DOI 10.1515/hmbci-2013-0033 Horm Mol Biol Clin Invest 2013; 15(2): 59–69
Emmanuelle Kuhn and Marc Lombès*
The mineralocorticoid receptor: a new player controlling energy homeostasis Abstract: Numerous studies have demonstrated the interaction that exists between adipocyte differentiation, energy balance and factors involved in fluid and electrolyte homeostasis, such as the renin-angiotensin-aldosterone system. More specifically, a potential impact of aldosterone on the function of several organs implicated in the control of energy homeostasis, such as adipose tissue, liver, skeletal muscle or pancreas, has been recently described. In addition, the mineralocorticoid receptor (MR, NR3C2), a transcription factor, was shown to play a crucial role on white and brown adipocyte differentiation and function, mediating the effects of both mineralocorticoid and glucocorticoid hormones on adipose tissues. Transgenic mouse models as well as pharmacological inactivation of MR signaling provided compelling evidence that MR is an important control point for energy homeostasis. Herein, we review recent findings on the involvement of aldosterone but also of MR on energy metabolism and discuss the therapeutic potential of manipulating MR signaling for the management of metabolic disorders in humans. Keywords: adipocyte; aldosterone; energy homeostasis; mineralocorticoid receptor. *Corresponding author: Dr. Marc Lombès, MD, PhD, Inserm U693, Le Kremlin-Bicêtre, F-94276, France; Univ Paris-Sud, Faculté de Médecine Paris-Sud, UMR-S693, Le Kremlin-Bicêtre, F-94276, France; and Assistance Publique-Hôpitaux de Paris, Hôpital de Bicêtre, Service d’Endocrinologie et des Maladies de la Reproduction, Le Kremlin Bicêtre F-94275, France, Phone: +33 1 49 59 67 02, E-mail: [email protected] Emmanuelle Kuhn: Inserm U693, Le Kremlin-Bicêtre, F-94276, France; and Univ Paris-Sud, Faculté de Médecine Paris-Sud, UMR-S693, Le Kremlin-Bicêtre, F-94276, France
Introduction The increased incidence of obesity in our industrialized societies is related to changes in nutritional and physical habits that had occurred during the last decades. There is a causal relationship between the obesity-associated remodeling of adipose tissue (AT) and the development
of insulin resistance, type 2 diabetes, and cardiovascular diseases [1–3] referred to as the metabolic syndrome (MetS) [1]. Other co-morbidities are also associated, such as pro-thrombotic and pro-inflammatory state and non-alcoholic steatohepatitis (NASH). MetS is the consequence of three alterations: (i) fat ectopic accumulation in muscle and liver, (ii) alteration of adipocyte endocrine function including modification of adipokine production (decreased adiponectine secretion, increased levels of adipokines involved in insulin resistance) and then (iii) development of macrophage infiltration and inflammatory state (inappropriate TNF-α and IL-1 secretion), that leads to insulin resistance and hyperinsulinemia [4]. Various studies have demonstrated the interaction existing between adipocyte differentiation, energy balance and factors involved in fluid and electrolyte homeostasis, such as the renin-angiotensin-aldosterone system. Indeed, besides its role in the control of sodium and water balance, recent studies suggest that the mineralocorticoid receptor (MR) plays a key role in adipocyte differentiation and function, relaying the effects of gluco- and mineralocorticoid hormones on AT. In this review, we describe the involvement of MR on energy metabolism, particularly focusing on organs implicated in the control of energy balance (such as AT, liver, pancreas and skeletal muscle) and its potential implication in the development of metabolic disorders in humans.
The mineralocorticoid receptor Gene, ARNm, protein MR (NR3C2) belongs to the nuclear receptor superfamily and steroid receptor subfamily. This transcription factor plays a key role in hydro-electrolytic homeostasis in epithelial tissues, such as kidney and colon. In humans, aldosterone is the main physiological ligand for MR, especially in sodium-transporting epithelial cells. Cortisol (or corticosterone in rodents) is another physiological ligand of MR. It can bind MR with the same affinity than aldosterone and is found in circulating levels 100–1000-times
60 Kuhn and Lombès: MR, adipocyte function and energy balance higher than that of aldosterone [5]. However, there are several mechanisms conferring the aldosterone selectivity for MR in epithelial cells. In humans, the gene for MR (hMR) is located on the long arm of chromosome 4 at locus 4q31.1–31.2 and spans approximately 450 kb [6, 7]. This gene contains 10 exons and 8 introns; the first two exons (1α, 1β) are transcribed but not translated and spliced into the common exon 2. Exons 2–9 encode the entire protein of 984 amino acids (aa) and of 107 kDa. MR has the classic three domains of nuclear receptors: the N-terminal domain (NTD) is encoded by exon 2, the DNA binding domain (DBD) has two zinc fingers encoded by exons 3 and 4, and the ligand binding domain (LBD) is encoded by exons 5–9. The 5′-flanking regions of the first two exons are two promoters that direct the expression of MR, named P1 for the region upstream of exon 1α and P2 for the region upstream of exon 1β, respectively. The P1 promoter has a higher basal transcriptional activity than that of the P2 promoter both in vitro and in vivo [8–10]. Moreover, it has been shown in vivo that the P1 promoter is active in all tissues expressing the MR while the promoter P2 is rather active during development [11]. Post-translational modifications have been identified and could modulate MR action. Most notably, phosphorylation sites on threonine and serine are present on the hMR LBD and could participate in ligand-dependent conformational changes. In addition, serine/threonine-rich sequence, located in the nuclear localization signal (NLS), is involved in the receptor subcellular trafficking. Furthermore, MR-sumoylation or acetylation could interfere in protein-protein interactions (such as in transcriptional complexes). MR could also be targeted to the proteasome by ubiquitylation [12], and thus modulating transcriptional activation [13].
Mechanism and sites of action Classically, in the absence of hormone, the MR is located in the cytoplasm where it interacts mainly through the LBD with a multiprotein complex of chaperone proteins (heat shock protein, hsp 90 and 70), of immunophillines and/or cyclophillines [14, 15]. Ligand binding induces a conformational change in the receptor (folding helix H12) and the dissociation of the protein complex [16], which then exposes the nuclear localization sequences. After dimerization, MR translocates to the nucleus where it binds via its DBD to palindromic sequences glucocorticoid responsive element (GRE ) located approximately 10 kb usptream of the transcription initiation sites of target
genes [17]. The exact nature as well as the precise selectivity of hormone response elements are still a matter of debate [13, 18], however, new technologies such as chomatin immunoprecipitation sequencing (ChiP Seq) should rapidly bring novel useful information. The transcriptional machinery as well as various coregulators involved in chromatin remodeling and histone acetylation (SRC-1 and CBP/p300) are recruited. Since its initial description in the 1990s, it has been demonstrated that several coregulators interact, modulate the activity and ensure the selectivity of MR. Thus, PPAR Gamma Coactivator-1 alpha (PGC-1α) is a major coactivator regulation of energy metabolism [19]. MR is currently considered as a ubiquitous receptor [13], but initially MR expression was restricted to epithelial tissues with tight junctions, such as the kidney and colon where it regulates the transepithelial ion transport. However, thereafter its expression has been demonstrated in non-epithelial tissues: AT, central nervous system, and cardiovascular system [13]. Identification of cardiac MR [20, 21] led to important therapeutic issues for the management of cardiovascular diseases. Extensive clinical trials (EPHESUS, RALES, EMPHASIS HF) demonstrated the beneficial effects of MR antagonists (spironolactone and eplerenone) for improving the prognosis and mortality of heart failure patients [22, 23] and during the post-infarction period [24].
Aldosterone selectivity MR is able to bind mineralo- (aldosterone) or glucocorticoids (cortisol or corticosterone in rodents) with the same affinity [5]. Because the plasma concentration of cortisol is 100–1000 times higher than that of aldosterone, there are several mechanisms to ensure aldosterone selectivity for MR [5]. One of these is provided by the expression and activity of an enzyme in epithelial tissues, the 11 β-hydroxysteroid dehydrogenase type 2 (11HDS2), which converts cortisol to cortisone, an inactive metabolite unable to bind MR [25–27]. However, this enzyme is not expressed in other aldosterone target tissues. Other mechanisms exist to confer the selectivity of aldosterone for MR. In contrast, several non-epithelial tissues, including the liver and AT, express substantial amounts of the 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) that regenerates active cortisol from inactive cortisone, and thus reinforces local glucocorticoid action [28]. As a result, cortisol is most likely the physiological ligand of MR in tissues expressing 11HSD1 (Figure 1). The aldosterone selectivity for MR is also ensured by specific conformational changes and recruitment of different specific
Kuhn and Lombès: MR, adipocyte function and energy balance 61
CoR MT MR MR
HRE Chaperone proteins
MR
OH
Cortisol O HO
Chaperone proteins
MR
OH
O
NAD+
11HSDI NADH
OH
O
O
OH
OH
O
O
O
OH
OH O
Aldosterone
HO
[0.35 nmol/L]plasmatic O
O
Cortisone
OH
Cortisol [400 nmol/L]plasmatic
Figure 1 Mechanism of MR action in adipocytes. MR, mineralocorticoid receptor; HRE, hormone responsive element; TM, transcriptional machinery; 11HSD1, 11β-hydroxysteroid dehydrogenase type 1; NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide.
coregulators [13]. The ligand-dependent conformational changes, specifically induced by the nature of the ligand (mineralocorticoid vs. glucocorticoid; agonist vs. antagonist), lead to different transactivation capabilities. Moreover, aldosterone dissociates from MR more slowly than cortisol, accounting for a more stable aldosterone-MR interaction than that of cortisol-MR complex. Thus, MR is able to discriminate between aldosterone and glucocorticoids providing an additional level of MR selectivity [5]. At the post-receptor level, the mineralocorticoid selectivity of MR is ensured by specific recruitment of coregulators, facilitating transcriptional activation of target genes. Along this line, the NTD appears to be as a key element of the mineralocorticoid selectivity. Identification of specific interacting coregulators represents a crucial step forward
better understanding of the cell-specific MR-mediated effects.
Distinction between an “aldosterone effect” and a “MR effect” Most aldosterone actions are mediated by MR, but as mentioned above, MR binds both aldosterone and glucocorticoids with the same affinity. In tissues expressing 11HSD2, the physiological ligand is aldosterone. In contrast, in tissues lacking 11HSD2 expression (and a fortiori those expressing 11HSD1, which increases the local cortisol availability), MR mediates glucocorticoid effects. In most tissues and cells involved in the control of energy
62 Kuhn and Lombès: MR, adipocyte function and energy balance balance (such as liver or AT), MR effects are very likely related to glucocorticoid binding rather than aldosterone binding. Thus, one has to always keep in mind the difference between the effects exerted by aldosterone to those exerted by the MR (which may mediate the effects of both aldosterone and glucocorticoids as well as ligand-independent, MR-mediated actions).
Aldosterone and energy metabolism Aldosterone and insulin resistance A direct functional relationship between aldosterone and insulin resistance was first suggested 40 years ago, in patients with primary hyperaldosteronism (PA) who presented with impaired glucose tolerance [29]. The high aldosterone levels are correlated with insulin resistance, independently of other MetS components [30–32]. In addition, medical (MR antagonists) or surgical treatment (resection of the adrenal adenoma) of PA significantly reduces plasma levels of insulin and glucose, in favor of an insulin sensitivity improvement [32, 33]. In patients with PA, a decrease in the number of insulin receptors and their affinity for insulin has been reported [34]. Moreover, there is a higher prevalence of MetS or glucose intolerance in these patients compared to those with essential hypertension [35]. However, these findings remain controversial. Indeed, recent studies showed that AT insulin sensitivity was similar between patients with aldosterone producing adenoma and those with non-functioning adenomas [36, 37]. Aldosterone could induce insulin resistance by modulating the expression of key proteins involved in the insulin signaling pathway. Using the 3T3-L1 adipocyte model, Wada et al. showed that aldosterone inhibited insulin-induced-glucose uptake in adipocytes by increasing IRS1 and IRS2 degradation via reactive oxygen species (ROS) production. Surprisingly, this effect was not mediated by the MR but rather by the GR [38]. The physiological relevance of this aldosterone effect is still very questionable considering the high, pharmacological aldosterone concentrations necessary to alter glucose transport (1–10 μM). Aldosterone was also shown to induce insulin resistance in skeletal muscle by down-regulating insulin receptor expression in this major insulin target tissue [39]. Moreover, in skeletal muscular cells of male adult rats, aldosterone was also able to reduce GLUT4 translocation to plasma membrane, leading to a decrease of glucose uptake, contributing to insulin resistance in this tissue [40]. However, direct aldosterone effects via the MR, as
well as the physiological relevance of these findings await further validation.
Aldosterone and AT inflammation The nutritional and hormonal environment exerts a fundamental role in the physiology of AT. Steroid hormones are well known to play key roles in adipocyte metabolism and in the adipogenesis processes. It has been reported that obesity is associated with aldosterone excess [41–43]. Moreover, obesity is also associated with inflammation in AT [44–46]. Indeed visceral AT is a source of proinflammatory cytokines that induce a systemic and chronic low grade inflammatory state, an oxidative stress and an insulin resistance [3]. In a culture of 3T3-L1 preadipocytes, aldosterone stimulated expression of pro-inflammatory cytokines and reduced adiponectin and PPARγ expression [47]. Similarly Hirata et al. have shown that aldosterone increased the intracellular ROS levels in 3T3-L1 adipocytes [48]. In vivo, aldosterone increased pro-inflammatory cytokines expression in rodent AT, leading to a reduction of insulin receptor expression and altered insulin-induced glucose transportation [49]. Thus, during obesity, aldosterone may participate to the induction of AT inflammation state by enhancing expression of pro-inflammatory factors. Collectively, it has been proposed that aldosterone may induce insulin resistance by inducing AT inflammation and by altering insulin signaling and action in adipocytes but also in skeletal muscle. However, as mentioned above, whether such aldosterone effects are physiologically relevant and directly mediated by the MR remains to be clarified.
Aldosterone and insulin secretion Other mechanisms have been suggested to explain the role of aldosterone in glucose intolerance. It has been demonstrated that aldosterone may affect insulin secretion through hypokalemia or through fibrosis induction [50]. In studies demonstrating an association between aldosterone and decreased β pancreatic mass, plasma aldosterone levels was inversely correlated with C-peptide levels and the HOMA-B [51]. These effects seemed to be independent of potassium levels, suggesting a specific effect of aldosterone on the pancreatic β cell function. It has been subsequently shown that deleterious effects of aldosterone on the structure and function of the pancreas were related to islet inflammation and oxidative stress [52]. Finally, using the aldosterone synthase-deficient (AS KO)
Kuhn and Lombès: MR, adipocyte function and energy balance 63
mouse model, in which knockout mice are unable to synthesize aldosterone, it was demonstrated that AS KO mice presented with an higher glucose induced-insulin secretion compared to WT mice during a hyperglycemic glucose clamp. This effect was independent of potassium level and angIotensin II, however, there was no difference in insulin sensitivity during euglycemic-hyperinsulinemic clamps [53]. In the same study, the authors showed, that in vitro aldosterone inhibited insulin secretion in MIN6 pancreatic β cells and in isolated pancreatic islets. This effect was not inhibited by an MR antagonist but prevented in the presence of an inhibitor of ROS production. These surprising findings suggested that aldosterone modulates insulin secretion via ROS production, but yet independently of MR [53]. More recently, the same group demonstrated that AS KO mice were relatively protected against HFD-induced beta cell dysfunction, hepatic steatosis, suggesting that aldosterone could be involved in the development of these obesity-associated diseases [54]. Thus, aldosterone could alter insulin secretion through hypokaliemia, induction of islet inflammation or oxidative stress. The underlying mechanisms remain however to be established.
Aldosterone and liver The liver is one of the main tissues involved in the regulation of energy balance by controlling carbohydrate, fat and protein metabolism. Among the key hepatic metabolic functions, gluconeogenesis seems to be influenced by aldosterone. Indeed, Yashimata et al. showed that expression of genes involved in gluconeogenesis (glucose-6-phosphatase; G6Pase, Fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase; PEPCK) was increased by aldosterone in a dose-dependent manner in a primary culture of mouse hepatocytes. The inhibitory effect of insulin on G6Pase was suppressed in part by aldosterone, such aldosterone-dependent effect being inhibited by mifepristone (RU 486), a GR antagonist but not by spironolactone an MR antagonist. It was concluded that this impaired hepatic glucose metabolism could account for glucose intolerance observed in primary hyperaldosteronism [55]. Given that MR is not expressed in hepatocytes, it is very likely that aldosterone-dependent impact on hepatic gluconeogenesis may be compromised through alternative and indirect pathways including the glucocorticoid receptor and/or other specific target cells. Taken together, these observations indicate that aldosterone, a key regulatory hormone on salt and water balance, also plays a role in the regulation of energy
homeostasis, by controlling both insulin secretion and action at different levels (liver, pancreas, skeletal muscle, AT). Aldosterone may exert direct effects o insulin receptor and its associated signaling pathway, on hepatic gluconeogenesis and on glucose/sodium co-transport [51, 52, 56]. It remains to be seen whether these reported effects are mediated by MR or by alternative signaling pathways either genomic or non-genomic that need to be further investigated.
MR and energy balance MR in white and brown adipocytes Our laboratory made the pioneering observation that MR is expressed in brown AT. A targeted oncogenesis strategy was developed to identify tissues in which MR was expressed. A transgenic mouse model P1-TAg, expressing the SV40 large T antigen under the control of the P1 proximal promoter of MR, has been generated. Remarkably, these animals developed hibernoma from brown AT, suggesting that the P1 promoter is transcriptionally active in brown fat cells [57]. MR expression studies confirmed that brown adipocytes did express this transcription factor. We subsequently showed that aldosterone was able to stimulate differentiation of brown fat cells. Aldosterone treatment of T37i cells derived from a hibernoma of transgenic mice P1-TAg, induced accumulation of lipid droplets and expression of adipogenic markers such as lipoprotein lipase (LPL) and PPAR-γ2, a key transcription factor of adipogenesis. This effect was mediated by MR. Indeed, this aldosterone-induced adipocyte differentiation process was inhibited by spironolactone, an MR antagonist but not in the presence of a GR antagonist [58]. Moreover, in brown fat cells, aldosterone was able to modulate adipokine secretion (leptin, adiponectin, resistin) [59] and had an anti-thermogenic effect by inhibiting expression of the uncoupling protein UCP1 [60], a mitochondrial protein involved in thermogenesis and energy expenditure (Figure 2). Conversely, aldosterone also exerts pro-adipogenic effects on white preadipocyte models (3T3-L1 and 3T3-F442A) in a dose-, time- and MR-dependent manner [61, 62] (Figure 2). It was subsequently established that the selective knockdown of MR expression by a RNA interference strategy, inhibits 3T3-L1 adipocyte differentiation [61] whereas downregulation of the GR appears to exert little or no effect on adipogenesis. More recently, it was also found that drospirenone, another MR antagonist, impaired adipogenesis [63]. This finding follows original
64 Kuhn and Lombès: MR, adipocyte function and energy balance same study, the authors provided immunohistological evidence for MR expression in pancreatic islets, more notably in the delta and pancreatic polypeptide producing cells [53]. However, the precise role of MR in the endocrine pancreas remains unclear. Further studies are clearly required to determine whether and how MR could regulate the function of pancreatic islet cells and if insulin secretion could be controlled by MR-mediated transcriptional events.
Preadipocyte
MR in other insulin target tissues (skeletal muscle and liver)
White adipocyte
Brown adipocyte
Adipogenesis (↑ PPARγ)
Adipogenesis Anti-thermogenic (↓ UCP1)
Figure 2 MR effects on white and brown adipocytes. MR is a pro-adipogenic factor in white preadipocytes. In brown fat cells, aldosterone through MR has a pro-adipogenic effect and an anti-thermogenic action by inhibiting expression of the uncoupling protein 1 (UCP1).
reports using genetically obese (ob/ob) and diabetic (db/ db) mice, which demonstrated that blocking MR by means of selective antagonists (eplerenone) decreased expression of pro-inflammatory and pro-thrombotic adipokines (TNF-α, PAI-1) and increased adiponectin and PPAR-γ expression [47, 48]. In these mouse models of obesity and type 2 diabetes, antagonizing MR with eplerenone reduced triglycerides levels, pro-inflammatory cytokines expression, macrophage infiltration and ROS expression in AT. Finally, a recent study showed a divergent effect between MR and GR on expression profile of pro-inflammatory genes (TNF-α, IL-6, MCP-1) in mature adipocytes. Indeed, MR activation by aldosterone stimulated expression of these genes while GR activation by dexamethasone induced opposite effects [64]. In summary, adipocyte MR exerts a pro-adipogenic effect but also could act as a proinflammatory factor enhancing inflammatory cytokine production by adipocytes.
MR in endocrine pancreas As mentioned above in the previous section, it has been reported that aldosterone could impair pancreatic insulin secretion [53], yet independently of MR activation. In the
Skeletal muscle represents an important insulin target tissue and the one of main organs responsible for glucose uptake. Dysfunction of insulin signaling in this tissue contributes to global insulin resistance. Administration of spironolactone (MR antagonist) in a model of reninangiotensin-aldosterone hyperactivity [TG(mREN2)27) rats] reduces oxidative stress (ROS production and activity of NADPH oxidase) while it partially restores mitochondrial structure and thus improves insulin sensitivity in skeletal muscle of these animals [65]. Finally, it has been further demonstrated that inhibition of MR signaling by using pharmacological (spironolactone) and RNA interference (siRNA) strategies, decreased expression of enzymes involved in hepatic glucose production (PEPCK, G6Pase, fructose-1,6-bisphosphatase) in primary cultured hepatocytes and in HepG2 cells [66]. Along this line, it was shown that administration of high doses of eplerenone, a selective MR antagonist, attenuated hepatic fibrosis induced by bile duct ligation, by reducing the oxidative stress in conjunction of decreased levels of circulating angiotensin II [67]. Similarly, Wada et al. also demonstrated that spironolactone treatment allowed improved glucose tolerance, normalized hepatic glucose production during pyruvate test, and reduced the hepatic triglycerides accumulation in mouse model of obesity and insulin resistance induced by a high fat (60%) and high fructose (30%) diet [68]. These obese mice also exhibited hepatic overexpression of pro-inflammatory genes (TNFα, IL-6, MCP-1), and of PEPCK. Such abnormalities were attenuated in animals treated with spironolactone, consistent with beneficial effects of MR blockage in improving dyslipidemia, fatty liver, probably through reducing insulin resistance and hepatic inflammation. Despite these observations that have been confirmed by some authors [69] but not by others [70], potential therapeutic use of MR antagonists needed to be further validated by appropriate clinical trials for the management of human metabolic disorders.
Kuhn and Lombès: MR, adipocyte function and energy balance 65
MR in other cell types (macrophages, brain) We recently characterized the metabolic consequences of global overexpression of human MR (hMR) in transgenic (Tg) mice, previously generated in our laboratory [71]. Unexpectedly, these Tg mice exhibited a marked resistance to high fat diet (HFD)-induced obesity. This was associated with a decrease of fat mass, an increased population of smaller adipocytes and an improved glucose tolerance compared to WT animals. This paradoxical resistance to HFD-induced obesity was not explained by impaired adipogenesis. Indeed, the differentiation ability of Tg preadipocytes isolated from inguinal stroma vascular fractions was unaffected (Figure 3), suggesting that other non-adipocyte factors might compromise AT expansion. We showed that, while AT macrophage infiltration was not different between genotypes, Tg mice exhibited a distinct macrophage polarization. Given that myeloid MR was previously showed to be an important control point for macrophage polarization [72] and owing to MR overexpression in these innate immune cells observed in our Tg model, we propose that the modification of M1 (proinflammatory)/M2 (alternative) macrophage polarization could account in part for the metabolic phenotype of Tg mice. Collectively, our results provide evidence that MR plays a pivotal role in energetic metabolism by directly controlling adipocyte differentiation but also indirectly through macrophage polarization regulation (Kuhn E.
Basal condition
Fatty acid synthase (FAS)
WT Differentiation cocktail Tg
Relative expression (FAS/18S)
3.0
*
2.5 2.0 1.5 1.0 0.5 0.0
WT
Tg
Figure 3 In vitro differentiation ability of preadipocytes isolated from inguinal AT of WT and Tg mice. Isolated stroma vascular fractions of inguinal adipose depot were composed of immature undifferentiated preadipocytes (upper panel). Under differentiation cocktail exposure for 7 days, preadipocytes undergo differentiation into mature adipocyte (middle and down panels). The adipocyte differentiation ability of hMR overexpressing preadipocytes was higher compared to WT preadipocytes, as evidenced by appearance of cytoplasmic lipid droplets (middle and lower panels), and fatty acid synthase (FAS) gene expression profile determined by real-time quantitative PCR.
et al, 2013 American Journal of Physiology, Endocrinology and metabolism, in revision). Finally, it is well established that MR is transcriptionally active in the central nervous system, particularly in hippocampus and hypothalamus [13], where it regulates sodium appetite and intake, stress responses, and memorization processes among other MR-regulated functions [73]. It is very likely that MR also plays an important role in the central regulation of food intake and energy expenditure, but further studies are required for analyzing this issue.
MR: a link between obesity and hypertension It is well known that obesity is an important cardiovascular risk factor and is associated with increased plasma aldosterone levels. It has been reported that adipocytes may secrete mineralocorticoid-releasing factors that enhance steroidogenesis and aldosterone production in human and rodent adrenocortical cells [49, 74–76]. Furthermore, aldosterone seems to be a novel adipocytederived factor and could regulate vascular function [77, 78]. It has also been suggested that endothelial MR could be implicated in obesity-induced endothelial dysfunction. In aortic endothelial cells isolated from mice subjected to a HFD during 14 weeks, eplerenone (MR antagonist) decreased expression of NADPH oxidase whereas it increased antioxidative genes expression [79]. Moreover, in obese mice (exhibiting a high aldosterone level) and in aldosterone-infused lean mice, the specific deletion of MR in endothelial cells precluded endothelial dysfunction [79]. Furthermore, another group reported that eplerenone could improve the acetylcholine-induced relaxation in db/ db mesenteric arteries containing perivascular fat [77]. Accordingly, obesity-induced endothelial dysfunction seems to be dependent on endothelial MR and is probably mediated to an activation of oxidative stress. Very recently in obese humans, Hwang et al. evaluated vascular endothelial function (brachial artery flow-mediates dilatation) and oxidative stress in 22 older adults (55–79 years) free of cardiovascular diseases but varying in adiposity (body mass index 20–45 kg/m2), during 1 month treatment with MR blockade (eplerenone, 100 mg/day). There is no difference between the treated group and placebo group considering vascular endothelial dysfunction and oxidative stress. However, in the sub-group with higher total body fat and abdominal fat, eplerenone ameliorates endothelial function [80]. Thus, many reports suggest
66 Kuhn and Lombès: MR, adipocyte function and energy balance Table 1 Summary of aldosterone and MR effects on energy homeostasis. Tissue
Effects
References
Adipose tissue
Adipogenesis in brown and white adipocyte
Zennaro et al., 1998 [57]; Penfornis et al., 2000 [58]; Rondinone et al., 1993 [62]; Caprio et al., 2007 [61] and 2011 [63] Viengchareun et al., 2001 [60] Guo et al., 2008 [47]; Hirata et al., 2009 [48]; Hoppman et al., 2010 [64] Wada et al., 2009 [38] Hayden and Sowers, 2008 [52]; Luther et al., 2011 [53] Yamashita et al., 2004 [55]; Liu et al., 2006 [66] Wada et al., 2010 [68] Calle et al., 2003 [39]; Selvaraj et al., 2009 [40] Kuhn E et al., 2013
Anti-thermogenic Inflammation (cytokines, ROS production)
Pancreas Liver Skeletal muscle Macrophage
Insulin resistance (insulin signaling) Insulin secretion alteration (ROS production, islet inflammation) Gluconeogenese activation Inflammation Insulin resistance (insulin signaling) Macrophage polarization, immunometabolic modulation
that aldosterone and MR could represent a link between obesity and vascular insulin resistance and dysfunction, this was discussed by Bender et al. in a recent review [81].
Conclusion Taken together, there is now accumulating evidence demonstrating that MR is involved not only in the control of fluid and electrolyte balance, but also in the regulation of energy homeostasis, mediating both mineralo- and glucocorticoids effects. Table 1 summarizes the main MR and aldosterone-mediated effects of energy homeostasis. MR
could represent a potential link between energy meta bolism, blood pressure regulation, salt and diet intake. Particularly, MR seems to be a potential candidate involved in the development of obesity, insulin resistance and metabolic complications. This specifies MR implication in the control of energy metabolism, its possible involvement in the pathophysiology of metabolic disorders in humans, but also opens innovative therapeutic strategies such as the use of selective and tissue-specific MR modulators in the management of metabolic diseases in humans. Received July 1, 2013; accepted July 18, 2013; previously published online August 12, 2013
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Emmanuelle Kuhn and Marc Lombès*
The mineralocorticoid receptor: a new player controlling energy homeostasis Abstract: Numerous studies have demonstrated the interaction that exists between adipocyte differentiation, energy balance and factors involved in fluid and electrolyte homeostasis, such as the renin-angiotensin-aldosterone system. More specifically, a potential impact of aldosterone on the function of several organs implicated in the control of energy homeostasis, such as adipose tissue, liver, skeletal muscle or pancreas, has been recently described. In addition, the mineralocorticoid receptor (MR, NR3C2), a transcription factor, was shown to play a crucial role on white and brown adipocyte differentiation and function, mediating the effects of both mineralocorticoid and glucocorticoid hormones on adipose tissues. Transgenic mouse models as well as pharmacological inactivation of MR signaling provided compelling evidence that MR is an important control point for energy homeostasis. Herein, we review recent findings on the involvement of aldosterone but also of MR on energy metabolism and discuss the therapeutic potential of manipulating MR signaling for the management of metabolic disorders in humans. Keywords: adipocyte; aldosterone; energy homeostasis; mineralocorticoid receptor. *Corresponding author: Dr. Marc Lombès, MD, PhD, Inserm U693, Le Kremlin-Bicêtre, F-94276, France; Univ Paris-Sud, Faculté de Médecine Paris-Sud, UMR-S693, Le Kremlin-Bicêtre, F-94276, France; and Assistance Publique-Hôpitaux de Paris, Hôpital de Bicêtre, Service d’Endocrinologie et des Maladies de la Reproduction, Le Kremlin Bicêtre F-94275, France, Phone: +33 1 49 59 67 02, E-mail: [email protected] Emmanuelle Kuhn: Inserm U693, Le Kremlin-Bicêtre, F-94276, France; and Univ Paris-Sud, Faculté de Médecine Paris-Sud, UMR-S693, Le Kremlin-Bicêtre, F-94276, France
Introduction The increased incidence of obesity in our industrialized societies is related to changes in nutritional and physical habits that had occurred during the last decades. There is a causal relationship between the obesity-associated remodeling of adipose tissue (AT) and the development
of insulin resistance, type 2 diabetes, and cardiovascular diseases [1–3] referred to as the metabolic syndrome (MetS) [1]. Other co-morbidities are also associated, such as pro-thrombotic and pro-inflammatory state and non-alcoholic steatohepatitis (NASH). MetS is the consequence of three alterations: (i) fat ectopic accumulation in muscle and liver, (ii) alteration of adipocyte endocrine function including modification of adipokine production (decreased adiponectine secretion, increased levels of adipokines involved in insulin resistance) and then (iii) development of macrophage infiltration and inflammatory state (inappropriate TNF-α and IL-1 secretion), that leads to insulin resistance and hyperinsulinemia [4]. Various studies have demonstrated the interaction existing between adipocyte differentiation, energy balance and factors involved in fluid and electrolyte homeostasis, such as the renin-angiotensin-aldosterone system. Indeed, besides its role in the control of sodium and water balance, recent studies suggest that the mineralocorticoid receptor (MR) plays a key role in adipocyte differentiation and function, relaying the effects of gluco- and mineralocorticoid hormones on AT. In this review, we describe the involvement of MR on energy metabolism, particularly focusing on organs implicated in the control of energy balance (such as AT, liver, pancreas and skeletal muscle) and its potential implication in the development of metabolic disorders in humans.
The mineralocorticoid receptor Gene, ARNm, protein MR (NR3C2) belongs to the nuclear receptor superfamily and steroid receptor subfamily. This transcription factor plays a key role in hydro-electrolytic homeostasis in epithelial tissues, such as kidney and colon. In humans, aldosterone is the main physiological ligand for MR, especially in sodium-transporting epithelial cells. Cortisol (or corticosterone in rodents) is another physiological ligand of MR. It can bind MR with the same affinity than aldosterone and is found in circulating levels 100–1000-times
60 Kuhn and Lombès: MR, adipocyte function and energy balance higher than that of aldosterone [5]. However, there are several mechanisms conferring the aldosterone selectivity for MR in epithelial cells. In humans, the gene for MR (hMR) is located on the long arm of chromosome 4 at locus 4q31.1–31.2 and spans approximately 450 kb [6, 7]. This gene contains 10 exons and 8 introns; the first two exons (1α, 1β) are transcribed but not translated and spliced into the common exon 2. Exons 2–9 encode the entire protein of 984 amino acids (aa) and of 107 kDa. MR has the classic three domains of nuclear receptors: the N-terminal domain (NTD) is encoded by exon 2, the DNA binding domain (DBD) has two zinc fingers encoded by exons 3 and 4, and the ligand binding domain (LBD) is encoded by exons 5–9. The 5′-flanking regions of the first two exons are two promoters that direct the expression of MR, named P1 for the region upstream of exon 1α and P2 for the region upstream of exon 1β, respectively. The P1 promoter has a higher basal transcriptional activity than that of the P2 promoter both in vitro and in vivo [8–10]. Moreover, it has been shown in vivo that the P1 promoter is active in all tissues expressing the MR while the promoter P2 is rather active during development [11]. Post-translational modifications have been identified and could modulate MR action. Most notably, phosphorylation sites on threonine and serine are present on the hMR LBD and could participate in ligand-dependent conformational changes. In addition, serine/threonine-rich sequence, located in the nuclear localization signal (NLS), is involved in the receptor subcellular trafficking. Furthermore, MR-sumoylation or acetylation could interfere in protein-protein interactions (such as in transcriptional complexes). MR could also be targeted to the proteasome by ubiquitylation [12], and thus modulating transcriptional activation [13].
Mechanism and sites of action Classically, in the absence of hormone, the MR is located in the cytoplasm where it interacts mainly through the LBD with a multiprotein complex of chaperone proteins (heat shock protein, hsp 90 and 70), of immunophillines and/or cyclophillines [14, 15]. Ligand binding induces a conformational change in the receptor (folding helix H12) and the dissociation of the protein complex [16], which then exposes the nuclear localization sequences. After dimerization, MR translocates to the nucleus where it binds via its DBD to palindromic sequences glucocorticoid responsive element (GRE ) located approximately 10 kb usptream of the transcription initiation sites of target
genes [17]. The exact nature as well as the precise selectivity of hormone response elements are still a matter of debate [13, 18], however, new technologies such as chomatin immunoprecipitation sequencing (ChiP Seq) should rapidly bring novel useful information. The transcriptional machinery as well as various coregulators involved in chromatin remodeling and histone acetylation (SRC-1 and CBP/p300) are recruited. Since its initial description in the 1990s, it has been demonstrated that several coregulators interact, modulate the activity and ensure the selectivity of MR. Thus, PPAR Gamma Coactivator-1 alpha (PGC-1α) is a major coactivator regulation of energy metabolism [19]. MR is currently considered as a ubiquitous receptor [13], but initially MR expression was restricted to epithelial tissues with tight junctions, such as the kidney and colon where it regulates the transepithelial ion transport. However, thereafter its expression has been demonstrated in non-epithelial tissues: AT, central nervous system, and cardiovascular system [13]. Identification of cardiac MR [20, 21] led to important therapeutic issues for the management of cardiovascular diseases. Extensive clinical trials (EPHESUS, RALES, EMPHASIS HF) demonstrated the beneficial effects of MR antagonists (spironolactone and eplerenone) for improving the prognosis and mortality of heart failure patients [22, 23] and during the post-infarction period [24].
Aldosterone selectivity MR is able to bind mineralo- (aldosterone) or glucocorticoids (cortisol or corticosterone in rodents) with the same affinity [5]. Because the plasma concentration of cortisol is 100–1000 times higher than that of aldosterone, there are several mechanisms to ensure aldosterone selectivity for MR [5]. One of these is provided by the expression and activity of an enzyme in epithelial tissues, the 11 β-hydroxysteroid dehydrogenase type 2 (11HDS2), which converts cortisol to cortisone, an inactive metabolite unable to bind MR [25–27]. However, this enzyme is not expressed in other aldosterone target tissues. Other mechanisms exist to confer the selectivity of aldosterone for MR. In contrast, several non-epithelial tissues, including the liver and AT, express substantial amounts of the 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) that regenerates active cortisol from inactive cortisone, and thus reinforces local glucocorticoid action [28]. As a result, cortisol is most likely the physiological ligand of MR in tissues expressing 11HSD1 (Figure 1). The aldosterone selectivity for MR is also ensured by specific conformational changes and recruitment of different specific
Kuhn and Lombès: MR, adipocyte function and energy balance 61
CoR MT MR MR
HRE Chaperone proteins
MR
OH
Cortisol O HO
Chaperone proteins
MR
OH
O
NAD+
11HSDI NADH
OH
O
O
OH
OH
O
O
O
OH
OH O
Aldosterone
HO
[0.35 nmol/L]plasmatic O
O
Cortisone
OH
Cortisol [400 nmol/L]plasmatic
Figure 1 Mechanism of MR action in adipocytes. MR, mineralocorticoid receptor; HRE, hormone responsive element; TM, transcriptional machinery; 11HSD1, 11β-hydroxysteroid dehydrogenase type 1; NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide.
coregulators [13]. The ligand-dependent conformational changes, specifically induced by the nature of the ligand (mineralocorticoid vs. glucocorticoid; agonist vs. antagonist), lead to different transactivation capabilities. Moreover, aldosterone dissociates from MR more slowly than cortisol, accounting for a more stable aldosterone-MR interaction than that of cortisol-MR complex. Thus, MR is able to discriminate between aldosterone and glucocorticoids providing an additional level of MR selectivity [5]. At the post-receptor level, the mineralocorticoid selectivity of MR is ensured by specific recruitment of coregulators, facilitating transcriptional activation of target genes. Along this line, the NTD appears to be as a key element of the mineralocorticoid selectivity. Identification of specific interacting coregulators represents a crucial step forward
better understanding of the cell-specific MR-mediated effects.
Distinction between an “aldosterone effect” and a “MR effect” Most aldosterone actions are mediated by MR, but as mentioned above, MR binds both aldosterone and glucocorticoids with the same affinity. In tissues expressing 11HSD2, the physiological ligand is aldosterone. In contrast, in tissues lacking 11HSD2 expression (and a fortiori those expressing 11HSD1, which increases the local cortisol availability), MR mediates glucocorticoid effects. In most tissues and cells involved in the control of energy
62 Kuhn and Lombès: MR, adipocyte function and energy balance balance (such as liver or AT), MR effects are very likely related to glucocorticoid binding rather than aldosterone binding. Thus, one has to always keep in mind the difference between the effects exerted by aldosterone to those exerted by the MR (which may mediate the effects of both aldosterone and glucocorticoids as well as ligand-independent, MR-mediated actions).
Aldosterone and energy metabolism Aldosterone and insulin resistance A direct functional relationship between aldosterone and insulin resistance was first suggested 40 years ago, in patients with primary hyperaldosteronism (PA) who presented with impaired glucose tolerance [29]. The high aldosterone levels are correlated with insulin resistance, independently of other MetS components [30–32]. In addition, medical (MR antagonists) or surgical treatment (resection of the adrenal adenoma) of PA significantly reduces plasma levels of insulin and glucose, in favor of an insulin sensitivity improvement [32, 33]. In patients with PA, a decrease in the number of insulin receptors and their affinity for insulin has been reported [34]. Moreover, there is a higher prevalence of MetS or glucose intolerance in these patients compared to those with essential hypertension [35]. However, these findings remain controversial. Indeed, recent studies showed that AT insulin sensitivity was similar between patients with aldosterone producing adenoma and those with non-functioning adenomas [36, 37]. Aldosterone could induce insulin resistance by modulating the expression of key proteins involved in the insulin signaling pathway. Using the 3T3-L1 adipocyte model, Wada et al. showed that aldosterone inhibited insulin-induced-glucose uptake in adipocytes by increasing IRS1 and IRS2 degradation via reactive oxygen species (ROS) production. Surprisingly, this effect was not mediated by the MR but rather by the GR [38]. The physiological relevance of this aldosterone effect is still very questionable considering the high, pharmacological aldosterone concentrations necessary to alter glucose transport (1–10 μM). Aldosterone was also shown to induce insulin resistance in skeletal muscle by down-regulating insulin receptor expression in this major insulin target tissue [39]. Moreover, in skeletal muscular cells of male adult rats, aldosterone was also able to reduce GLUT4 translocation to plasma membrane, leading to a decrease of glucose uptake, contributing to insulin resistance in this tissue [40]. However, direct aldosterone effects via the MR, as
well as the physiological relevance of these findings await further validation.
Aldosterone and AT inflammation The nutritional and hormonal environment exerts a fundamental role in the physiology of AT. Steroid hormones are well known to play key roles in adipocyte metabolism and in the adipogenesis processes. It has been reported that obesity is associated with aldosterone excess [41–43]. Moreover, obesity is also associated with inflammation in AT [44–46]. Indeed visceral AT is a source of proinflammatory cytokines that induce a systemic and chronic low grade inflammatory state, an oxidative stress and an insulin resistance [3]. In a culture of 3T3-L1 preadipocytes, aldosterone stimulated expression of pro-inflammatory cytokines and reduced adiponectin and PPARγ expression [47]. Similarly Hirata et al. have shown that aldosterone increased the intracellular ROS levels in 3T3-L1 adipocytes [48]. In vivo, aldosterone increased pro-inflammatory cytokines expression in rodent AT, leading to a reduction of insulin receptor expression and altered insulin-induced glucose transportation [49]. Thus, during obesity, aldosterone may participate to the induction of AT inflammation state by enhancing expression of pro-inflammatory factors. Collectively, it has been proposed that aldosterone may induce insulin resistance by inducing AT inflammation and by altering insulin signaling and action in adipocytes but also in skeletal muscle. However, as mentioned above, whether such aldosterone effects are physiologically relevant and directly mediated by the MR remains to be clarified.
Aldosterone and insulin secretion Other mechanisms have been suggested to explain the role of aldosterone in glucose intolerance. It has been demonstrated that aldosterone may affect insulin secretion through hypokalemia or through fibrosis induction [50]. In studies demonstrating an association between aldosterone and decreased β pancreatic mass, plasma aldosterone levels was inversely correlated with C-peptide levels and the HOMA-B [51]. These effects seemed to be independent of potassium levels, suggesting a specific effect of aldosterone on the pancreatic β cell function. It has been subsequently shown that deleterious effects of aldosterone on the structure and function of the pancreas were related to islet inflammation and oxidative stress [52]. Finally, using the aldosterone synthase-deficient (AS KO)
Kuhn and Lombès: MR, adipocyte function and energy balance 63
mouse model, in which knockout mice are unable to synthesize aldosterone, it was demonstrated that AS KO mice presented with an higher glucose induced-insulin secretion compared to WT mice during a hyperglycemic glucose clamp. This effect was independent of potassium level and angIotensin II, however, there was no difference in insulin sensitivity during euglycemic-hyperinsulinemic clamps [53]. In the same study, the authors showed, that in vitro aldosterone inhibited insulin secretion in MIN6 pancreatic β cells and in isolated pancreatic islets. This effect was not inhibited by an MR antagonist but prevented in the presence of an inhibitor of ROS production. These surprising findings suggested that aldosterone modulates insulin secretion via ROS production, but yet independently of MR [53]. More recently, the same group demonstrated that AS KO mice were relatively protected against HFD-induced beta cell dysfunction, hepatic steatosis, suggesting that aldosterone could be involved in the development of these obesity-associated diseases [54]. Thus, aldosterone could alter insulin secretion through hypokaliemia, induction of islet inflammation or oxidative stress. The underlying mechanisms remain however to be established.
Aldosterone and liver The liver is one of the main tissues involved in the regulation of energy balance by controlling carbohydrate, fat and protein metabolism. Among the key hepatic metabolic functions, gluconeogenesis seems to be influenced by aldosterone. Indeed, Yashimata et al. showed that expression of genes involved in gluconeogenesis (glucose-6-phosphatase; G6Pase, Fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase; PEPCK) was increased by aldosterone in a dose-dependent manner in a primary culture of mouse hepatocytes. The inhibitory effect of insulin on G6Pase was suppressed in part by aldosterone, such aldosterone-dependent effect being inhibited by mifepristone (RU 486), a GR antagonist but not by spironolactone an MR antagonist. It was concluded that this impaired hepatic glucose metabolism could account for glucose intolerance observed in primary hyperaldosteronism [55]. Given that MR is not expressed in hepatocytes, it is very likely that aldosterone-dependent impact on hepatic gluconeogenesis may be compromised through alternative and indirect pathways including the glucocorticoid receptor and/or other specific target cells. Taken together, these observations indicate that aldosterone, a key regulatory hormone on salt and water balance, also plays a role in the regulation of energy
homeostasis, by controlling both insulin secretion and action at different levels (liver, pancreas, skeletal muscle, AT). Aldosterone may exert direct effects o insulin receptor and its associated signaling pathway, on hepatic gluconeogenesis and on glucose/sodium co-transport [51, 52, 56]. It remains to be seen whether these reported effects are mediated by MR or by alternative signaling pathways either genomic or non-genomic that need to be further investigated.
MR and energy balance MR in white and brown adipocytes Our laboratory made the pioneering observation that MR is expressed in brown AT. A targeted oncogenesis strategy was developed to identify tissues in which MR was expressed. A transgenic mouse model P1-TAg, expressing the SV40 large T antigen under the control of the P1 proximal promoter of MR, has been generated. Remarkably, these animals developed hibernoma from brown AT, suggesting that the P1 promoter is transcriptionally active in brown fat cells [57]. MR expression studies confirmed that brown adipocytes did express this transcription factor. We subsequently showed that aldosterone was able to stimulate differentiation of brown fat cells. Aldosterone treatment of T37i cells derived from a hibernoma of transgenic mice P1-TAg, induced accumulation of lipid droplets and expression of adipogenic markers such as lipoprotein lipase (LPL) and PPAR-γ2, a key transcription factor of adipogenesis. This effect was mediated by MR. Indeed, this aldosterone-induced adipocyte differentiation process was inhibited by spironolactone, an MR antagonist but not in the presence of a GR antagonist [58]. Moreover, in brown fat cells, aldosterone was able to modulate adipokine secretion (leptin, adiponectin, resistin) [59] and had an anti-thermogenic effect by inhibiting expression of the uncoupling protein UCP1 [60], a mitochondrial protein involved in thermogenesis and energy expenditure (Figure 2). Conversely, aldosterone also exerts pro-adipogenic effects on white preadipocyte models (3T3-L1 and 3T3-F442A) in a dose-, time- and MR-dependent manner [61, 62] (Figure 2). It was subsequently established that the selective knockdown of MR expression by a RNA interference strategy, inhibits 3T3-L1 adipocyte differentiation [61] whereas downregulation of the GR appears to exert little or no effect on adipogenesis. More recently, it was also found that drospirenone, another MR antagonist, impaired adipogenesis [63]. This finding follows original
64 Kuhn and Lombès: MR, adipocyte function and energy balance same study, the authors provided immunohistological evidence for MR expression in pancreatic islets, more notably in the delta and pancreatic polypeptide producing cells [53]. However, the precise role of MR in the endocrine pancreas remains unclear. Further studies are clearly required to determine whether and how MR could regulate the function of pancreatic islet cells and if insulin secretion could be controlled by MR-mediated transcriptional events.
Preadipocyte
MR in other insulin target tissues (skeletal muscle and liver)
White adipocyte
Brown adipocyte
Adipogenesis (↑ PPARγ)
Adipogenesis Anti-thermogenic (↓ UCP1)
Figure 2 MR effects on white and brown adipocytes. MR is a pro-adipogenic factor in white preadipocytes. In brown fat cells, aldosterone through MR has a pro-adipogenic effect and an anti-thermogenic action by inhibiting expression of the uncoupling protein 1 (UCP1).
reports using genetically obese (ob/ob) and diabetic (db/ db) mice, which demonstrated that blocking MR by means of selective antagonists (eplerenone) decreased expression of pro-inflammatory and pro-thrombotic adipokines (TNF-α, PAI-1) and increased adiponectin and PPAR-γ expression [47, 48]. In these mouse models of obesity and type 2 diabetes, antagonizing MR with eplerenone reduced triglycerides levels, pro-inflammatory cytokines expression, macrophage infiltration and ROS expression in AT. Finally, a recent study showed a divergent effect between MR and GR on expression profile of pro-inflammatory genes (TNF-α, IL-6, MCP-1) in mature adipocytes. Indeed, MR activation by aldosterone stimulated expression of these genes while GR activation by dexamethasone induced opposite effects [64]. In summary, adipocyte MR exerts a pro-adipogenic effect but also could act as a proinflammatory factor enhancing inflammatory cytokine production by adipocytes.
MR in endocrine pancreas As mentioned above in the previous section, it has been reported that aldosterone could impair pancreatic insulin secretion [53], yet independently of MR activation. In the
Skeletal muscle represents an important insulin target tissue and the one of main organs responsible for glucose uptake. Dysfunction of insulin signaling in this tissue contributes to global insulin resistance. Administration of spironolactone (MR antagonist) in a model of reninangiotensin-aldosterone hyperactivity [TG(mREN2)27) rats] reduces oxidative stress (ROS production and activity of NADPH oxidase) while it partially restores mitochondrial structure and thus improves insulin sensitivity in skeletal muscle of these animals [65]. Finally, it has been further demonstrated that inhibition of MR signaling by using pharmacological (spironolactone) and RNA interference (siRNA) strategies, decreased expression of enzymes involved in hepatic glucose production (PEPCK, G6Pase, fructose-1,6-bisphosphatase) in primary cultured hepatocytes and in HepG2 cells [66]. Along this line, it was shown that administration of high doses of eplerenone, a selective MR antagonist, attenuated hepatic fibrosis induced by bile duct ligation, by reducing the oxidative stress in conjunction of decreased levels of circulating angiotensin II [67]. Similarly, Wada et al. also demonstrated that spironolactone treatment allowed improved glucose tolerance, normalized hepatic glucose production during pyruvate test, and reduced the hepatic triglycerides accumulation in mouse model of obesity and insulin resistance induced by a high fat (60%) and high fructose (30%) diet [68]. These obese mice also exhibited hepatic overexpression of pro-inflammatory genes (TNFα, IL-6, MCP-1), and of PEPCK. Such abnormalities were attenuated in animals treated with spironolactone, consistent with beneficial effects of MR blockage in improving dyslipidemia, fatty liver, probably through reducing insulin resistance and hepatic inflammation. Despite these observations that have been confirmed by some authors [69] but not by others [70], potential therapeutic use of MR antagonists needed to be further validated by appropriate clinical trials for the management of human metabolic disorders.
Kuhn and Lombès: MR, adipocyte function and energy balance 65
MR in other cell types (macrophages, brain) We recently characterized the metabolic consequences of global overexpression of human MR (hMR) in transgenic (Tg) mice, previously generated in our laboratory [71]. Unexpectedly, these Tg mice exhibited a marked resistance to high fat diet (HFD)-induced obesity. This was associated with a decrease of fat mass, an increased population of smaller adipocytes and an improved glucose tolerance compared to WT animals. This paradoxical resistance to HFD-induced obesity was not explained by impaired adipogenesis. Indeed, the differentiation ability of Tg preadipocytes isolated from inguinal stroma vascular fractions was unaffected (Figure 3), suggesting that other non-adipocyte factors might compromise AT expansion. We showed that, while AT macrophage infiltration was not different between genotypes, Tg mice exhibited a distinct macrophage polarization. Given that myeloid MR was previously showed to be an important control point for macrophage polarization [72] and owing to MR overexpression in these innate immune cells observed in our Tg model, we propose that the modification of M1 (proinflammatory)/M2 (alternative) macrophage polarization could account in part for the metabolic phenotype of Tg mice. Collectively, our results provide evidence that MR plays a pivotal role in energetic metabolism by directly controlling adipocyte differentiation but also indirectly through macrophage polarization regulation (Kuhn E.
Basal condition
Fatty acid synthase (FAS)
WT Differentiation cocktail Tg
Relative expression (FAS/18S)
3.0
*
2.5 2.0 1.5 1.0 0.5 0.0
WT
Tg
Figure 3 In vitro differentiation ability of preadipocytes isolated from inguinal AT of WT and Tg mice. Isolated stroma vascular fractions of inguinal adipose depot were composed of immature undifferentiated preadipocytes (upper panel). Under differentiation cocktail exposure for 7 days, preadipocytes undergo differentiation into mature adipocyte (middle and down panels). The adipocyte differentiation ability of hMR overexpressing preadipocytes was higher compared to WT preadipocytes, as evidenced by appearance of cytoplasmic lipid droplets (middle and lower panels), and fatty acid synthase (FAS) gene expression profile determined by real-time quantitative PCR.
et al, 2013 American Journal of Physiology, Endocrinology and metabolism, in revision). Finally, it is well established that MR is transcriptionally active in the central nervous system, particularly in hippocampus and hypothalamus [13], where it regulates sodium appetite and intake, stress responses, and memorization processes among other MR-regulated functions [73]. It is very likely that MR also plays an important role in the central regulation of food intake and energy expenditure, but further studies are required for analyzing this issue.
MR: a link between obesity and hypertension It is well known that obesity is an important cardiovascular risk factor and is associated with increased plasma aldosterone levels. It has been reported that adipocytes may secrete mineralocorticoid-releasing factors that enhance steroidogenesis and aldosterone production in human and rodent adrenocortical cells [49, 74–76]. Furthermore, aldosterone seems to be a novel adipocytederived factor and could regulate vascular function [77, 78]. It has also been suggested that endothelial MR could be implicated in obesity-induced endothelial dysfunction. In aortic endothelial cells isolated from mice subjected to a HFD during 14 weeks, eplerenone (MR antagonist) decreased expression of NADPH oxidase whereas it increased antioxidative genes expression [79]. Moreover, in obese mice (exhibiting a high aldosterone level) and in aldosterone-infused lean mice, the specific deletion of MR in endothelial cells precluded endothelial dysfunction [79]. Furthermore, another group reported that eplerenone could improve the acetylcholine-induced relaxation in db/ db mesenteric arteries containing perivascular fat [77]. Accordingly, obesity-induced endothelial dysfunction seems to be dependent on endothelial MR and is probably mediated to an activation of oxidative stress. Very recently in obese humans, Hwang et al. evaluated vascular endothelial function (brachial artery flow-mediates dilatation) and oxidative stress in 22 older adults (55–79 years) free of cardiovascular diseases but varying in adiposity (body mass index 20–45 kg/m2), during 1 month treatment with MR blockade (eplerenone, 100 mg/day). There is no difference between the treated group and placebo group considering vascular endothelial dysfunction and oxidative stress. However, in the sub-group with higher total body fat and abdominal fat, eplerenone ameliorates endothelial function [80]. Thus, many reports suggest
66 Kuhn and Lombès: MR, adipocyte function and energy balance Table 1 Summary of aldosterone and MR effects on energy homeostasis. Tissue
Effects
References
Adipose tissue
Adipogenesis in brown and white adipocyte
Zennaro et al., 1998 [57]; Penfornis et al., 2000 [58]; Rondinone et al., 1993 [62]; Caprio et al., 2007 [61] and 2011 [63] Viengchareun et al., 2001 [60] Guo et al., 2008 [47]; Hirata et al., 2009 [48]; Hoppman et al., 2010 [64] Wada et al., 2009 [38] Hayden and Sowers, 2008 [52]; Luther et al., 2011 [53] Yamashita et al., 2004 [55]; Liu et al., 2006 [66] Wada et al., 2010 [68] Calle et al., 2003 [39]; Selvaraj et al., 2009 [40] Kuhn E et al., 2013
Anti-thermogenic Inflammation (cytokines, ROS production)
Pancreas Liver Skeletal muscle Macrophage
Insulin resistance (insulin signaling) Insulin secretion alteration (ROS production, islet inflammation) Gluconeogenese activation Inflammation Insulin resistance (insulin signaling) Macrophage polarization, immunometabolic modulation
that aldosterone and MR could represent a link between obesity and vascular insulin resistance and dysfunction, this was discussed by Bender et al. in a recent review [81].
Conclusion Taken together, there is now accumulating evidence demonstrating that MR is involved not only in the control of fluid and electrolyte balance, but also in the regulation of energy homeostasis, mediating both mineralo- and glucocorticoids effects. Table 1 summarizes the main MR and aldosterone-mediated effects of energy homeostasis. MR
could represent a potential link between energy meta bolism, blood pressure regulation, salt and diet intake. Particularly, MR seems to be a potential candidate involved in the development of obesity, insulin resistance and metabolic complications. This specifies MR implication in the control of energy metabolism, its possible involvement in the pathophysiology of metabolic disorders in humans, but also opens innovative therapeutic strategies such as the use of selective and tissue-specific MR modulators in the management of metabolic diseases in humans. Received July 1, 2013; accepted July 18, 2013; previously published online August 12, 2013
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