has broadened with the discovery of other key players such as the enzyme ACE2, other angiotensin peptides Ang 1–7 and Ang 1–9 and the Mas receptor (108). The Ang II-AT1R axis increases blood pressure and insulin resistance, whereas the Ang II-AT2R and Ang 1–7-Mas axes lower both parameters. In addition to the systemic RAS, several local ones exist in organs such as the brain, kidney, pancreas, muscle, and adipose tissue (98).

Adipose tissue’s major function is to store excess energy as fat. However, with the discovery that this tissue also secretes numerous hormones, it is now well recognized as an endocrine organ (69). Since RAS exhibits autonomous regulation, adipose tissue RAS emerged an important regulator not only of adipose tissue metabolism, but also of whole body energy, blood pressure and glucose homeostasis (68). Moreover, components of the adipose RAS are overexpressed in obesity, while RAS blockade improves glucose homeostasis. These findings highlight the importance of this system in energy homeostasis, especially in the context of obesity (26). This overview article provides a summary of components and functions of the RAS, genetic and pharmacological manipulations of RAS components and an update on the functions of adipose RAS on adipose tissue physiology, adipogenesis, and whole body metabolism.

White and Brown Adipose Tissue

Adipose tissue, a loose connective tissue, was traditionally classified into two distinct types, viz., white adipose tissue (WAT) and brown adipose tissue (BAT). The main function of WAT, which contains white adipocytes, was considered to be the storage of excess energy in the form of fat. The predominant function of BAT, which contains brown adipocytes, is to maintain body temperature via nonshivering thermogenesis, especially in newborns. Recent evidence, however, has uncovered many new roles for these two types of adipose tissue. Moreover, a third type of adipocyte named beige adipocyte was discovered recently, which exhibits intermediate morphological and physiological features to that of the aforementioned types of adipocytes (81). Excessive fat deposition and dysfunction of WAT is linked with obesity and its associated metabolic comorbidities such as Type-2 diabetes mellitus (66). In contrast, BAT and beige adipocytes are emerging as novel therapeutic targets for the treatment of obesity.

Brown fat cells are derived from precursor cells of embryonic mesoderm origin, which also generates skeletal muscle cells and a subpopulation of white fat cells (110). Brown adipocytes express several markers, including Ucp1, Pgc1α, Cidea, and classical brown-selective markers, such as Zic1. White adipocytes originate from mesodermal stem cells (Fig. 1). The origin of beige fat cells is not fully understood. Beige fall cells are derived from beige precursors or from transdifferentiation of mature white adipocytes (103). Beige adipocytes express several markers, including Ucp1, Pgc1α, and Cidea, and beige-selective markers, such as Cd137, Tbx1, Tmed26, and Cited1. Several environmental factors such as cold exposure, physical activity, and PPARγ ligands may control differentiation of beige fat cells.

Figure 1.

Origin of different adipocyte types and their association with obesity. Brown adipocytes originate from a subset of dermomyotomal precursors (A). White adipocytes originate from mesodermal stem cells (B and C). Beige adipocytes are hypothesized to be derived from beige precursors (C) or transdifferentiation from mature white adipocytes (B). Beige adipocyte differentiation is induced by cold exposure and beta-agonists.

White Adipose Tissue Composition and Function

WAT comprises adipocytes, an extracellular matrix (ECM), vascular, and neural tissues, other cells such as stem cells or fat cell precursors, fibroblasts, endothelial cells, and various blood cells including macrophages and T cells. WAT produces various substances such as growth factors, cytokines, and hormones called adipocytokines or adipokines that play key roles in energy homeostasis and inflammation. Proinflammatory adipokines [leptin resistin, monocyte chemoattractant protein-1 (MCP-1), angiotensin II (Ang II) and others] as well as anti-inflammatory adipokines (such as IL-10; adiponectin) (66). These properties make adipose tissue a dynamic endocrine tissue with key functions that impact energy homeostasis, blood pressure regulation, macronutrient metabolism, and whole body immune functions.

Excess energy storage in adipose tissue lead to adipocyte hypertrophy, and dysregulation of adipokine secretory patterns. Moreover, it leads to change in immune cell populations within WAT. Thus, it is now established that obesity is associated with a chronic low-grade inflammation (referred as sterile inflammation) in WAT. Adipose tissue associated inflammation in obesity is characterized by macrophage infiltration. The Adipose tissue macrophages (ATM) are classified into two main types. M1 or classically activated macrophages are stimulated by interferon gamma (IFN-γ) and lipopolysaccharide (LPS) to produce pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1 and reactive oxygen species such as NO. M2 or alternatively activated macrophages are activated by IL-4 and IL-13 and express anti-inflammatory factors such as IL-10, TGF-β, IL-1 receptor antagonists, IL-4, and arginases (93).

In obesity, macrophage population in adipose tissue leans toward more of an M1 type, leading to an increased ratio of proinflammatory versus anti-inflammatory cytokines produced form adipose tissue. This remodeling and changes in macrophage profiles in adipose tissue during obesity may be accounted for by increased M1 macrophage infiltration into fat tissue and/or a shift in macrophage phenotype from M2 to M1 (93). This shift may be due to excess lipid and lipotoxicity in macrophages. Evidence also points toward involvement of other immune cells like T cells in obesity-associated adipose tissue inflammation (83). WAT from lean individuals have a large number of regulatory (Treg) T cells. In obese individuals, these Treg numbers are reduced while CD8+ effector T cells numbers increase. Moreover, the T helper 1 (Th1) to T helper 2 (Th2) ratio is increased in obesity promoting IFN-γ secretion from adipose tissue. The cytokine profile of these T cells could play an important role in determining the M1/M2 phenotype of ATM. T cell cytokine profile may be a determining factor in the phenotype of adipose tissue macrophages.

What sets off adipose inflammation is yet to be identified but a few ideas have been suggested. Adipose tissue expands during positive energy balance allowing excess energy to be stored. Dynamic changes and remodeling take place through two key processes, which involves degradation of the ECM, and adipogenesis. Defective adipose tissue expansion could lead to adipocyte injury, death, and inflammation. Adipose tissue hypoxia (96), oxidative stress, and endoplasmic reticulum (ER) stress (10) are other factors, which are likely to play a role in the trigger of adipose tissue inflammation. Furthermore, the role of RAS in adipose tissue inflammation will be discussed later.

While many studies have documented that adipose growth and adipocyte hypertrophy may trigger the inflammatory cascades that accompany obesity, others suggest that dietary lipids also trigger these processes (72). Especially saturated fatty acids are ligands for Toll-like receptors (TLRs) 2 and 4, which activate the nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-kb) pathway. This is consistent with our recent findings that omega-3 fatty acids can reverse high-fat induced metabolic disorders and adipose inflammation (65,79). Moreover, adipose tissue inflammation is not completely resolved following caloric restriction with diets containing a high-fat content (70). On the other hand, other studies have demonstrated that changes in adipose tissue inflammation can take place without changes in fat mass. These findings indicate that dietary lipids play an important role in adipose tissue inflammation, leading to changes in adipo/cytokine secretion from WAT, which induces insulin resistance in glucose homeostatic organs such as the skeletal muscle and liver leading to insulin resistance, which is considered to be a key link between obesity and metabolic syndrome.

Brown and Beige Fat

Brown and beige (also known as brite) adipocytes are important in non-shivering thermogenesis and have distinct anatomical features from that of white adipocytes. In humans, brown adipocytes are found in the interscapular brown adipose depots of infants, which gradually disappear with age (81). In contrast, BAT depots of rodents remain throughout life. While BAT was considered to be absent in adults, a landmark study in 2007 showed that functional BAT is present in adults as well (91). Using fluorodeoxyglucose positron emission tomography, these investigators discovered functional BAT depots in the cervical, supraclavicular, axillary, and paravertebral areas. Although both are thermogenic, the localization of the brown and beige adipocytes is distinct. The interscapular and perirenal BAT depots mainly comprise classical brown adipocytes containing multilocular lipid droplets. It is separated from the WATby a layer of connective tissue (81). While beige and brown fat cells share several biochemical features such as high number of lipid droplets and mitochondria with expression of UCP1, they originate from distinct progenitors. There are a number of positive and negative regulators of brown/beige adipocyte development (116). Chronic cold exposure is one of the most potent stimuli for both brown and beige adipocyte development. Upon cold stimulation, browning of WAT occurs, giving rise to beige adipocytes. Norepinephrine released from the sympathetic nerves is a powerful stimulator of WAT browning. In fact, clusters of beige adipocytes emerge in areas with tyrosine hydroxylase–expressing noradrenergic nerve fiber innervated areas. It activates BAT thermogenic program via protein kinase A (PKA) and p38-mitogen-activated protein kinase (MAPK) signaling, followed by the production of free fatty acid (FFA) by lipolysis for Ucp1-mediated proton uncoupling. Exercise also induces WAT browning via activation of the transcriptional coactivator Pgc1α. Myokines (hormones secreted by skeletal muscle) such as irisin and meteorin-like also activate beige adipocyte development. Pathological conditions like cancer cachexia can also be associated with WAT browning and increased energy expenditure, leading to weight loss. Another activator of BAT is omega-3 fatty acids, which increases thermogenic markers in BAT from high-fat diet fed obese mice as demonstrated by our lab recently (94).

An association between BAT activity and leanness exists (77). Moreover, cold-induced thermogenesis is proportional to BAT perfusion and activity. Further, BAT activation is estimated to increase energy expenditure by 10% to 27% in humans (135). Finally, recent studies have shown that adult BAT contains beige-like adipocytes, which can be induced to differentiate from white adipocytes (19). Taken together, beige adipocytes are an attractive therapeutic target for metabolic diseases and a viable alternative to BAT activation that is limited in amount compared to WAT.

The Renin Angiotensin System

The RAS is more complex than it was originally regarded and is an evolving endocrine system (128). Two main branches of the RAS are currently known, Ang II-AT receptor and Ang 1–7-Mas receptor branch (Fig. 2). In the classic RAS pathway, circulating Agt (produced by the liver) is cleaved by renin (released by kidney) into Angiotensin I, which is rapidly hydrolyzed by angiotensin converting enzyme (ACE) to produce the active hormone, Angiotensin II. Ang II then binds to G protein coupled receptors AT1R and AT2R to mediate its effects. Most of the effects are mediated by AT1R, which includes vasoconstriction, inflammation and fluid retention (74), while AT2R exerts opposing effects. ACE2, homologue of ACE divides this axis into another branch and cleaves Ang I and Ang II into Ang 1–9 which is then converted to Ang 1–7, the active peptide. Ang 1–7 upon binding to the Mas receptor opposes the actions of Ang II and reverses some of the effects of Ang II (36,42). The prorenin/renin receptor [(P)RR] is a component of the RAS which upon binding with renin induces nonproteolytic activation of prorenin and increases the efficiency of the Agt cleavage to Ang I.

Figure 2.

Components of the RAS. In the classical RAS pathway, angiotensinogen (Agt) is cleaved by renin into Angiotensin I (Ang I), which is cleaved by angiotensin converting enzyme (ACE) to produce the active hormone, Angiotensin II. Agt is produced mainly by the liver, while WAT also contributes to Agt levels, especially in obese individuals. Ang II binds to G protein coupled receptors ATR1 and ATR2 to mediate its effects. ACE2, homolog of ACE divides this axis into other branch and cleaves Ang I and Ang II into Ang 1–9 which is then converted to Ang 1–7, the active peptide. Ang 1–7 binds to the Mas receptor and opposes the actions of Ang II. The prorenin/renin receptor [(P)RR] is another component of the RAS which upon binding with renin induces nonproteolytic activation of pro/renin and increases the efficiency of the Agt cleavage to Ang I.

RAS Components in WAT

RAS components were identified in the late 1980s in the adipose tissue and were found to be present both in human and rodent adipose tissue as well as cultured adipocytes (16, 24, 33, 63). Specifically, human WAT expresses Agt, renin, ACE, ACE2, AT1R, AT2R, (P)RR, and Mas receptor (33,71). The main precursor of RAS, Agt is expressed in WAT in both animal and human models (131). As adipose tissue contains other celi types apart from adipocytes, studies were performed in clonai adipocytes to confirm expression of Agt in cultured human and mouse adipocytes (34, 60). More interestingly, adipose tissue produces 30% of total circulating Agt with the liver being the primary source of Agt (35,86).

Renin was detected at relatively low levels in earlier studies involving rodent models but was found to be expressed at similar levels to other RAS components in human adipose tissue with higher levels present in visceral compared to subcutaneous adipose depot, with its expression regulated posttranslationally (2). WAT also expresses (P)RR, which upon binding to its ligand renin, increases the conversion of Agt to Ang I, thereby increasing Ang II levels and its subsequent actions in WAT. Renin expression and its enzymatic activity are undetectable in 3T3-L1 preadipocytes. During differentiation, renin expression increases and its enzymatic activity also increases both intra and extracellularly (38). ACE is also present in both visceral and subcutaneous human adipose tissue (64). Murine WAT also expresses ACE2, which is important in production of Ang 1–7 and is dysregulated in obesity (52).

Angiotensin receptors are present in WAT of both humans and rodents. The expression levels vary according to the WAT depot (29). In rodents, AT1R is encoded by two genes, Atla and At1b. Of these, At1a is expressed in kidney, liver, adrenal gland, ovary, brain, testes, lung, heart, placenta, and adipose tissue. Atlb is present in brain, testes, and adrenal gland (14). There are contrasting reports to the level of expression of AT1R and AT2R in murine adipocytes. We have found that AT2R predominates in 3T3L-1 adipocytes, while others have reported that AT1R predominates in the same cell line (84). AT1R is well studied for its biological effects compared to AT2R. AT2R is activated only under specific conditions when AT1R is blocked with antagonists (28,29). Lastly, the components of the Ang 1–7- Mas receptor are also present in the WAT (129). Human WAT Ang II is removed by proteolytic cleavage by endopeptidases and aminopeptidases such as neprilysin (107). Ang II increases aldosterone production by the adrenal cortex. Recent evidence shows that adipocytes express aldosterone synthase and produce aldosterone in an Ang II-AT1R dependent manner (12). Thus, physiological significance of the adipose-derived aldosterone merits further investigation.

Adipose RAS and its Regulation

RAS and its effects on lipid metabolism

Agt expression in WAT is nutritionally and hormonally regulated (Fig. 3). WAT Agt mRNA expression decreases with fasting and increases following feeding both in vivo and in vitro studies (39). This is in contrast to Agt expression in the liver, which does not exhibit changes with feeding status. Long-chain fatty acids also activate Agt gene expression in adipocytes (104). Hyperglycemia is also known to induce Agt expression in WAT of rodents. Additionally, we have shown that Ang II increases lipogenesis in 3T3-L1 adipocytes as well as human adipose cells via increased transcriptional activation of the lipogenic enzymes, fatty acid synthase, and glycerol-3-phosphate dehydrogenase in an AT2R-dependent manner (62).Taken together, this suggests that increased nutrient availability following feeding increases Agt expression and Ang II production from adipocytes which in turn promotes lipid storage. Other than the activation of lipogenic enzymes, Ang II also promotes lipid transport into adipocytes by translocation of the scavenger receptor class B, Type I into the adipocyte cell membrane from the cytoplasm (124). Moreover, Ang II increases lipoprotein lipase expression in subcutaneous (but not visceral) WAT, potentially increasing the hydrolysis of triglycerides from plasma lipoproteins and increasing delivery of fatty acids to adipocytes (126). The important role of Ang II in modulating lipogenesis is also highlighted from studies where RAS is dysregulated in conditions of lipid storage. For example, perilipin-1 (Plin1) is a protein, which coats lipid droplets in adipocytes and regulates triglyceride storage and hydrolysis in adipocytes. In Plin1 deficient mice, there is increased production of Ang II from perivascular WAT that leads to hypertension (139).

Figure 3.

Mechanisms of WAT RAS effects. WAT expresses all components of the RAS. Agt and Ang II production are stimulated by nutrients such as long-chain fatty acids and glucose, hormones such as insulin and cytokines. Ang II binds to AT1R and activates NADPH oxidase to increase reactive oxygen species (ROS) production, which in turn activates the Nf-kb pathway, leading to transcription of proinflammatory genes such as Resistin, PAI-1 and MCP-1. Ang II binding to AT1R also has an inhibitory effect on adipogenesis and lipolysis. Ang II also binds to AT2R, which increases production of key lipogenic enzymes and leads to lipogenesis. Thus, the net effect of RAS on WAT is to increase lipid storage. Renin binds to the pro/renin receptor [(P)RR] which increases the efficiency of Ang II production, thus indirectly promoting lipid storage. Ang 1–7, produced by subsequent cleavages of Ang I and Ang II, binds to the Mas receptor and exerts anti-inflammatory actions in WAT.

In addition to its lipogenic function in adipose tissue, Ang II also inhibits lipolysis in human WAT (11) in an AT1R-dpendent manner (45). Using microdialysis technique, this antilipolytic effect of Ang II was shown to be partially attributed to reductions in local WAT blood flow (46). Agt expression is also decreased by beta-adrenergic stimulation (63), potentially aiding the lipolytic actions of the latter. While long-chain fatty acids activate Agt expression in adipocytes, we have shown that the long-chain omega 3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) does not increase Agt secretion from cultured 3T3-L1 adipocytes. EPA also prevents arachidonic acid-mediated increases in Agt secretion (117).

RAS regulation by hormones and obesity

Several hormones such as insulin, glucocorticoids, and androgens control the WAT RAS. The effect of insulin on Agt expression in WAT is controversial. We have shown that Agt mRNA expression is increased by treatment with insulin in 3T3-L1 adipocytes in vitro (63). Similar effects are found in isolated human subcutaneous adipocytes in vitro (54, 55). Others, however, have shown that insulin downregulates Agt expression in Ob1771 and 3T3-F442A adipose cells in vitro (8) and in lean rats in vivo (41). Glucocorticoids increase Agt production, probably via transcriptional regulation (7). Similarly, androgens increase Agt expression both in vitro and in vivo in rat adipocytes (111). Cytokines such as TNF-α can increase Agt production from adipocytes (54).

Agt expression in WAT is increased in obese animal models such as the obese (fa/fa) Zucker rat (53) and diet-induced obese C57BL/6J mice (102), resulting in increased systemic RAS (39,127,131). Some, but not all studies, report an increase in Agt expression in human WAT in obesity. Agt expression is also reduced in human WAT following weight reduction (32). The best evidence for changes in WAT RAS in chronic energy excess originates from a study in which 44 healthy men were overfed with a lipid-enriched diet for 2 months (5). Subcutaneous abdominal adipose tissue microarray analyses showed that Agt gene expression increased by 1.5 and 1.4 fold at the end of 2 and 8 weeks respectively. ACE expression was increased by 1.2- and 1.5-fold compared to baseline at the same two time points. These expression levels were validated by reverse transcription quantitativepolymerasechain reaction (RT-qPCR). This study clearly indicates that adipose RAS in overactivated in chronic energy excess. In addition to the effect of high-fat feeding on WAT RAS of individuals, recent evidence also indicates that maternal feeding of high-fat diets can lead to activation of the WAT RAS of the offspring (51). WAT renin receptor expression also increases with high-fat feeding in rodents (3).

Endocrine functions of WAT RAS

Several lines of evidence support the hypothesis that WAT RAS contributes to whole body metabolism and homeostasis, that is, an endocrine function of WAT RAS. Arteriovenous difference in Ang II in blood draining subcutaneous abdominal adipose tissue suggests that human WAT is a significant source of systemic Ang II (54). We have shown that overactivation of adipose RAS can lead to increased expression of Agt and AT1R in the kidneys of mice (73). Moreover, adipose tissue specific overexpression of Agt leads to systemic insulin resistance and hypertension (67,86). Conversely, adipose-specific Agt deletion prevents high-fat diet induced hypertension and glucose intolerance (80, 134). Taken together these findings suggest that in addition to para/autocrine effects, WAT RAS has important endocrine functions in regulation of blood pressure, energy balance, glucose homeostasis, and inflammation. Because of the dominant effects of the systemic RAS, dissecting out the individual contribution of WAT RAS to whole body homeostasis can be difficult. It is essential to interpret the data from numerous genetic/pharmacological manipulations of RAS components to get a holistic view of the contribution of systemic versus WAT RAS to these functions, which are summarized in Table 1.

Table 1.

RAS Manipulations, Body Weight, and Metabolic Changes in Rodents

Experiment	BP	Body weight/ adiposity	Insulin sensitivity	EI	EE	Ref.
Ang II infusion	↑	↓	↓	↓	↑	(25,31)
Intracerebroventricular Ang II infusion		↓		↓	↑	(99,100)
Ang 1 –7 infusion	↓		↑			(44)
Ang 1 –7 overexpression		↓	↑	NC	↑	(105)
WAT Agt overexpression	↑	↑	↓			(67,86)
Agt deficiency		↓		NC	↑	(87)
Adipocyte Agt deficiency	↓	NC	↑			(80,133)
Hepatocyte Agt deficiency	↓					(134)
ACE inhibition		↓	↑	NC/↓		(30,88,114)
AT1R deficiency		↓	↑	NC	↑	(75)
AT2R deletion		↓	↑			(136)
Renin deficiency		↓	↑	NC	↓	(119)
Hepatic renin overexpression	↑	↓	↓			(37)
Renin receptor inhibition		↓	↑			(121)
ACE deficiency		↓	↑	NC	↓	(61)
ACE2 deficiency		↓	↓			(97)
Mas receptor deficiency		↑	↓			(106)
AT2R deletion with WAT Agt overexpression	↑	↓	↓			(137)
Adipose-specific Renin receptor deletion	↑	↓	↑	NC	↑	(112,130)

RAS and Blood Pressure

The systemic RAS has been traditionally known for its role in regulation of blood pressure. Overactivation of systemic RAS leads to hypertension and is implicated in the obesity-associated hypertension (Table 1). These include systemic Ang II infusion and genetic renin overexpression studies. Conversely, systemic RAS blockade either genetically or pharmacologically prevents high-fat diet-induced hypertension. Agt deficiency, renin or ACE knockout, and ACE inhibitors all prevent hypertension in rodent models. The Ang II-AT1R axis is the major contributor to hypertension, as evident by studies showing that AT1R deficiency protects rodents from hypertension, whereas AT2R deficiency has no protective effect. The Ang 1–7-Mas axis seems to have an anti-hypertensive effect, since Ang 1–7 infusion prevents and Mas deletion induces hypertension.

In addition to the systemic RAS, adipose RAS is also a contributor to systemic blood pressure. To test the hypothesis that WAT RAS overactivation can induce hypertension, Massiera et al. generated a mouse model, in which the adipose specific aP2 promoter drives the expression Agt in WAT. These mice (aP2-Agt mice) have a 30% increase in plasma Agt compared to wild-type mice (86). They also develop hypertension, confirming the aforementioned hypothesis. Indeed, the adipose derived Agt is sufficient to normalize blood pressure in global Agt-deficient hypotensive mice. More recently, the cre-loxP technology was used to generate adipose-specific Agt knockout mice. While wild-type mice had increased plasma Ang II levels and hypertension following high-fat feeding, the adipose Agt knockout mice are protected from these changes indicating that adipose RAS is important in the development of high-fat diet-induced hypertension (133).

The (P)RR increases the conversion of Agt to Ang I. Therefore, adipose (P)RR inhibition was considered a potential target to prevent obesity-induced hypertension. Paradoxically, adipose-specific (P)RR deletion in adiponectin-Cre mice leads to increased blood pressure (130). Therefore, the role of (P)RR in WAT especially mediating obesity-induced hypertension is likely to be complex, and merits further investigation.

RAS and Energy Balance

RAS blockade induces weight loss in rodents (30). Moreover, genetic knockdown of several RAS genes (Agt, renin, ACE and angiotensin receptors) have protective effects against obesity in rodents (61). Conversely, interruption of the Ang 1–7-Mas axis via deletion of the Mas receptor leads to excessive weight gain (106) and Ang 1–7 overexpression leads to reduced abdominal fat content (105) (Table 1). Taken together, these evidence point toward a role of RAS in regulating energy balance.

Although RAS blockade by the above methods leads to antiobesity effects, systemic RAS activation by either Ang II infusions or overexpression of renin have yielded inconsistent results: Ang II infusions lead to weight loss in rodents (25). While we have shown that renin overexpression in mice also leads to lower adiposity (37), others have shown that renin overexpression leads to weight gain in rats (49). Taken together, this suggests that local adipose RAS, rather than systemic RAS, maybe implicated in obesity. Indeed, WAT-specific Agt overexpression leads to excessive adiposity in mice (80). However, while WAT-specific Agt deletion does not protect mice from high-fat diet-induced obesity (87), WAT-specific (P)RR deletion leads to a phenotype with almost undetectable WAT (130) highlighting the complexity of the relationship between RAS and body weight.

Body weight is determined by the balance between energy intake and expenditure. RAS blockade leads to increased energy expenditure in rodents. Mice lacking renin are protected from diet-induced obesity by having increased energy expenditure (119). Further, AT1R blocker telmisartan prevents obesity by increasing Ucp1 expression in BAT and increasing energy expenditure in high-fat fed mice (6). Activation of the Ang 1–7-Mas axis regulates body weight again via increasing energy expenditure (105). Therefore, it is likely that systemic RAS blockade and activation of the Ang 1–7-Mas axis both increase energy expenditure. Paradoxically, Ang II infusion also increases energy expenditure. However, this likely to be via a central mechanism. This is supported by the fact that brain RAS activation increases thermogenesis and energy expenditure, via increases in sympathetic stimulation leading to thermogenesis in BAT and WAT. Brain-specific RAS activity in mice produces a hyperphagic but lean phenotype, due to increased metabolic rate and thermogenesis (50). These mice also have suppression of systemic RAS. Interestingly, the metabolic rate in these mice can be normalized by restoring plasma Ang II, suggesting that brain RAS activation increases energy expenditure, potentially via downregulating systemic RAS. Additionally, brain RAS activation increases sympathetic activity in adipose tissue: For example, Ang II central administration increases beta-3-adrenergic receptor expression in BAT and WAT (31). Brain RAS-mediated increases in adipose tissue thermogenesis is attenuated via adipose tissue AT2R activation (82). Taken together, systemic, adipose and brain RAS are all important in energy homeostasis, and defects of one or more of these systems could lead to obesity.

RAS Dysfunction in Human Obesity

Genetic studies show that polymorphisms in RAS genes are associated with obesity. Several studies have shown an association between the ACE insertion/deletion (I/D) polymorphism and obesity (4, 78, 89). A few studies have also shown associations between polymorphisms of AGT, AT1R, and MAS1 genes and obesity (101, 138). Therefore, it is plausible that RAS is linked to the risk of obesity. Conversely, obesity can lead to overactivation of RAS as well. Indeed, obesity is associated with increased plasma Agt, renin, ACE, and Ang II levels (9, 27, 32, 109), suggesting that systemic RAS is overactivated in obesity (68). While some studies show that WAT RAS components are overexpressed in obesity as well (127,131), others do not support this assertion (32,48). Although there are genetic links between RAS genes and obesity, to date there is no evidence from human studies indicating that RAS blockade leads to weight loss. However, there are several clinical studies showing that RAS blockade can improve obesity-associated metabolic derangements such as insulin resistance and Type-2 diabetes as discussed below.

RAS and Glucose Homeostasis

Both systemic and WAT RAS are important in the regulation of glucose homeostasis. Several clinical studies have shown that individuals on ACEI or ARBs are protected from Type-2 diabetes compared to patients on other anti-hypertensives (1). Genetic and pharmacological studies show that systemic RAS overactivation leads to insulin resistance. Moreover, RAS blockade via ARBs or ACEI and deletion of components of the Ang II-AT1/AT2 axis protects from high-fat diet-induced insulin resistance (Table 1). We have reviewed the detailed mechanisms of RAS-mediated insulin resistance previously (68).

To test the hypothesis that WAT RAS is causally linked to the pathogenesis of obesity-associated insulin resistance we employed two different strategies. First, we overexpressed Agt in the adipose tissue in the aP2-Agt mouse model (67). As predicted, male transgenic micebecome glucose intolerant even on a low-fat diet. These mice exhibited adipose tissue inflammation. Hyperinsulinemic-euglycemic clamp studies show that these mice are insulin resistant as well. Interestingly, these mice have reduced skeletal muscle glucose uptake indicating that WAT-specific Agt overexpression can lead to skeletal muscle insulin resistance and dysregulation of whole body glucose homeostasis. The second approach we employed was to generate adipose-specific Agt knockout mice, using the Cre-LoxP technology (80). These mice had no change in body weight or fat mass compared to wild-type controls both on low and high-fat diets. However, the knockout mice had better glucose tolerance and reduced WAT macrophage infiltration compared to wild-type controls (80). Additionally, adipose-specific (P)RR deletion leads to a leaner phenotype, especially in male mice, and protects against high-fat diet-induced insulin resistance confirming that RAS pathway is important in the pathogenesis of glucose intolerance in obesity (121).

WAT RAS and Adipogenesis

Adipogenesis is the process of cell differentiation where new adipocytes are formed from precursors such as preadipocytes or mesenchymal stem cells. WAT is the site for storage of excess energy in the body. When its storage capacity/function is dysregulated, ectopic fat deposition in liver and skeletal muscle can occur, which can give rise to metabolic disorders such as insulin resistance and hepatic steatosis. During fat Storage, adipose tissue expansion occurs by adipocyte hypertrophy (enlargement of adipocytes) and hyperplasia (adipogenesis). Adipocyte hypertrophy is associated with deranged adipokine secretion and insulin resistance, whereas adipocyte hypotrophy with increased cell number is associated with better insulin sensitivity. Therefore, the normal adipogenic capacity is vital for WAT function as well as whole body metabolism.

During differentiation, adipocytes produce Agt, probably via differentiation-dependent transcriptional activation (120). It is likely that this transcriptional activation occurs via the proximal region (position −96 to −52) in the Agt promotor (120). Agt knockdown reduces differentiation of human visceral preadipocytes in vitro (132). Moreover, we have shown that Agt knockdown leads to reduced lipid accumulation and lower expression of genes involved in adipogenesis such as Ppar-g, Cebpb, Srebf1 and Adig in murine adipocytes in vitro (20). Therefore, it is likely that adipocyte-derived Agt plays a role in adipocyte differentiation. The Ang II-AT2R and Ang 1–7-Mas axes promote adipocyte differentiation (59,115,122). Ang II acting via the AT2R increases adipocyte differentiation through increasing prostacyclin in a paracrine fashion. The Ang 1–7 Mas axis promotes adipocyte differentiation via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways (122).

While Adipose-derived Agt is important in adipogenesis, higher levels of Ang II suppress adipogenesis in both human and animal models (13,60,95). Ang II exerts a greater anti-adipogenic effect in adipocytes from obese individuals (13). The Ang II induced suppression of adipogenesis is mediated via the AT1R and is through activation of ERK 1/2 and MAPK kinase pathways (122) and phosphorylation of Ppar-g (40). Since WAT RAS is overexpressed in obesity, the Ang II-mediated impairment of adipogenic capacity could lead to adipocyte hypertrophy, insulin resistance and ectopic fat deposition. Additionally, cell differentiation is in part mediated by AT2R, and blocking this receptor increased cell differentiation via epidermal growth factor (82).

Adipose RAS and Inflammation

Obesity is characterized by chronic low-grade inflammation and adipose tissue dysfunction. Adipose tissue dysfunction leads to an increase in proinflammatory markers such as TNF-α and MCP-1 along with an increase in Ang II levels. Various inhibitors of the RAS pathway have been tested in clonal adipocytes, rodent models, and clinical studies, which consistently demonstrate that reducing angiotensin levels decrease inflammation. RAS blockage leads to weight loss in rodents, and since weight loss and reductions in adiposity alleviate adipose inflammation, the anti-inflammatory effects of RAS blockade could be an indirect effect. For example, rats when treated with ACEI, had lower levels of leptin as well as fat mass indicating that the reduced fat mass caused by ACE inhibition could contribute to lower leptin levels (88). However, other lines of evidence points toward a direct effect of RAS on inflammation. Ang II induces inflammatory markers like MCP-1 and reduces adiponectin specifically secreted by adipose tissue (56). AT1R inhibitors have beneficial effects in terms of reducing inflammation. Specifically, irbesartan when fed to mice lowers levels of proinflammatory cytokines like TNF-α and CRP levels thereby reducing inflammation and also improves anti-inflammatory marker IL-10 specifically in the adipose tissue (56). Further, these inhibitors also reduce macrophage infiltration. Mice treated with other AT1R inhibitors such as valsartan, telmisartan also exhibit normalization of adipocytokines (123). Clinical studies with patients treated with AT1R inhibitors show improvements in adiponectin, which suggests that inhibiting angiotensin reduces inflammation. Other studies performed in patients with impaired glucose metabolism demonstrate reduced macrophage infiltration of adipose tissue (47). Although AT1R inhibitors have Ppar-g agonist properties (57), which also might lead to suppression of inflammation, other RAS blockers such as ACE inhibitors have similar anti-inflammatory properties (58), suggesting that suppression of the RAS is important in alleviating inflammation.

The reduced inflammation with inhibition of the RAS pathway is also evident from the knockout mouse models. We have shown that adipose–specific Agt knocked down leads to reduced adipose tissue macrophage along with reductions in proinflammatory cytokines and increased in anti-inflammatory cytokines (80). While AT1R activation increased WAT inflammation, AT2R activation appears to alleviate inflammation. For example, AT2R receptor agonists improve adiponectin secretion and reduce proinflammatory cytokines in mice (92).

While the Ang II-AT1R axis is proinflammatory in the WAT, the Ang 1–7 Mas axis exerts anti-inflammatory properties. For example, Ang 1–7 reverses the proinflammatory phenotype in obese db/db mice (90). Furthermore, increasing Ang 1–7 levels lead to reduced activation of Nf-kb and increases adiponectin secretion in primary adipocytes (85,105). In line with this, ACE2 whole body knockout mice have worsening of epicardiac fat inflammation and polarization of macrophages to a proinflammatory phenotype (97). Also, AT2R inactivation reverses adipose specific Agt over expression on inflammation but not on blood pressure (137).

There are several mechanisms by which the Ang II-AT1R pathway promotes inflammation. Ang II treatment induces MCP-1 in cultured adipocytes, which can be inhibited by AT1R antagonists through an Nf-kb-dependent mechanism (125). Furthermore, Ang II stimulates IL-6 and IL-8 release from human adipocytes also by an Nf-kb-dependent mechanism through the AT1R receptor (118). The Ang II effects on Nf-kb pathway is mainly mediated via the AT1R, because blocking the AT1R receptor completely inhibits the Nf-kb pathway, while blocking AT2R only partially inhibits it (118). It is likely that Ang II activates NADPH oxidase, leading to increased production of reactive oxygen species, which in turn activates the NF-kb pathway (67).

RAS in Brown Adipose Tissue

BAT expresses all components of the RAS similar to WAT (43,98). In the late 1980s, Agt expression was reported in peri-aortic BAT (15). Ang peptides (Ang I, Ang II, Ang 1–9, Ang 1–7, and Ang 2–8) are detectable in rodent BAT (17,18, 113). While ACE, ACE2 are expressed in peri-aortic BAT, at levels comparable to that in mesenteric WAT (43), renin levels are expressed at a higher level in the peri-aortic BAT (43). On the contrary, AT1R and AT2R are expressed at lower levels in BAT compared to mesenteric WAT (43). The presence of renin activity in isolated interscapular BAT and its production of Ang peptides ex vivo, demonstrates the presence of a functional local RAS in BAT (113).

While the exact function of RAS in BAT is yet to be elucidated, it appears to play a role in sympathetic-stimulated thermogenesis (Fig. 4). Sympathetic activation leads to increased Ang II levels in BAT (23). Furthermore, cold exposure (4°C for 7 days) increases Ang II levels in interscapular BAT in rodents, which is independent of plasma renin levels (22). While cold exposure increases norepinephrine levels in BAT (Fig. 4), this increase is prevented by treatment with losartan, suggesting that cold-induced increases in norepinephrine is mediated via Ang II in an AT1R-dependent manner (23). It is postulated that Ang II facilitates presynaptic norepinephrine release as well as reduces neuronal uptake enhancing sympathetic-mediated cold-induced thermogenesis of BAT (22). There is evidence that this sympatheticenhancing effect of Ang II is abolished in obesity, as the Ang II-stimulated sympathetic effects are reduced in adult obese, but not in young zucker rats (21). Whether this is a possible contributory factor to aging-associated obesity remains to be explored. In addition to the aforementioned effects of local BAT RAS, central actions of Ang II is also important in modulating BAT function. Central administration of Ang II stimulates the sympathetic system in both BAT and WAT, leading to increased energy expenditure and thermogenesis. This is characterized by increased Ucp1 expression in BAT as well (31).

Figure 4.

RAS function in BAT. Brown adipocytes and some white adipocytes respond to cold exposure through actions of the sympathetic nervous system (SNS) to increase Ang II production. Ang II facilitates presynaptic norepinephrine release as well as reduces neuronal uptake, enhancing sympathetic-mediated cold-induced thermogenesis of BAT. This contributes to increased Ucp1 expression, nonshivering thermogenesis in BAT, increased energy expenditure, metabolic rate, and fatty acid oxidation.

Summary

Adipose tissue plays a critical role both in health maintenance and in metabolic diseases. This is best demonstrated though its endocrine function. Here, we reviewed the functions of RAS in adipose tissue to illustrate local and systemic impacts of adipose-derived hormones.

This review provides a summary of documented effects of RAS, especially Ang II and Agt in adipose tissue and whole body metabolism, in cell, animal and human studies. These demonstrate that RAS is expressed in both white and brown fat where it regulates local metabolism. Further, adipose RAS impacts whole body homeostasis through secretion of Agt and Ang II into the bloodstream, ultimately increasing systemic inflammation and insulin resistance and contributing to the development/progression of metabolic diseases. However, further studies are needed to dissect mechanisms of depot specific effects of RAS in obesity and diabetes. Moreover, given that other organs also express RAS, especially the Ang II receptors, the contribution of adipose RAS must be evaluated in the context of cross-talk across various organs.

The RAS is even more important given that is has been extensively studied clinically and inhibitors of RAS are successfully used not only for treating hypertension but also to increase insulin sensitivity in patients with diabetes. Thus, it is important to understand and identify additional and broader effects of RAS beyond hypertension. Given the expression and effects of RAS in adipose tissue as reviewed here, further studies in adipose and other local RASs would enhance our understanding of tissue specific effects of RAS and help understand identify potential new strategies to prevent and/or treat metabolic disease.

Didactic Synopsis.

Major teaching points

1. Understanding the role of renin angiotensin system (RAS) beyond regulation of blood pressure, fluid and electrolyte balance.

2. In addition to the classically known systemic RAS, several local RAS exist in other tissues including both white and brown adipose tissue.

3. Adipose tissue RAS is an important regulator not only of adipose tissue metabolism, but also of whole body energy, blood pressure and glucose homeostasis.

4. Adipose tissue dysfunction is associated with increased Ang II levels and obesity. Various inhibitors of the RAS pathway have been tested in clonal adipocytes, rodent models, and clinical studies, which consistently demonstrate that reducing angiotensin levels decreases obesity-associated inflammation.