Adipocyte-Specific Mineralocorticoid Receptor Overexpression in Mice Is Associated With Metabolic Syndrome and Vascular Dysfunction: Role of Redox-Sensitive PKG-1 and Rho Kinase

Aldosterone classically acts via the renal epithelial mineralocorticoid receptors (MR), leading to salt and volume homeostasis and, consequently, blood pressure regulation. MR is also expressed in nonepithelial cells, including cardiomyocytes (1), vascular cells (2,3) and macrophages (4,5), and has been implicated in pathological conditions such as heart failure, endothelial dysfunction, hypertension, insulin resistance, and obesity (6–10). Recently, MR has been identified in adipocytes (11–14). The (patho)-physiological significance of adipocyte MR is unclear, although experimental and human studies have demonstrated increased expression of adipocyte MR in obesity, metabolic syndrome, and diabetes (6,14,15). Adipose tissue has long been considered primarily as simple fat storage of triglycerides to regulate several important functions, such as energy balance and thermogenesis, but is now recognized as a metabolically active endocrine/paracrine/autocrine organ that synthesizes, stores, and secretes hormones, vasoactive factors, and proteins, termed adipo(cyto)kines (16,17). MR mediates effects of aldosterone and glucocorticoids, which in adipocytes regulate adipogenesis, adipocyte maturation, and adipokines production (12,18–20). We recently demonstrated that adipocytes possess functionally active MR and have the machinery to produce aldosterone, processes that are increased in obesity-associated diabetes and influence vascular function (6,12,13).

To better understand the relevance of the adipocyte MR, we generated a conditional transgenic mouse model allowing restricted and inducible MR overexpression (MROE) in an adipocyte-specific manner. These mice exhibit features of metabolic syndrome, including weight gain, insulin resistance, and dyslipidemia, with increased activation of prostaglandin D2 synthase (14). Considering the growing evidence that adipose tissue and adipokines influence vascular tone and that the aldosterone/MR system is involved in vascular pathology associated with cardiovascular disease, we questioned whether upregulation of adipocyte MR in mice leads to vascular dysfunction.

The study was approved by the University of Ottawa Animal Ethics Committee. All studies in animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures followed were in accordance with institutional guidelines.

We generated mice with targeted overexpression of human MR (hMR) in adipocytes using the conditional tetracycline-inducible system, as we recently reported (14) and summarized in Supplementary Fig. 1A and B. Double-transgenic adipocyte MROE (Adipo-MROE) mice were obtained by mating two strains: the monotransgenic tetO-hMR mouse and the monotransgenic transactivator aP2-rtTA mouse. In Adipo-MROE mice, hMR expression is switched on in adipocytes by administering doxycycline (Dox) (2 g/L for 4 weeks) (200 mg/kg Dox diet; Harlan Laboratories, Mississauga, Ontario, Canada). The litters consisting of Adipo-MROE, monotransgenic tetO-hMR, monotransgenic aP2-rtTA, and wild-type mice were born in the expected mendelian ratio (25% for each genotype). The last three groups, which displayed no molecular or functional differences among them, were considered as controls (control-MR). Blood pressure was measured by the tail-cuff method in 2- to 3-month-old mice. Mice were killed to recover mesenteric arteries and adipose tissues used for the subsequent manipulations of cell culture, histology, or biomolecular analysis.

Mouse abdominal epididymal visceral adipose tissue (EVAT) was obtained from 4-month-old male Adipo-MROE and their littermate control-MR mice. Mature adipocytes fraction was isolated and cultured at 37°C for 24 h, as previously described (12).

The conditioned culture medium, collected from mature adipocytes (ACM) 24 h after isolation (12,21), was concentrated in a speed vacuum system (Vacufuge, Eppendorf) and kept at −20°C for further determination of aldosterone, corticosterone, angiotensin II, and hydrogen peroxide (H₂O₂) levels.

Second-order branches of mesenteric artery without perivascular fat (PVAT) were isolated from 3- to 4-month-old Adipo-MROE and their littermate control-MR mice and mounted on a wire myograph (DMT myograph; ADInstruments Ltd., Oxford, U.K.) as previously described (13). Endothelium-dependent and -independent relaxations were assessed by measuring dilatory responses to acetylcholine (Ach) (10⁻⁹ to 10⁻⁵ mol/L) and sodium nitroprusside (SNP) (10⁻⁹ to 10⁻⁵ mol/L), respectively, in arteries precontracted with phenylephrine (Phe) to similar degrees to achieve ∼80% of maximal response. Dose-response curves for insulin-induced relaxation (10⁻¹⁰ to 10⁻⁵ mol/L) were also obtained in arteries with intact endothelium in the absence or presence of a general reactive oxygen species (ROS) scavenger, N-acetyl-cysteine (10⁻⁶ mol/L) or the H₂O₂ scavenger, polyethylene glycol (PEG)-catalase (100 units/mL), incubated 30 min before precontraction with Phe. Contraction curves to cumulative increasing doses of Phe, noradrenaline, and serotonin (5-hydroxytryptamine [5-HT]; 10⁻⁹ to 10⁻⁵ mol/L) were performed in arteries with and without intact endothelium.

Healthy PVAT and other fat depots display a protective anticontractile effect on the vasculature that is lost in obesity. This anticontractile property remained with the transfer of the culture bath to vessels without adipose tissue and suggested the action of secreted relaxing substances. In that way, we investigated the effects of visceral fat using ACM (from EVAT) from control-MR and Adipo-MROE mice on contractile responses to Phe. A small piece of EVAT is incubated in 10 mL of Krebs solution 30 min before the concentration-response curve to Phe. It has been suggested that PVAT behaves similarly to visceral fat in secretion of vasoactive factors (22). We also evaluated the effects of a specific inhibitor of protein kinase G type 1 (PKG-1) on vascular reactivity. Arteries were pretreated with Rp-8-Br-PET-cGMPS (3 × 10⁻⁵ mol/L) or vehicle (DMSO) for 30 min before exposing vessels to Phe (endothelium-denuded mesenteric arteries) or H₂O₂ (10⁻⁶ to 10⁻³ mol/L) (endothelium-intact arteries).

Arterial stiffness was studied with a pressure myograph (Living Systems, Burlington, VT), as previously described (13). The artery was set to an internal pressure of 45 mmHg, pressure-fixed (30 min, 10% formalin, at 37°C), and stored embedded in paraffin until histomorphometry studies. Vascular structure and mechanical parameters were calculated as previously described (13).

Deparaffinized sections (5 μm) were rehydrated in sequential alcohol baths and washed in distilled water. For elastin content, tissue sections were stained with Miller’s Elastin (VWR, Cat. #351154S) and Van Gieson solutions (Sigma-Aldrich, Cat. #HT25A). Elastic fibers and mast cell granules are stained black/purple, collagen in red, and cytoplasm and muscle in yellow. Mesenteric arteries were visualized under an Axio Observer-Z1 Zeiss microscope and captured with a color camera under identical conditions of light intensity and exposure time settings. For all sections, total area and area of elastin content were measured on calibrated images at original magnification ×40 using ImageJ software. All data are presented as mean ± SE percentage of elastin content of nine mice per group.

To interrogate some molecular mechanisms underlying altered vascular function in Adipo-MROE mice, we also studied cultured vascular smooth muscle cells (VSMCs) (passages 4 to 6) from control mice (C57/Bl6 mice) mesenteric arteries, as described in detail previously (12). VSMCs were stimulated with H₂O₂ (10⁻⁶ to 10⁻³ mol/L) for 24 h to recapitulate vascular oxidative stress observed in Adipo-MROE mice. In a second set of experiments, VSMCs were incubated with PEG-catalase (1,000 units/mL) or the PKG-1 antagonist (DT-3, 10⁻⁸ mol/L) for 30 min and then stimulated with H₂O₂ (10⁻³ mol/L) for 24 h. The concentrations of antagonists used, which effectively inhibited respective receptors, were based on preliminary dose-response studies (data not shown).

The inhibitors DT-3 and Rp-8-Br-PET-cGMPS used in our studies are both highly potent, permeable, and selective for PKG-1α, and reduced cyclic guanosine monophosphate (cGMP)-stimulated PKG activity (23).

Aldosterone (#10004377), corticosterone (#500651), and angiotensin II (#A05880) concentrations were determined in plasma and ACM from cultured adipocytes from Adipo-MROE and control-MR mice by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. Standard curves were derived using nonconditioned medium, and blank measurement of nonconditioned medium was subtracted from all samples. Blood samples were collected into heparinized tubes, centrifuged at 2,000g at 4°C for 10 min, and plasma was further stored at −80°C until analysis.

Enzymatic activity of Rho kinase was evaluated using a Rho-associated protein kinase (ROCK) Activity Assay Kit (#CSA001; Merck Millipore, Watford, Hertfordshire, U.K.), and the experiments were performed in mesenteric arteries and aortic protein lysates, according to the manufacturer’s instructions.

The Amplex Red assay (Invitrogen, Life Technologies, Paisley, U.K.) involves measurement of H₂O₂ by the horseradish peroxidase–catalyzed oxidation of the colorless and nonfluorescent molecule N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red) to resorufin, which, when excited at 530 nm, strongly emits light at 590 nm. The assay was performed in vessels and adipose tissues from Adipo-MROE and control-MR mice according to the manufacturer’s instructions.

Phosphorylation of insulin receptor substrate 1 (IRS1) at the serine-307 was evaluated using an ELISA kit (Merck Millipore #17-459), and the experiments were performed in mesenteric arteries protein lysates, according to the manufacturer’s instructions. Serine-307 phosphorylation of IRS1 has been shown to mediate insulin resistance (24–26).

Mesenteric arteries, aorta from Adipo-MROE and control-MR mice, and VSMCs were prepared, and Western blotting analysis was performed as previously described (12). Antibodies were as follows: anti–phospho-MYPT1 (Thr-696) (1:1000; #sc-17556, Santa Cruz Biotechnology, Santa Cruz, CA), anti-MYPT1 (H-130) (1:1000, #sc-25618), anti–phospho-ezrin (Thr-567)-radixin (Thr-564)-moesin (Thr-558) (p-ERM, 1:1000; #3142, Cell Signaling Technology, Danvers, MA) and anti-ERM (1:1000, #3142), anti–phospho-MLC 2 (Thr18/Ser19) (1:1000; #3674, Cell Signaling Technology), and anti-MLC (1:1000, #3672), anti–phospho-endothelial nitric oxide (NO) synthase (eNOS) (Ser-1177) (1:1000; #9571, Cell Signaling Technology), anti-eNOS (1:1000, #9572), anti-ROCK isoform 2 (ROCK2; 1:1000; #8236, Cell Signaling Technology), anti-RhoA (26C4) (1:500; #sc-418, Santa Cruz Biotechnology), and anti–PKG-1 (1:1000; 3248, Cell Signaling Technology). Blots were analyzed densitometrically using ImageJ software.

Total RNA was extracted from mature adipocytes, EVAT, PVAT, and mesenteric arteries, as previously described (13,14). Real-time PCR was performed on a 7900HT Fast Real-Time PCR machine (Applied Biosystems) using gene-specific primers to quantify the relative abundance with SYBR Green I as the fluorescent molecule. The primers were designed using software Primer 3 and are listed in Supplementary Table 1. Ubiquitin C (Ubc) housekeeping gene was used as the reference gene for normalization. The relative copies number of the target genes were calculated with the 2^–ΔΔCt method after assessment that PCR efficiency was 100%.

ACh, SNP, Phe, 3-isobutyl-1-methylxanthine (IBMX), insulin, dexamethasone, H₂O₂, N-acetyl-cysteine, and PEG-catalase were obtained from Sigma-Aldrich Ltd. (Dorset, U.K.). DT-3 was obtained from Merck Millipore (Calbiochem Ltd., Nottingham, U.K.). Rp-8-Br-PET-cGMPS was obtained from Tocris (Cederlane Corp., Burlington, Ontario, Canada). Dexamethasone was dissolved in 100% ethanol. IBMX was dissolved in 0.35N KOH. ACh, SNP, Phe, H₂O₂, N-acetyl-cysteine, and insulin were dissolved in distilled water. DT-3, PEG-catalase, and Rp-8-Br-PET-cGMPS were dissolved in DMSO.

Values reported are means ± SE. Differences between groups were assessed with the nonparametric Mann-Whitney test. Vascular reactivity results were assessed with one- and two-way ANOVA with repeated measures, followed by the Bonferroni multiple comparison test, as appropriate. Morphometry analyses were assessed with one-way ANOVA. An independent two-tailed t test assuming equal variances was used for comparing the mean percentage of elastin content between the different groups. Values of P < 0.05 were considered significant.

As previously described (14), conditional MROE for 4 weeks leads to a three- to fourfold increase in MR expression in mature adipocytes isolated from EVAT from Adipo-MROE mice compared with adipocytes from littermate control-MR mice (Supplementary Fig. 2A). MR expression is increased in whole EVAT as well as in PVAT around the mesentery but not in mesenteric arteries (Supplementary Table 2). Gene expression of a known downstream target of aldosterone/MR activation, serum and glucocorticoid regulated kinase 1 (Sgk1), was increased threefold in adipocytes from Adipo-MROE versus control-MR mice but was not changed in mesenteric arteries (Supplementary Fig. 2B), indicating that hMR is functionally active and that its activation is increased in Adipo-MROE mice. Supplementary Table 3 summarizes the general characteristics of the control-MR and Adipo-MROE mice. Systolic blood pressure and heart rate were similar between Adipo-MROE and control-MR mice. Body weight was increased by 21.4% in Adipo-MROE mice. Heart and kidney weights relative to tibia length were comparable between both groups, whereas EVAT weight relative to tibia length was increased by 21.7% in Adipo-MROE mice. Plasma levels of aldosterone, corticosterone, and angiotensin II were similar between Adipo-MROE and control-MR mice. Levels of these hormones in ACM were also similar between the groups (Table 1).

ACh- and insulin-induced vasorelaxation was assessed in mesenteric arteries from Adipo-MROE and control-MR mice. ACh-induced vasorelaxation, indicative of endothelium-dependent vasodilation, and SNP-induced vasorelaxation, indicative of endothelium-independent vasodilation, were similar between the groups (Supplementary Fig. 3A and B). However, the ability of insulin to cause dose-dependent vasorelaxation was significantly more impaired in vascular segments from Adipo-MROE mice than in those from control-MR littermates (15.7 ± 4.4% vs. 30.3 ± 8.3% to 10⁻⁶ mol/L insulin; P < 0.05), indicating an impaired insulin sensitivity in vessels from Adipo-MROE mice (Supplementary Fig. 3C). However, this was improved ex vivo by both inhibitors of ROS, N-acetyl-cysteine or PEG-catalase (Supplementary Fig. 3D). These results were confirmed with increased phosphorylation of IRS1 at serine-307 (+154% from Control-MR) (Supplementary Fig. 3E). IRS1 phosphorylation at serine-307 has been highlighted as a molecular event that causes insulin resistance (25,26). Thus, Adipo-MROE mice displayed vascular insulin resistance.

Maximum responses to a high concentration of potassium chloride (KCl) are identical between control-MR and Adipo-MROE mice (2.2 ± 0.1 vs. 2.1 ± 0.2 mN/mm, respectively; n = 10 mice per group, not significant) (Supplementary Fig. 4A). Phe-induced contraction of arteries with intact endothelium was significantly reduced in Adipo-MROE mice versus control counterparts (Fig. 1A). Similar contractile alterations were observed in Adipo-MROE mice in response to increasing doses of noradrenaline and 5-HT in which the endothelium had been mechanically denuded (Fig. 1B and Supplementary Fig. 4B and C). The difference in contractile responses between the experimental and control groups was greater when the endothelium was denuded (Phe 10⁻⁵ mol/L, mN/mm: control-MR + endothelium, 2.5 ± 0.2; Adipo-MROE + endothelium, 2.0 ± 0.1, Δ = 0.5; control-MR − endothelium, 2.7 ± 0.2; Adipo-MROE − endothelium, 1.7 ± 0.3, Δ = 1.0; n = 8–10 mice per group, P < 0.05).

The experimental evidence supports a pathological effect of PVAT on the vasculature with increasing adiposity. To test whether this is the case in our conditional model that displays visceral adiposity, we examined the effect of adipose tissue–secreted factors using mouse ACM (from EVAT) on contractility of mesenteric arteries of control-MR or Adipo-MROE mice. Contractile responses to Phe are decreased by ACM from control-MR mice but not by the ACM from Adipo-MROE mice (Fig. 1C and D), suggesting that ACM from Adipo-MROE loses its anticontractile effect. Interestingly, ACM from Adipo-MROE mice increases contractility in arteries of control-MR by secreting possibly vasoconstrictor factors that influence vascular tone in a paracrine way (Fig. 1C).

Small GTPases of the Rho family influence vascular tone primarily by regulating VSMC contraction. Considering the significant alteration of vascular contractility in Adipo-MROE mice, we explored the possibility that RhoA/ROCK may be involved. ROCK activity was significantly reduced in mesenteric arteries from Adipo-MROE versus control-MR mice (% control-MR: 100 ± 12.9 vs. 41 ± 7.5; n = 7 mice per group, P < 0.01). This was associated with decreased phosphorylation of downstream targets of ROCK signaling, specifically the myosin phosphatase subunit 1 (MYPT1) (Fig. 2A), the ERM (proteins linking the actin cytoskeleton with the plasma membrane) (Fig. 2B), and the myosin light chain (MLC) (Fig. 2C).

Protein levels of RhoA and ROCK2 were similar in experimental and control groups (Supplementary Fig. 5A). To evaluate whether contractile alterations relate to eNOS, we assessed phosphorylation status of eNOS on the active site, serine-1177, which was not significantly different in Adipo-MROE and control mice (Supplementary Fig. 5B).

To investigate molecular mechanisms involved in decreased vascular ROCK activity in mice with adipocytes-targeted MROE, we focused on cGMP-dependent PKG-1, which is activated by ROS such as H₂O₂ in VSMCs and has been shown to attenuate vasoconstriction and promote vasodilation (27,28). As shown in Fig. 3A, expression of PKG-1 was significantly higher in arteries from Adipo-MROE mice compared with controls. To explore potential molecular mechanisms whereby PKG-1 is activated, we focused on diffusible factors, such as H₂O₂, that are involved in regulation of vascular tone. Production of H₂O₂ in ACM from mature adipocytes (EVAT) of Adipo-MROE mice showed a 2.8-fold increase versus control-MR mice, as well as in extracts from EVAT (2.3-fold increase) (Fig. 3B). The accumulation of H₂O₂ in adipose tissue is supported by the 2.5-fold decrease in catalase mRNA levels in EVAT from Adipo-MROE mice (control-MR, 1.3 ± 0.2 vs. Adipo-MROE, 0.52 ± 0.05 arbitrary units; n = 6 mice per group, P < 0.01), whereas Sod (superoxide dismutase) mRNA levels were unchanged (control-MR, 0.9 ± 0.1 vs. Adipo-MROE, 1.22 ± 0.2 arbitrary units; n = 6 mice per group, not significant). Moreover, we evaluated the mRNA levels of NADPH oxidase (Nox) isoforms 1, 2, and 4 to define the source of ROS. Interestingly, Nox-4 mRNA levels are increased, whereas Nox-1 and Nox-2 are unchanged in adipose tissues from Adipo-MROE mice (Supplementary Fig. 6A and B). Thus, increased generation of H₂O₂ by adipocytes of Adipo-MROE mice may influence vascular contractility of Adipo-MROE mice and the RhoA/ROCK system through PKG-1 activation.

To understand the mechanistic basis of the relationship between PKG-1 and ROCK activity in VSMCs exposed to oxidative stress conditions, we examined ROCK activity in primary cultured mouse VSMCs exposed to exogenous H₂O₂. H₂O₂ dose-dependently increased expression of PKG-1 (Fig. 4A). We next analyzed the effect of H₂O₂ on ROCK activity in the absence and presence of a specific inhibitor of PKG-1 (DT-3) or an antioxidant, PEG-catalase. Exogenous H₂O₂ induced a significant decrease in ROCK activity (Fig. 4B) and its downstream signaling (Fig. 4C). This response was reversed when cells were pretreated with DT-3 and PEG-catalase (Fig. 4B), indicating that H₂O₂ is a negative regulator of ROCK activation through processes that involve PKG-1.

To analyze whether modulating PKG-1 expression affects vascular functional responses, we analyzed contractile responses in the presence of Rp-8-Br-PET-cGMPS, a specific inhibitor of PKG-1. Phe-induced vasoconstriction in endothelium-denuded arteries from Adipo-MROE was comparable with control-MR mice when PKG-1 was inhibited (Fig. 5A). Endothelium-dependent relaxation induced by H₂O₂ after PKG-1 inhibition was similar in arteries between Adipo-MROE and control-MR mice (Fig. 5B).

To evaluate potential factors contributing to vascular hypocontractility in Adipo-MROE mice, we examined elasticity and elastin status of mesenteric arteries from the different groups. As shown in Supplementary Fig. 7A, the stress-strain relationship curve was shifted to the right, indicating that the mesenteric arteries from Adipo-MROE mice are more elastic than those from the control-MR mice.

Using elastic Van Gieson stain we show that elastin content was higher, as assessed qualitatively and semiquantitatively, in mesenteric arteries from Adipo-MROE mice compared with the control-MR mice (Supplementary Fig. 7B and C). This is further supported by upregulation of elastin mRNA levels in mesenteric arteries from Adipo-MROE mice compared with the control-MR mice (Supplementary Fig. 7D).

Another molecular mechanism that could be involved in the hypocontractility is the counterregulatory pathway of the renin-angiotensin-aldosterone system, the ACE2/Ang (1-7)/Mas receptor system, which has been reported to contribute to visceral obesity (29–31). Ace2 and Mrga (Mas receptor) mRNA levels were upregulated in EVAT of Adipo-MROE mice by fivefold and twofold, respectively. Moreover, Mrga transcripts levels were increased in mesenteric arteries of Adipo-MROE mice, but not Ace2 (Supplementary Table 4).

Insulin resistance and visceral obesity are commonly associated with the chronic, low-grade inflammatory state. Thus the mechanisms whereby adipose tissue causes alterations in insulin sensitivity could include the secretion of proinflammatory factors by the adipose tissue. Moreover, these adipo(cyto)kines and chemokines would also represent candidates/mediators through which adipose tissue can modulate vascular function.

We evaluated by real-time PCR mRNA levels of specific markers of macrophages. In EVAT and PVAT from Adipo-MROE mice versus control-MR mice, levels of transcripts of F4/80 and Cd-68 were approximately twofold increased, suggesting that adipose tissues from Adipo-MROE contain more macrophages. We then assessed the two activation states of macrophages: M1 (proinflammatory; e.g., interleukin [IL]-6, IL-12, monocyte chemotactic protein 1 [MCP-1], tumor necrosis factor α, regulated on activation, normal T-cell expressed and secreted [RANTES]) and M2 (anti-inflammatory; e.g., adiponectin, IL-10, Cd-206 [mannose receptor]). Interestingly, in EVAT and PVAT from Adipo-MROE, mRNA levels of some proinflammatory markers (Il-6, Mcp-1, and Rantes) are increased, whereas mRNA levels of anti-inflammatory markers are decreased (Adiponectin and Cd-206). Results are summarized in Supplementary Table 5.

We previously demonstrated that MR overactivation, specifically in adipocytes, leads to significant metabolic abnormalities, including increased visceral adiposity, insulin resistance, and dyslipidemia (14). Here, we examined the potential vascular comorbidities that might accompany these metabolic alterations. These studies were prompted by our previous findings where we demonstrated that increased expression of adipocyte MR in obese mice and humans with diabetes was associated with vascular dysfunction (13), but a causal relationship was not established. Using mice that conditionally overexpress MR in an adipocyte-specific manner, we found that vascular contractility was markedly reduced, whereas endothelial function was normal. These phenomena were associated with increased H₂O₂ generation by adipocytes leading to upregulation of redox-sensitive PKG-1 and downregulation of vascular ROCK (Fig. 6). Adipose tissue from Adipo-MROE lost its anticontractile properties, possibly by increasing secretion of contractile factors, as evidenced by our experiments with ACM on contractility of healthy arteries of control-MR. Serine-307 phosphorylation of IRS1 has been highlighted as a molecular event that causes insulin resistance (24,26). In our studies, the increase in phosphorylation IRS1 at serine-307 confirms the insulin resistant phenotype of the Adipo-MROE mice.

Together, these findings indicate that upregulation of adipocyte MR has a significant effect not only on metabolic parameters but also on vascular function. Increasing evidence supports a role for adipocyte MR overactivation in pathophysiological conditions associated with metabolic disorders and cardiovascular diseases; for example:

1. There is increased expression and activation of MR in adipose tissue in experimental and human obesity, diabetes, and cardiovascular diseases (6,14,15,32).

2. MR antagonism with eplerenone or spironolactone improves metabolic disorders and insulin resistance in obese and diabetic mice (9,18,19,33).

3. Adipo-MROE mice exhibit features of metabolic syndrome (14).

4. MR antagonism in obese mice improves vascular function (10,13).

Here, we further support this thesis by showing that increased activation of adipocyte-specific MR influences functional properties of arteries. The vascular phenotype identified in Adipo-MROE mice (which are obese and have features of metabolic syndrome but no hypertension) is unusual because it shows no apparent endothelial dysfunction but exhibits hypocontractility of small arteries. Our transgenic mouse model has some special characteristics that differ from other models of obesity such as the db/db mice. Our model did not display global obesity, did not have increased levels of aldosterone, and did not exhibit altered MR signaling in the vasculature. The mice did, however, show features of hyperelasticity and upregulation of the protective axis of the renin-angiotensin-system, namely Ang-(1-7)/Mas, which may partly explain why the Adipo-MROE mice do not have endothelial dysfunction associated with the weight gain and why the vessels are hypocontractile.

Other models have also demonstrated hypocontractility of vessels in pathological conditions; for example, in portal hypertension, ROS (in particular H₂O₂) levels are increased and vascular contractility is reduced. In Ccl4-induced cirrhotic rats, H₂O₂ has been directly implicated in regulating mesenteric hypocontractility to noradrenaline through the Rho kinase signaling pathway (34). Another study in splanchnic vessels of patients and rats with cirrhosis suggested a role for the ACE2/Ang(1-7)/Mas axis to explain the vascular hypocontractility (35). In our study, Ace2 and Mrga (Mas receptor) gene expression were upregulated in EVAT of Adipo-MROE mice, supporting other studies that have demonstrated hypocontractility (35).

One of the major signaling pathways regulating vascular smooth muscle contraction is the RhoA/ROCK signaling pathway and its downstream targets (36). The contractile state of vascular smooth muscle is driven by phosphorylation of the regulatory protein, MLC, and reflects the balance of the Ca²⁺-calmodulin–dependent MLC kinase and MLC phosphatase activities (37). Phosphorylation of MYPT1 at Thr-696 plays a dominant role in MLC phosphate inhibition and is increased in hypertension (38,39). The ERM proteins are also downstream targets of RhoA and are involved in cytoskeletal organization. We found a significant decrease in ROCK activity and decreased activation of ROCK-dependent signaling proteins, including MLC, MYPT1, and ERM, in vessels from Adipo-MROE mice. Downregulation of vascular RhoA/ROCK signaling may contribute to impaired contractility in an endothelium-independent manner, as previously reported (40–42). This is unusual as well, because in the context of metabolic syndrome, obesity, or diabetes, animals displayed an increase in MR activity associated with increased ROCK activity (6,43,44). In addition, plasma aldosterone levels are increased in obese humans and animals with diabetes, whereas in Adipo-MROE mice, aldosterone plasma levels are unchanged, and MR overactivation is restricted to the adipocytes.

Adipocyte MR overactivation leads to an increase in ROS production by adipocytes, which is also evident in adipose tissue from obese mice (45) and humans (46). Functionally, increased H₂O₂ has been associated with vasoconstriction (47–49) or vasodilation (50–52) through activation of PKG-1. Activation of vascular PKG-1 was increased in Adipo-MROE mice, which may also affect dysregulated VSMC contractility in these mice. Indeed, pharmacological inhibition of PGK-1 activity blunted mesenteric hypocontractility in Adipo-MROE mice. Hence, we demonstrated a direct role of MR-dependent ROS production by adipocytes on the vasculature. In vitro studies in cultured VSMCs indicated that exposure to H₂O₂ induced significant downregulation of ROCK and increased activation of PKG-1 pathways involved in VSMC contraction/dilation. The ex vivo studies recapitulated what we observed in Adipo-MROE mice, unraveling a key mechanism underlying the cross talk between adipose tissue and vascular function.

Putative mechanisms whereby adipocyte MR-induced H₂O₂ generation regulates vascular function through PKG-1 likely involve cGMP because PKG-1 regulates NOS activation and NO production, which in turn activates guanylyl cyclase and results in elevated cGMP, a potent regulator of VSMC dilation (53). In addition to this classical pathway, recent evidence indicates that H₂O₂ can directly activate cGMP via compound I, which activates PKG-1 and results in changes in activity or function of various serine/threonine proteins (54–56). Moreover, H₂O₂-dependent oxidation/activation of PKG-1 is associated with vasodilation (57). Accordingly, cGMP may be a possible link between adipocyte-derived H₂O₂ and PKG-1–associated changes in vascular function.

In summary, our in vivo study demonstrates that mice with conditional upregulation of adipocyte MR, which have metabolic syndrome and obesity, exhibit impaired vascular contractility through processes that involve adipocyte-derived H₂O₂, which influences vascular redox-sensitive PKG-1 and Rho kinase pathways. Mechanisms associated with these vascular changes may relate to increased elastin content and upregulation of the protective axis of the renin-angiotensin system. Our study further highlights important cross talk between adipocytes and vascular cells and indicates that activation of the adipocyte MR system leads to production of vasoactive adipocyte-derived factors, such as H₂O₂, which may affect vascular dysfunction in conditions associated with metabolic disorders. These novel findings emphasize the functional importance of adipocyte MR in cardiovascular complications associated with obesity/adiposity, insulin resistance, and metabolic syndrome.

Acknowledgments. The authors thank Elisabeth Beattie, Carol Jenkins, and Andrew Carswell, from Cardiovascular Research and Medical Sciences Institute, University of Glasgow, Glasgow, U.K., for their technical assistance.

Funding. This work was supported by grants from the British Heart Foundation (RG/13/7/30099, CH/12/429762) and the Canadian Institutes of Health Research (CIHR 44018). A.N.D.C. held a postdoctoral fellowship from the CIHR. F.J. was supported through INSERM and Fondation de France (2014-00047968). R.M.T. was supported through a Canada Research Chair/Canadian Foundation for Innovation award and a British Heart Foundation Chair. R.M.T. and F.J. benefited from support of the European COST-ADMIRE 1301 network.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. A.N.D.C. designed and performed experiments, analyzed data, interpreted results of experiments, prepared figures and tables, and drafted, edited, revised, and approved the final version of the manuscript. T.T.A., G.E.C., and A.S. performed experiments, analyzed data, interpreted results of experiments, and reviewed and approved the final version of the manuscript. S.T., M.G.D.-L., A.A., and Y.H. performed experiments and reviewed and approved the final version of the manuscript. A.C.M., F.J., and R.M.T. provided guidance on design of experiments, interpreted results of experiments, and reviewed and approved the final version of the manuscript. R.M.T. is the guarantor of this work and, as such, had full access to all the data in this study and takes responsibility for the integrity of the data and the accuracy of the data analysis.