Aurelie Nguyen Dinh Cat,1 Tayze T. Antunes,2 Glaucia E. Callera,2 Ana Sanchez,3

Sofia Tsiropoulou,1 Maria G. Dulak-Lis,1 Aikaterini Anagnostopoulou,1 Ying He,2

Augusto C. Montezano,1 Frederic Jaisser,4,5 and Rhian M. Touyz1,2

Adipocyte-Specific MineralocorticoidReceptor Overexpression in Mice IsAssociated With Metabolic Syndromeand Vascular Dysfunction: Role ofRedox-Sensitive PKG-1 and RhoKinaseDiabetes 2016;65:2392–2403 | DOI: 10.2337/db15-1627

Mineralocorticoid receptor (MR) expression is increasedin adipose tissue from obese individuals and animals.We previously demonstrated that adipocyte-MR over-expression (Adipo-MROE) in mice is associated with meta-bolic changes. Whether adipocyte MR directly influencesvascular function in these mice is unknown. We tested thishypothesis in resistant mesenteric arteries from Adipo-MROE mice using myography and in cultured adipocytes.Molecular mechanisms were probed in vessels/vascularsmooth muscle cells and adipose tissue/adipocytes andfocused on redox-sensitive pathways, Rho kinase ac-tivity, and protein kinase G type-1 (PKG-1) signaling.Adipo-MROE versus control-MR mice exhibited reducedvascular contractility, associated with increased genera-tion of adipocyte-derived hydrogen peroxide, activationof vascular redox-sensitive PKG-1, and downregulationof Rho kinase activity. Associated with these vascularchanges was increased elastin content in Adipo-MROE.Inhibition of PKG-1 with Rp-8-Br-PET-cGMPS normalizedvascular contractility in Adipo-MROE. In the presence ofadipocyte-conditioned culture medium, anticontractileeffects of the adipose tissue were lost in Adipo-MROEmice but not in control-MRmice. In conclusion, adipocyte-MR upregulation leads to impaired contractility with pre-served endothelial function and normal blood pressure.

Increased elasticity may contribute to hypocontractility.We also identify functional cross talk between adipo-cyte MR and arteries and describe novel mechanismsinvolving redox-sensitive PKG-1 and Rho kinase. Ourresults suggest that adipose tissue from Adipo-MROEsecrete vasoactive factors that preferentially influ-ence vascular smooth muscle cells rather than endo-thelial cells. Our findings may be important in obesity/adiposity where adipocyte-MR expression/signalingis amplified and vascular risk increased.

Aldosterone classically acts via the renal epithelial miner-alocorticoid receptors (MR), leading to salt and volume ho-meostasis and, consequently, blood pressure regulation. MRis also expressed in nonepithelial cells, including cardiomyo-cytes (1), vascular cells (2,3) and macrophages (4,5), and hasbeen implicated in pathological conditions such as heartfailure, endothelial dysfunction, hypertension, insulinresistance, and obesity (6–10). Recently, MR has beenidentified in adipocytes (11–14). The (patho)-physiologicalsignificance of adipocyte MR is unclear, although experi-mental and human studies have demonstrated increasedexpression of adipocyte MR in obesity, metabolic syndrome,and diabetes (6,14,15). Adipose tissue has long been

1Cardiovascular Research and Medical Sciences Institute, University of Glasgow,Glasgow, U.K.2Kidney Research Centre, Ottawa Hospital Research Institute, University of Ot-tawa, Ottawa, Ontario, Canada3Departamento de Fisiología, Facultad de Farmacia, Universidad Complutense,Madrid, Spain4INSERM Unit 1138 Team 1, Centre de Recherche des Cordeliers, UniversityPierre and Marie Curie, Paris, France5INSERM, Clinical Investigation Centre 1430, APHP, Henri Mondor Hospital, PoleVERDI, Paris East University, Creteil, France

Corresponding authors: Aurelie Nguyen Dinh Cat, cattuong.ndc@gmail.com, andRhian M. Touyz, rhian.touyz@glasgow.ac.uk.

Received 1 December 2015 and accepted 9 April 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1627/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

See accompanying article, p. 2127.

2392 Diabetes Volume 65, August 2016

PATHOPHYSIO

LOGY

considered primarily as simple fat storage of triglyceridesto regulate several important functions, such as energybalance and thermogenesis, but is now recognized as ametabolically active endocrine/paracrine/autocrine organthat synthesizes, stores, and secretes hormones, vasoactivefactors, and proteins, termed adipo(cyto)kines (16,17). MRmediates effects of aldosterone and glucocorticoids, whichin adipocytes regulate adipogenesis, adipocyte maturation,and adipokines production (12,18–20). We recently dem-onstrated that adipocytes possess functionally active MRand have the machinery to produce aldosterone, processesthat are increased in obesity-associated diabetes and in-fluence vascular function (6,12,13).

To better understand the relevance of the adipocyteMR, we generated a conditional transgenic mouse modelallowing restricted and inducible MR overexpression (MROE)in an adipocyte-specific manner. These mice exhibit featuresof metabolic syndrome, including weight gain, insulinresistance, and dyslipidemia, with increased activation ofprostaglandin D2 synthase (14). Considering the growingevidence that adipose tissue and adipokines influencevascular tone and that the aldosterone/MR system isinvolved in vascular pathology associated with cardiovas-cular disease, we questioned whether upregulation ofadipocyte MR in mice leads to vascular dysfunction.

RESEARCH DESIGN AND METHODS

The study was approved by the University of OttawaAnimal Ethics Committee. All studies in animals wereconducted in accordance with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals.All procedures followed were in accordance with institu-tional guidelines.

Generation of Mice Expressing Human MRin AdipocytesWe generated mice with targeted overexpression of humanMR (hMR) in adipocytes using the conditional tetracycline-inducible system, as we recently reported (14) and summa-rized in Supplementary Fig. 1A and B. Double-transgenicadipocyte MROE (Adipo-MROE) mice were obtained bymating two strains: the monotransgenic tetO-hMR mouseand the monotransgenic transactivator aP2-rtTA mouse.In Adipo-MROE mice, hMR expression is switched onin adipocytes by administering doxycycline (Dox) (2 g/Lfor 4 weeks) (200 mg/kg Dox diet; Harlan Laboratories,Mississauga, Ontario, Canada). The litters consisting ofAdipo-MROE, monotransgenic tetO-hMR, monotransgenicaP2-rtTA, and wild-type mice were born in the expectedmendelian ratio (25% for each genotype). The last threegroups, which displayed no molecular or functional differ-ences among them, were considered as controls (control-MR). Blood pressure was measured by the tail-cuff methodin 2- to 3-month-old mice. Mice were killed to recovermesenteric arteries and adipose tissues used for the sub-sequent manipulations of cell culture, histology, or bio-molecular analysis.

Isolation and Culture of Mature Adipocytes FromMouse Adipose Tissue

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

Conditioned Culture MediumThe conditioned culture medium, collected from ma-ture adipocytes (ACM) 24 h after isolation (12,21), wasconcentrated in a speed vacuum system (Vacufuge,Eppendorf) and kept at 220°C for further determinationof aldosterone, corticosterone, angiotensin II, and hydro-gen peroxide (H2O2) levels.

Vascular StudiesSecond-order branches of mesenteric artery without peri-vascular fat (PVAT) were isolated from 3- to 4-month-oldAdipo-MROE and their littermate control-MR miceand mounted on a wire myograph (DMT myograph;ADInstruments Ltd., Oxford, U.K.) as previously de-scribed (13). Endothelium-dependent and -independentrelaxations were assessed by measuring dilatory responsesto acetylcholine (Ach) (1029 to 1025 mol/L) and sodiumnitroprusside (SNP) (1029 to 1025 mol/L), respectively,in arteries precontracted with phenylephrine (Phe) tosimilar degrees to achieve ;80% of maximal response.Dose-response curves for insulin-induced relaxation (10210

to 1025 mol/L) were also obtained in arteries with intactendothelium in the absence or presence of a generalreactive oxygen species (ROS) scavenger, N-acetyl-cysteine(1026 mol/L) or the H2O2 scavenger, polyethylene glycol(PEG)-catalase (100 units/mL), incubated 30 min beforeprecontraction with Phe. Contraction curves to cumulativeincreasing doses of Phe, noradrenaline, and serotonin(5-hydroxytryptamine [5-HT]; 1029 to 1025 mol/L) wereperformed in arteries with and without intact endothelium.

Healthy PVAT and other fat depots display a protectiveanticontractile effect on the vasculature that is lost inobesity. This anticontractile property remained with thetransfer of the culture bath to vessels without adipose tissueand suggested the action of secreted relaxing substances. Inthat way, we investigated the effects of visceral fat usingACM (from EVAT) from control-MR and Adipo-MROE miceon contractile responses to Phe. A small piece of EVAT isincubated in 10 mL of Krebs solution 30 min before theconcentration-response curve to Phe. It has been suggestedthat PVAT behaves similarly to visceral fat in secretion ofvasoactive factors (22). We also evaluated the effects of aspecific inhibitor of protein kinase G type 1 (PKG-1) onvascular reactivity. Arteries were pretreated with Rp-8-Br-PET-cGMPS (3 3 1025 mol/L) or vehicle (DMSO) for30 min before exposing vessels to Phe (endothelium-denuded mesenteric arteries) or H2O2 (10

26 to 1023 mol/L)(endothelium-intact arteries).

diabetes.diabetesjournals.org Nguyen Dinh Cat and Associates 2393

Arterial stiffness was studied with a pressure myo-graph (Living Systems, Burlington, VT), as previouslydescribed (13). The artery was set to an internal pressureof 45 mmHg, pressure-fixed (30 min, 10% formalin, at37°C), and stored embedded in paraffin until histomorph-ometry studies. Vascular structure and mechanical parame-ters were calculated as previously described (13).

Elastin Content Assessment by Elastic Van GiesonDeparaffinized sections (5 mm) were rehydrated in sequen-tial alcohol baths and washed in distilled water. For elastincontent, tissue sections were stained with Miller’s Elastin(VWR, Cat. #351154S) and Van Gieson solutions (Sigma-Aldrich, Cat. #HT25A). Elastic fibers and mast cell granulesare stained black/purple, collagen in red, and cytoplasmand muscle in yellow. Mesenteric arteries were visualizedunder an Axio Observer-Z1 Zeiss microscope and capturedwith a color camera under identical conditions of light in-tensity and exposure time settings. For all sections, totalarea and area of elastin content were measured on cali-brated images at original magnification 340 using ImageJsoftware. All data are presented as mean 6 SE percentageof elastin content of nine mice per group.

Cell Culture of Mouse Vascular Smooth Muscle CellsTo interrogate some molecular mechanisms underlyingaltered vascular function in Adipo-MROE mice, we alsostudied cultured vascular smooth muscle cells (VSMCs)(passages 4 to 6) from control mice (C57/Bl6 mice) mesen-teric arteries, as described in detail previously (12). VSMCswere stimulated with H2O2 (10

26 to 1023 mol/L) for 24 hto recapitulate vascular oxidative stress observed in Adipo-MROE mice. In a second set of experiments, VSMCs wereincubated with PEG-catalase (1,000 units/mL) or the PKG-1antagonist (DT-3, 1028 mol/L) for 30 min and then stimu-lated with H2O2 (10

23 mol/L) for 24 h. The concentrationsof antagonists used, which effectively inhibited respectivereceptors, were based on preliminary dose-response studies(data not shown).

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

Enzymatic Immunoassays

Aldosterone, Corticosterone, and Angiotensin IIMeasurementsAldosterone (#10004377), corticosterone (#500651), andangiotensin II (#A05880) concentrations were determinedin plasma and ACM from cultured adipocytes from Adipo-MROE and control-MR mice by enzyme immunoassay(Cayman Chemical, Ann Arbor, MI) according to themanufacturer’s instructions. Standard curves were de-rived using nonconditioned medium, and blank measure-ment of nonconditioned medium was subtracted from allsamples. Blood samples were collected into heparinizedtubes, centrifuged at 2,000g at 4°C for 10 min, and plasmawas further stored at 280°C until analysis.

Rho Kinase ActivityEnzymatic activity of Rho kinase was evaluated using aRho-associated protein kinase (ROCK) Activity Assay Kit(#CSA001; Merck Millipore, Watford, Hertfordshire,U.K.), and the experiments were performed in mesentericarteries and aortic protein lysates, according to the man-ufacturer’s instructions.

Amplex Red AssayThe Amplex Red assay (Invitrogen, Life Technologies, Paisley,U.K.) involves measurement of H2O2 by the horseradishperoxidase–catalyzed oxidation of the colorless and non-fluorescent 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 per-formed in vessels and adipose tissues from Adipo-MROEand control-MR mice according to the manufacturer’sinstructions.

Phosphorylation of Insulin Receptor Substrate 1Phosphorylation of insulin receptor substrate 1 (IRS1) atthe serine-307 was evaluated using an ELISA kit (MerckMillipore #17-459), and the experiments were performedin mesenteric arteries protein lysates, according to themanufacturer’s instructions. Serine-307 phosphorylation ofIRS1 has been shown to mediate insulin resistance (24–26).

Western Blot AnalysisMesenteric arteries, aorta from Adipo-MROE and control-MR mice, and VSMCs were prepared, and Western blottinganalysis was performed as previously described (12). Anti-bodies 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) andanti-ERM (1:1000, #3142), anti–phospho-MLC 2 (Thr18/Ser19) (1:1000; #3674, Cell Signaling Technology), andanti-MLC (1:1000, #3672), anti–phospho-endothelial nitricoxide (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 Signal-ing Technology), anti-RhoA (26C4) (1:500; #sc-418, SantaCruz Biotechnology), and anti–PKG-1 (1:1000; 3248, CellSignaling Technology). Blots were analyzed densitometri-cally using ImageJ software.

Quantitative Real-Time PCRTotal 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-TimePCR machine (Applied Biosystems) using gene-specific pri-mers to quantify the relative abundance with SYBR Green Ias the fluorescent molecule. The primers were designedusing software Primer 3 and are listed in SupplementaryTable 1. Ubiquitin C (Ubc) housekeeping gene was used asthe reference gene for normalization. The relative copiesnumber of the target genes were calculated with the 2–DDCt

method after assessment that PCR efficiency was 100%.

2394 Adipocyte MR Induces Vascular Dysfunction Diabetes Volume 65, August 2016

Drugs and SolutionsACh, SNP, Phe, 3-isobutyl-1-methylxanthine (IBMX), in-sulin, dexamethasone, H2O2, N-acetyl-cysteine, and PEG-catalase were obtained from Sigma-Aldrich Ltd. (Dorset,U.K.). DT-3 was obtained from Merck Millipore (CalbiochemLtd., Nottingham, U.K.). Rp-8-Br-PET-cGMPS was obtainedfrom Tocris (Cederlane Corp., Burlington, Ontario, Canada).Dexamethasone was dissolved in 100% ethanol. IBMX wasdissolved 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 inDMSO.

Data AnalysisValues reported are means 6 SE. Differences betweengroups were assessed with the nonparametric Mann-Whitneytest. Vascular reactivity results were assessed with one-and two-way ANOVA with repeated measures, followed bythe Bonferroni multiple comparison test, as appropriate.Morphometry analyses were assessed with one-way ANOVA.An independent two-tailed t test assuming equal varianceswas used for comparing the mean percentage of elastincontent between the different groups. Values of P , 0.05were considered significant.

RESULTS

Conditional MROE in Adipocytes in MiceAs previously described (14), conditional MROE for 4 weeksleads to a three- to fourfold increase in MR expression inmature adipocytes isolated from EVAT from Adipo-MROEmice compared with adipocytes from littermate control-MRmice (Supplementary Fig. 2A). MR expression is increased inwhole EVAT as well as in PVAT around the mesentery butnot in mesenteric arteries (Supplementary Table 2). Geneexpression 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 mes-enteric arteries (Supplementary Fig. 2B), indicating that hMRis functionally active and that its activation is increased inAdipo-MROE mice. Supplementary Table 3 summarizes thegeneral characteristics of the control-MR and Adipo-MROEmice. Systolic blood pressure and heart rate were similarbetween Adipo-MROE and control-MR mice. Body weightwas increased by 21.4% in Adipo-MROE mice. Heart andkidney weights relative to tibia length were comparable be-tween both groups, whereas EVAT weight relative to tibialength was increased by 21.7% in Adipo-MROE mice. Plasmalevels of aldosterone, corticosterone, and angiotensin IIwere similar between Adipo-MROE and control-MR mice.Levels of these hormones in ACM were also similar be-tween the groups (Table 1).

Endothelial Function and Vascular Contractilityin Adipo-MROE Mice

Vasodilatory ResponsesACh- and insulin-induced vasorelaxation was assessed inmesenteric 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. 3Aand B). However, the ability of insulin to cause dose-dependent vasorelaxation was significantly more impairedin vascular segments from Adipo-MROE mice than in thosefrom control-MR littermates (15.7 6 4.4% vs. 30.3 68.3% to 1026 mol/L insulin; P , 0.05), indicating animpaired insulin sensitivity in vessels from Adipo-MROEmice (Supplementary Fig. 3C). However, this was improvedex vivo by both inhibitors of ROS, N-acetyl-cysteine orPEG-catalase (Supplementary Fig. 3D). These results wereconfirmed with increased phosphorylation of IRS1 at ser-ine-307 (+154% from Control-MR) (Supplementary Fig.3E). IRS1 phosphorylation at serine-307 has been high-lighted as a molecular event that causes insulin resistance(25,26). Thus, Adipo-MROE mice displayed vascular insulinresistance.

Contractile ResponsesMaximum responses to a high concentration of potassiumchloride (KCl) are identical between control-MR andAdipo-MROE mice (2.2 6 0.1 vs. 2.1 6 0.2 mN/mm,respectively; n = 10 mice per group, not significant) (Supple-mentary Fig. 4A). Phe-induced contraction of arteries withintact endothelium was significantly reduced in Adipo-MROEmice versus control counterparts (Fig. 1A). Similar con-tractile alterations were observed in Adipo-MROE micein response to increasing doses of noradrenaline and5-HT in which the endothelium had been mechanicallydenuded (Fig. 1B and Supplementary Fig. 4B and C).The difference in contractile responses between the ex-perimental and control groups was greater when theendothelium was denuded (Phe 1025 mol/L, mN/mm:control-MR + endothelium, 2.5 6 0.2; Adipo-MROE +endothelium, 2.06 0.1, D = 0.5; control-MR2 endothelium,2.7 6 0.2; Adipo-MROE 2 endothelium, 1.7 6 0.3, D = 1.0;n = 8–10 mice per group, P , 0.05).

The experimental evidence supports a pathological effectof PVAT on the vasculature with increasing adiposity. Totest whether this is the case in our conditional model

Table 1—Aldosterone, corticosterone, and angiotensin IIlevels in plasma and ACM from EVAT from Adipo-MROE andlittermate control-MR mice

ParametersControl-MR, Adipo-MROE,

n = 7 n = 10

Plasma levelsAldosterone (pg/mL) 432 6 45 388 6 29Corticosterone (ng/mL) 1,064 6 86 1,105 6 84Angiotensin II (pg/mL) 476 6 59 573.6 6 70

ACM levels (pg/mL/mg RNA)Aldosterone 272 6 48 347 6 96Corticosterone 2,088 6 378 2,297 6 403Angiotensin II 852 6 295 705 6 217

Values are means 6 SE.

diabetes.diabetesjournals.org Nguyen Dinh Cat and Associates 2395

that displays visceral adiposity, we examined the effectof adipose tissue–secreted factors using mouse ACM(from EVAT) on contractility of mesenteric arteries ofcontrol-MR or Adipo-MROE mice. Contractile responsesto Phe are decreased by ACM from control-MR mice butnot by the ACM from Adipo-MROE mice (Fig. 1C and D),suggesting that ACM from Adipo-MROE loses its anti-contractile effect. Interestingly, ACM from Adipo-MROEmice increases contractility in arteries of control-MR bysecreting possibly vasoconstrictor factors that influencevascular tone in a paracrine way (Fig. 1C).

Adipocyte-Specific MROE Is Associated WithDownregulation of Vascular Rho Kinase SignalingSmall GTPases of the Rho family influence vascular toneprimarily by regulating VSMC contraction. Consideringthe significant alteration of vascular contractility in Adipo-MROE mice, we explored the possibility that RhoA/ROCKmay be involved. ROCK activity was significantly reducedin mesenteric arteries from Adipo-MROE versus control-MR mice (% control-MR: 100 6 12.9 vs. 41 6 7.5; n = 7mice per group, P , 0.01). This was associated with de-creased phosphorylation of downstream targets of ROCK

signaling, specifically the myosin phosphatase subunit 1(MYPT1) (Fig. 2A), the ERM (proteins linking the actincytoskeleton with the plasma membrane) (Fig. 2B), andthe myosin light chain (MLC) (Fig. 2C).

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

Adipocyte-Specific MROE Is Associated WithActivation of cGMP-Dependent PKG-1 and IncreasedLevels of Vascular ROS ProductionTo investigate molecular mechanisms involved in de-creased 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 hasbeen shown to attenuate vasoconstriction and promotevasodilation (27,28). As shown in Fig. 3A, expression ofPKG-1 was significantly higher in arteries from Adipo-MROE mice compared with controls. To explore potentialmolecular mechanisms whereby PKG-1 is activated, we

Figure 1—Effect of adipocyte-conditioned medium on contractility of mesenteric arteries from Adipo-MROE and control-MR mice. Con-tractile responses to cumulative and increasing doses of Phe (1029 to 1025 mol/L) in mesenteric arteries with (A) and without (B) intactendothelium were significantly decreased in Adipo-MROE vs. control-MR mice. Effect of ACM on contractile responses to Phe of arteriesfrom control-MR mice (C) and from Adipo-MROE mice (D). For A–D, the two-way ANOVA test was performed, followed by the Bonferronitest. Data were performed in repeated measures and are presented as mean6 SE (n = 8 to 10 mice per group). *P< 0.05, Adipo-MROE vs.control-MR. †P < 0.05, without ACM vs. ACM (control-MR). ‡P < 0.05, ACM (control-MR) vs. ACM (Adipo-MROE).

2396 Adipocyte MR Induces Vascular Dysfunction Diabetes Volume 65, August 2016

focused on diffusible factors, such as H2O2, that are in-volved in regulation of vascular tone. Production of H2O2

in ACM from mature adipocytes (EVAT) of Adipo-MROEmice 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 sup-ported by the 2.5-fold decrease in catalase mRNA levels inEVAT from Adipo-MROE mice (control-MR, 1.3 6 0.2 vs.Adipo-MROE, 0.52 6 0.05 arbitrary units; n = 6 miceper group, P , 0.01), whereas Sod (superoxide dismutase)mRNA levels were unchanged (control-MR, 0.9 6 0.1 vs.Adipo-MROE, 1.22 6 0.2 arbitrary units; n = 6 mice pergroup, not significant). Moreover, we evaluated themRNA levels of NADPH oxidase (Nox) isoforms 1, 2,and 4 to define the source of ROS. Interestingly, Nox-4mRNA levels are increased, whereas Nox-1 and Nox-2 areunchanged in adipose tissues from Adipo-MROE mice(Supplementary Fig. 6A and B). Thus, increased genera-tion of H2O2 by adipocytes of Adipo-MROE mice mayinfluence vascular contractility of Adipo-MROE mice andthe RhoA/ROCK system through PKG-1 activation.

Exogenous H2O2 Leads to Downregulationof ROCK ActivityTo understand the mechanistic basis of the relationshipbetween PKG-1 and ROCK activity in VSMCs exposed tooxidative stress conditions, we examined ROCK activity inprimary 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 activityin 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 wasreversed when cells were pretreated with DT-3 and PEG-catalase (Fig. 4B), indicating that H2O2 is a negative reg-ulator of ROCK activation through processes that involvePKG-1.

PKG-1 Inhibition Restores Normal Contraction inAdipo-MROE MiceTo analyze whether modulating PKG-1 expression af-fects vascular functional responses, we analyzed contrac-tile responses in the presence of Rp-8-Br-PET-cGMPS, aspecific inhibitor of PKG-1. Phe-induced vasoconstrictionin endothelium-denuded arteries from Adipo-MROE wascomparable with control-MR mice when PKG-1 wasinhibited (Fig. 5A). Endothelium-dependent relaxation in-duced by H2O2 after PKG-1 inhibition was similar in arter-ies between Adipo-MROE and control-MR mice (Fig. 5B).

Elasticity and Elastin Content Are Increased inMesenteric Arteries From Adipo-MROE MiceTo evaluate potential factors contributing to vascular hypo-contractility in Adipo-MROE mice, we examined elasticityand elastin status of mesenteric arteries from the differentgroups. As shown in Supplementary Fig. 7A, the stress-strain relationship curve was shifted to the right, indicatingthat the mesenteric arteries from Adipo-MROE mice aremore elastic than those from the control-MR mice.

Using elastic Van Gieson stain we show that elastin con-tent was higher, as assessed qualitatively and semiquanti-tatively, in mesenteric arteries from Adipo-MROE micecompared with the control-MR mice (Supplementary Fig. 7Band C). This is further supported by upregulation of elastinmRNA levels in mesenteric arteries from Adipo-MROE

Figure 2—ROCK signaling in mesenteric arteries from Adipo-MROE and control-MR mice. Downregulation of downstream targets of RhoA/Rho kinase activation. MYPT1 (A), ERM (B), and MLC20 (C) protein phosphorylations (p) were significantly decreased in mesenteric arteries fromAdipo-MROE vs. control-MRmice. Western blotting was performed usingMypt1 and p-Mypt1 (Thr696), ERM, and p-ERM (Thr567, Thr564, Thr558),MLC20, and p-MLC20 (Thr18/Ser19) antibodies. Integrated intensities were obtained by using ImageJ software. For A–C: The Mann-Whitneynonparametric test was performed. Data represent means 6 SE (n = 10 mice per group). *P < 0.05, **P < 0.01, Adipo-MROE vs. control-MR.

diabetes.diabetesjournals.org Nguyen Dinh Cat and Associates 2397

mice compared with the control-MR mice (SupplementaryFig. 7D).

Upregulation of ACE2/Ang (1-7)/Mas System inAdipo-MROE MiceAnother molecular mechanism that could be involved inthe hypocontractility is the counterregulatory pathway ofthe renin-angiotensin-aldosterone system, the ACE2/Ang(1-7)/Mas receptor system, which has been reported tocontribute to visceral obesity (29–31). Ace2 and Mrga (Masreceptor) mRNA levels were upregulated in EVAT ofAdipo-MROE mice by fivefold and twofold, respectively.

Moreover, Mrga transcripts levels were increased in mes-enteric arteries of Adipo-MROE mice, but not Ace2 (Sup-plementary Table 4).

Proinflammatory Phenotype in Adipose Tissue FromAdipo-MROE MiceInsulin resistance and visceral obesity are commonly asso-ciated with the chronic, low-grade inflammatory state. Thusthe mechanisms whereby adipose tissue causes alterationsin insulin sensitivity could include the secretion of proin-flammatory factors by the adipose tissue. Moreover, theseadipo(cyto)kines and chemokines would also represent

Figure 3—Adipocyte-specific MROE: increased vascular PKG-1 protein levels and H2O2 production by adipocytes. A: PKG-1 protein levelsare upregulated in mesenteric arteries from Adipo-MROE vs. control-MR mice. B: Levels of H2O2 in adipocytes culture medium and in EVATfrom Adipo-MROE vs. control-MR mice. For A and B: The Mann-Whitney nonparametric test was performed. Data represent means 6 SE(n = 6 to 7 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, Adipo-MROE vs. control-MR.

2398 Adipocyte MR Induces Vascular Dysfunction Diabetes Volume 65, August 2016

candidates/mediators through which adipose tissue canmodulate vascular function.

We evaluated by real-time PCR mRNA levels of specificmarkers of macrophages. In EVAT and PVAT from Adipo-MROE mice versus control-MR mice, levels of transcriptsof F4/80 and Cd-68 were approximately twofold increased,suggesting that adipose tissues from Adipo-MROE containmore macrophages. We then assessed the two activationstates of macrophages: M1 (proinflammatory; e.g., inter-leukin [IL]-6, IL-12, monocyte chemotactic protein 1[MCP-1], tumor necrosis factor a, regulated on activa-tion, normal T-cell expressed and secreted [RANTES])

and M2 (anti-inflammatory; e.g., adiponectin, IL-10, Cd-206 [mannose receptor]). Interestingly, in EVAT andPVAT from Adipo-MROE, mRNA levels of some proin-flammatory markers (Il-6, Mcp-1, and Rantes) are increased,whereas mRNA levels of anti-inflammatory markers aredecreased (Adiponectin and Cd-206). Results are summa-rized in Supplementary Table 5.

DISCUSSION

We previously demonstrated that MR overactivation, spe-cifically in adipocytes, leads to significant metabolic abnor-malities, including increased visceral adiposity, insulin

Figure 4—Exogenous H2O2 increases expression of PKG-1 and decreases activity of ROCK in mouse VSMCs. A: Dose-response curve.VSMCs from mouse mesenteric arteries were stimulated with H2O2 (1026 to 1023 mol/L) for 24 h. B: VSMCs from mouse mesentericarteries were stimulated with H2O2 (10

23 mol/L) in the absence and presence of a specific PKG-1 inhibitor or an antioxidant for 24 h: ROCKkinase activity in H2O2-stimulated VSMCs. Values are expressed as means 6 SE (n = 8 individual experiments). C: ROCK signaling inVSMCs. Protein levels of a marker of ROCK activation, ERM phosphorylation (p), are significantly decreased by H2O2 (1023 mol/L).Antioxidant: PEG-catalase, 1,000 units/mL; PKG-1 inhibitor: DT-3, 1028 mol/L. Veh, vehicle. For A–C: The one-way ANOVA test wasperformed, followed by the Bonferroni test. Data represent means 6 SE (n = 8 individual experiments). *P < 0.05, **P < 0.01, ***P < 0.001vs. vehicle; ††P < 0.01 vs. H2O2.

diabetes.diabetesjournals.org Nguyen Dinh Cat and Associates 2399

resistance, and dyslipidemia (14). Here, we examined thepotential vascular comorbidities that might accompanythese metabolic alterations. These studies were promptedby our previous findings where we demonstrated that in-creased expression of adipocyte MR in obese mice and hu-mans with diabetes was associated with vascular dysfunction(13), but a causal relationship was not established. Usingmice that conditionally overexpress MR in an adipocyte-specific manner, we found that vascular contractility wasmarkedly reduced, whereas endothelial function was normal.These phenomena were associated with increased H2O2 gen-eration by adipocytes leading to upregulation of redox-sen-sitive PKG-1 and downregulation of vascular ROCK (Fig. 6).Adipose tissue from Adipo-MROE lost its anticontractileproperties, possibly by increasing secretion of contractilefactors, as evidenced by our experiments with ACM oncontractility of healthy arteries of control-MR. Serine-307 phosphorylation of IRS1 has been highlighted as amolecular event that causes insulin resistance (24,26). Inour studies, the increase in phosphorylation IRS1 at ser-ine-307 confirms the insulin resistant phenotype of theAdipo-MROE mice.

Together, these findings indicate that upregulation ofadipocyte MR has a significant effect not only on metabolicparameters but also on vascular function. Increasing evi-dence supports a role for adipocyte MR overactivation inpathophysiological conditions associated with metabolicdisorders and cardiovascular diseases; for example:

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

2. MR antagonism with eplerenone or spironolactone im-proves metabolic disorders and insulin resistance inobese and diabetic mice (9,18,19,33).

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

4. MR antagonism in obese mice improves vascular func-tion (10,13).

Here, we further support this thesis by showing thatincreased activation of adipocyte-specific MR influencesfunctional properties of arteries. The vascular phenotype

Figure 5—Effect of PKG-1 inhibition (inh) on vascular reactivity in Adipo-MROE mice. Wire myography was used to evaluate contractile andvasodilatory responses of fat-free mesenteric resistant arteries from Adipo-MROE mice and their control-MR littermates. A: Preincubationwith a specific PKG-1 inhibitor, 30 min before concentration responses curves to Phe (1029 to 1025 mol/L), of endothelium-denudedmesenteric arteries from Adipo-MROE mice restores the contractile responses to levels comparable with control-MR mice. B: Preincuba-tion with a specific PKG-1 inhibitor 30 min before concentration responses curves to H2O2 (10

26 to 1023 mol/L) of mesenteric arteries withintact endothelium has similar effects in relaxant responses between Adipo-MROE and control-MR mice. PKG-1 inhibitor: Rp-8-Br-PET-cGMPS, 3 3 1025 mol/L. For A and B: the two-way ANOVA test was performed, followed by the Bonferroni test. Data were performed inrepeated measures and are presented as mean 6 SE. The parentheses indicate the number of animals per group. *P < 0.05, Adipo-MROEvs. control-MR. †P < 0.05, Adipo-MROE vs. Adipo-MROE+PKG-1 inh.

2400 Adipocyte MR Induces Vascular Dysfunction Diabetes Volume 65, August 2016

identified in Adipo-MROE mice (which are obese and havefeatures of metabolic syndrome but no hypertension) isunusual because it shows no apparent endothelial dys-function but exhibits hypocontractility of small arteries.Our transgenic mouse model has some special character-istics that differ from other models of obesity such asthe db/db mice. Our model did not display global obesity,did not have increased levels of aldosterone, and didnot exhibit altered MR signaling in the vasculature. Themice did, however, show features of hyperelasticity andupregulation of the protective axis of the renin-angiotensin-system, namely Ang-(1-7)/Mas, which may partly explainwhy the Adipo-MROE mice do not have endothelial dys-function associated with the weight gain and why thevessels are hypocontractile.

Other models have also demonstrated hypocontractil-ity of vessels in pathological conditions; for example, inportal hypertension, ROS (in particular H2O2) levels areincreased and vascular contractility is reduced. In Ccl4-induced cirrhotic rats, H2O2 has been directly implicatedin regulating mesenteric hypocontractility to noradrena-line through the Rho kinase signaling pathway (34).Another study in splanchnic vessels of patients and ratswith cirrhosis suggested a role for the ACE2/Ang(1-7)/Masaxis to explain the vascular hypocontractility (35). In ourstudy, Ace2 and Mrga (Mas receptor) gene expression were

upregulated in EVAT of Adipo-MROE mice, supporting otherstudies that have demonstrated hypocontractility (35).

One of the major signaling pathways regulating vascularsmooth muscle contraction is the RhoA/ROCK signalingpathway and its downstream targets (36). The contractilestate of vascular smooth muscle is driven by phosphoryla-tion of the regulatory protein, MLC, and reflects the bal-ance of the Ca2+-calmodulin–dependent MLC kinase andMLC phosphatase activities (37). Phosphorylation of MYPT1at Thr-696 plays a dominant role in MLC phosphate in-hibition and is increased in hypertension (38,39). TheERM proteins are also downstream targets of RhoAand are involved in cytoskeletal organization. We founda significant decrease in ROCK activity and decreasedactivation of ROCK-dependent signaling proteins, in-cluding MLC, MYPT1, and ERM, in vessels from Adipo-MROE mice. Downregulation of vascular RhoA/ROCKsignaling may contribute to impaired contractility in anendothelium-independent manner, as previously report-ed (40–42). This is unusual as well, because in the con-text of metabolic syndrome, obesity, or diabetes, animalsdisplayed an increase in MR activity associated with in-creased ROCK activity (6,43,44). In addition, plasma al-dosterone levels are increased in obese humans andanimals with diabetes, whereas in Adipo-MROE mice,aldosterone plasma levels are unchanged, and MR over-activation is restricted to the adipocytes.

Adipocyte MR overactivation leads to an increase inROS production by adipocytes, which is also evident inadipose tissue from obese mice (45) and humans (46). Func-tionally, increased H2O2 has been associated with vasocon-striction (47–49) or vasodilation (50–52) through activationof PKG-1. Activation of vascular PKG-1 was increased inAdipo-MROE mice, which may also affect dysregulatedVSMC contractility in these mice. Indeed, pharmacologicalinhibition of PGK-1 activity blunted mesenteric hypocon-tractility in Adipo-MROE mice. Hence, we demonstrated adirect role of MR-dependent ROS production by adipo-cytes on the vasculature. In vitro studies in culturedVSMCs indicated that exposure to H2O2 induced signifi-cant downregulation of ROCK and increased activation ofPKG-1 pathways involved in VSMC contraction/dilation.The ex vivo studies recapitulated what we observed inAdipo-MROE mice, unraveling a key mechanism underly-ing the cross talk between adipose tissue and vascularfunction.

Putative mechanisms whereby adipocyte MR-inducedH2O2 generation regulates vascular function throughPKG-1 likely involve cGMP because PKG-1 regulatesNOS activation and NO production, which in turn acti-vates guanylyl cyclase and results in elevated cGMP,a potent regulator of VSMC dilation (53). In additionto this classical pathway, recent evidence indicates thatH2O2 can directly activate cGMP via compound I, whichactivates PKG-1 and results in changes in activity orfunction of various serine/threonine proteins (54–56).Moreover, H2O2-dependent oxidation/activation of PKG-1

Figure 6—Hypothetical scheme of the signaling mechanisms inducedby aldosterone/MR overactivation in adipocytes. Adipocyte-specificMR overactivation leads to decrease in contractility of mesentericarteries from Adipo-MROE vs. control-MR mice. This may involvedownregulation of Rho kinase activity and signaling in arteriesthrough H2O2-induced PKG-1 activation.

diabetes.diabetesjournals.org Nguyen Dinh Cat and Associates 2401

is associated with vasodilation (57). Accordingly, cGMPmay 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, whichhave metabolic syndrome and obesity, exhibit impairedvascular contractility through processes that involveadipocyte-derived H2O2, which influences vascular redox-sensitive PKG-1 and Rho kinase pathways. Mechanismsassociated with these vascular changes may relate to in-creased elastin content and upregulation of the protectiveaxis of the renin-angiotensin system. Our study furtherhighlights important cross talk between adipocytes andvascular cells and indicates that activation of the adipo-cyte MR system leads to production of vasoactive adipocyte-derived factors, such as H2O2, which may affect vasculardysfunction in conditions associated with metabolic dis-orders. These novel findings emphasize the functionalimportance of adipocyte MR in cardiovascular complica-tions 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 SciencesInstitute, University of Glasgow, Glasgow, U.K., for their technical assistance.Funding. This work was supported by grants from the British HeartFoundation (RG/13/7/30099, CH/12/429762) and the Canadian Institutes of HealthResearch (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 forInnovation award and a British Heart Foundation Chair. R.M.T. and F.J. benefitedfrom support of the European COST-ADMIRE 1301 network.Duality of Interest. No potential conflicts of interest relevant to this articlewere 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 resultsof 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 approvedthe final version of the manuscript. A.C.M., F.J., and R.M.T. providedguidance on design of experiments, interpreted results of experiments, andreviewed and approved the final version of the manuscript. R.M.T. is theguarantor of this work and, as such, had full access to all the data in thisstudy and takes responsibility for the integrity of the data and the accuracy ofthe data analysis.

References1. Sainte-Marie Y, Nguyen Dinh Cat A, Perrier R, et al. Conditional glucocor-ticoid receptor expression in the heart induces atrio-ventricular block. FASEB J2007;21:3133–31412. Nguyen Dinh Cat A, Griol-Charhbili V, Loufrani L, et al. The endothelialmineralocorticoid receptor regulates vasoconstrictor tone and blood pressure.FASEB J 2010;24:2454–24633. McCurley A, Pires PW, Bender SB, et al. Direct regulation of blood pressure bysmooth muscle cell mineralocorticoid receptors. Nat Med 2012;18:1429–14334. Lim HY, Müller N, Herold MJ, van den Brandt J, Reichardt HM. Glucocor-ticoids exert opposing effects on macrophage function dependent on their con-centration. Immunology 2007;122:47–53

5. Usher MG, Duan SZ, Ivaschenko CY, et al. Myeloid mineralocorticoid re-ceptor controls macrophage polarization and cardiovascular hypertrophy andremodeling in mice. J Clin Invest 2010;120:3350–33646. Guo C, Ricchiuti V, Lian BQ, et al. Mineralocorticoid receptor blockadereverses obesity-related changes in expression of adiponectin, peroxisomeproliferator-activated receptor-gamma, and proinflammatory adipokines. Circu-lation 2008;117:2253–22617. Leopold JA, Dam A, Maron BA, et al. Aldosterone impairs vascular reactivityby decreasing glucose-6-phosphate dehydrogenase activity. Nat Med 2007;13:189–1978. Messaoudi S, Azibani F, Delcayre C, Jaisser F. Aldosterone, mineralocor-ticoid receptor, and heart failure. Mol Cell Endocrinol 2012;350:266–2729. Bender SB, McGraw AP, Jaffe IZ, Sowers JR. Mineralocorticoid receptor-mediated vascular insulin resistance: an early contributor to diabetes-relatedvascular disease? Diabetes 2013;62:313–31910. Schäfer N, Lohmann C, Winnik S, et al. Endothelial mineralocorticoid re-ceptor activation mediates endothelial dysfunction in diet-induced obesity. EurHeart J 2013;34:3515–352411. Caprio M, Fève B, Claës A, Viengchareun S, Lombès M, Zennaro MC. Pivotalrole of the mineralocorticoid receptor in corticosteroid-induced adipogenesis.FASEB J 2007;21:2185–219412. Nguyen Dinh Cat A, Briones AM, Callera GE, et al. Adipocyte-derived factorsregulate vascular smooth muscle cells through mineralocorticoid and glucocor-ticoid receptors. Hypertension 2011;58:479–48813. Briones AM, Nguyen Dinh Cat A, Callera GE, et al. Adipocytes produce al-dosterone through calcineurin-dependent signaling pathways: implications indiabetes mellitus-associated obesity and vascular dysfunction. Hypertension2012;59:1069–107814. Urbanet R, Nguyen Dinh Cat A, Feraco A, et al. Adipocyte mineralocorticoidreceptor activation leads to metabolic syndrome and induction of prostaglandinD2 synthase. Hypertension 2015;66:149–15715. Hirata A, Maeda N, Nakatsuji H, et al. Contribution of glucocorticoid-mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction.Biochem Biophys Res Commun 2012;419:182–187

16. Halberg N, Wernstedt-Asterholm I, Scherer PE. The adipocyte as an en-docrine cell. Endocrinol Metab Clin North Am 2008;37:753–768, x–xi

17. Karastergiou K, Mohamed-Ali V. The autocrine and paracrine roles of adi-pokines. Mol Cell Endocrinol 2010;318:69–78

18. Hirata A, Maeda N, Hiuge A, et al. Blockade of mineralocorticoid receptorreverses adipocyte dysfunction and insulin resistance in obese mice. CardiovascRes 2009;84:164–17219. Tirosh A, Garg R, Adler GK. Mineralocorticoid receptor antagonists and themetabolic syndrome. Curr Hypertens Rep 2010;12:252–257

20. Marzolla V, Armani A, Zennaro MC, et al. The role of the mineralocorticoidreceptor in adipocyte biology and fat metabolism. Mol Cell Endocrinol 2012;350:281–28821. Yarmo MN, Landry A, Molgat AS, Gagnon A, Sorisky A. Macrophage-conditionedmedium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes.Exp Cell Res 2009;315:411–41822. Omar A, Chatterjee TK, Tang Y, Hui DY, Weintraub NL. Proinflammatoryphenotype of perivascular adipocytes. Arterioscler Thromb Vasc Biol 2014;34:1631–163623. Taylor MS, Okwuchukwuasanya C, Nickl CK, Tegge W, Brayden JE, DostmannWR. Inhibition of cGMP-dependent protein kinase by the cell-permeable peptideDT-2 reveals a novel mechanism of vasoregulation. Mol Pharmacol 2004;65:1111–111924. Hirosumi J, Tuncman G, Chang L, et al. A central role for JNK in obesity andinsulin resistance. Nature 2002;420:333–33625. Sugita M, Sugita H, Kaneki M. Increased insulin receptor substrate 1 serinephosphorylation and stress-activated protein kinase/c-Jun N-terminal kinaseactivation associated with vascular insulin resistance in spontaneously hyper-tensive rats. Hypertension 2004;44:484–489

2402 Adipocyte MR Induces Vascular Dysfunction Diabetes Volume 65, August 2016

26. Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF. Phos-phorylation of Ser307 in insulin receptor substrate-1 blocks interactions with theinsulin receptor and inhibits insulin action. J Biol Chem 2002;277:1531–153727. Burgoyne JR, Madhani M, Cuello F, et al. Cysteine redox sensor in PKGIaenables oxidant-induced activation. Science 2007;317:1393–139728. Noma K, Oyama N, Liao JK. Physiological role of ROCKs in the cardiovas-cular system. Am J Physiol Cell Physiol 2006;290:C661–C66829. Cassis LA, Police SB, Yiannikouris F, Thatcher SE. Local adipose tissuerenin-angiotensin system. Curr Hypertens Rep 2008;10:93–9830. Bindom SM, Lazartigues E. The sweeter side of ACE2: physiological evi-dence for a role in diabetes. Mol Cell Endocrinol 2009;302:193–20231. Santos SH, Fernandes LR, Pereira CS, et al. Increased circulating angiotensin-(1-7) protects white adipose tissue against development of a proinflammatory statestimulated by a high-fat diet. Regul Pept 2012;178:64–7032. Caprio M, Antelmi A, Chetrite G, et al. Antiadipogenic effects of the min-eralocorticoid receptor antagonist drospirenone: potential implications for thetreatment of metabolic syndrome. Endocrinology 2011;152:113–12533. Makki K, Froguel P, Wolowczuk I. Adipose tissue in obesity-related in-flammation and insulin resistance: cells, cytokines, and chemokines. ISRN In-flamm 2013;2013:13923934. Chen W, Liu DJ, Huo YM, Wu ZY, Sun YW. Reactive oxygen species areinvolved in regulating hypocontractility of mesenteric artery to norepinephrine incirrhotic rats with portal hypertension. Int J Biol Sci 2014;10:386–39535. Grace JA, Klein S, Herath CB, et al. Activation of the MAS receptor byangiotensin-(1-7) in the renin-angiotensin system mediates mesenteric vasodi-latation in cirrhosis. Gastroenterology 2013;145:874–884.e536. Loirand G, Guilluy C, Pacaud P. Regulation of Rho proteins by phosphory-lation in the cardiovascular system. Trends Cardiovasc Med 2006;16:199–20437. Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rhoand Rho-associated kinase (Rho-kinase). Science 1996;273:245–24838. Seko T, Ito M, Kureishi Y, et al. Activation of RhoA and inhibition of myosinphosphatase as important components in hypertension in vascular smooth muscle.Circ Res 2003;92:411–41839. Guilluy C, Sauzeau V, Rolli-Derkinderen M, et al. Inhibition of RhoA/Rhokinase pathway is involved in the beneficial effect of sildenafil on pulmonaryhypertension. Br J Pharmacol 2005;146:1010–101840. Laufs U, Endres M, Stagliano N, et al. Neuroprotection mediated by changesin the endothelial actin cytoskeleton. J Clin Invest 2000;106:15–2441. Takemoto M, Sun J, Hiroki J, Shimokawa H, Liao JK. Rho-kinase mediateshypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation2002;106:57–6242. Wolfrum S, Dendorfer A, Rikitake Y, et al. Inhibition of Rho-kinase leads torapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardio-vascular protection. Arterioscler Thromb Vasc Biol 2004;24:1842–1847

43. Tokuyama H, Wakino S, Hara Y, et al. Role of mineralocorticoid receptor/Rho/Rho-kinase pathway in obesity-related renal injury. Int J Obes 2012;36:1062–107144. Noda K, Nakajima S, Godo S, et al. Rho-kinase inhibition amelioratesmetabolic disorders through activation of AMPK pathway in mice. PLoS One2014;9:e11044645. Furukawa S, Fujita T, Shimabukuro M, et al. Increased oxidative stress inobesity and its impact on metabolic syndrome. J Clin Invest 2004;114:1752–176146. Chattopadhyay M, Khemka VK, Chatterjee G, Ganguly A, Mukhopadhyay S,Chakrabarti S. Enhanced ROS production and oxidative damage in subcutaneouswhite adipose tissue mitochondria in obese and type 2 diabetes subjects. MolCell Biochem 2015;399:95–10347. Rodríguez-Martínez MA, García-Cohen EC, Baena AB, González R, SalaícesM, Marín J. Contractile responses elicited by hydrogen peroxide in aorta fromnormotensive and hypertensive rats. Endothelial modulation and mechanisminvolved. Br J Pharmacol 1998;125:1329–133548. Gao YJ, Lee RM. Hydrogen peroxide induces a greater contraction inmesenteric arteries of spontaneously hypertensive rats through thromboxane A(2)production. Br J Pharmacol 2001;134:1639–164649. Moreno JM, Rodriguez Gomez I, Wangensteen R, et al. Mechanisms ofhydrogen peroxide-induced vasoconstriction in the isolated perfused rat kidney.J Physiol Pharmacol 2010;61:325–33250. Yang Z, Zhang A, Altura BT, Altura BM. Hydrogen peroxide-induced endothelium-dependent relaxation of rat aorta involvement of Ca2+ and other cellularmetabolites. Gen Pharmacol 1999;33:325–33651. Matoba T, Shimokawa H, Nakashima M, et al. Hydrogen peroxide is anendothelium-derived hyperpolarizing factor in mice. J Clin Invest 2000;106:1521–153052. Fujimoto S, Asano T, Sakai M, et al. Mechanisms of hydrogen peroxide-induced relaxation in rabbit mesenteric small artery. Eur J Pharmacol 2001;459:291–30053. Waldman SA, Murad F. Biochemical mechanisms underlying vascularsmooth muscle relaxation: the guanylate cyclase-cyclic GMP system. J Cardio-vasc Pharmacol 1988;12(Suppl. 5):S115–S11854. Fujimoto S, Mori M, Tsushima H. Mechanisms underlying the hydrogenperoxide-induced, endothelium-independent relaxation of the norepinephrine-contraction in guinea-pig aorta. Eur J Pharmacol 2003;459:65–7355. Cai H. Hydrogen peroxide regulation of endothelial function: origins,mechanisms, and consequences. Cardiovasc Res 2005;68:26–3656. Francis SH, Busch JL, Corbin JD, Sibley D. cGMP-dependent protein kinasesand cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev2010;62:525–56357. Prysyazhna O, Rudyk O, Eaton P. Single atom substitution in mouse proteinkinase G eliminates oxidant sensing to cause hypertension. Nat Med 2012;18:286–290

diabetes.diabetesjournals.org Nguyen Dinh Cat and Associates 2403