Research Article The Risk of Heart Failure and...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 253657 16 pageshttpdxdoiorg1011552013253657

Research ArticleThe Risk of Heart Failure and Cardiometabolic Complicationsin Obesity May Be Masked by an Apparent Healthy Status ofNormal Blood Glucose

Shuchita Tiwari1 Manish Mishra1 Ashok Jadhav1 Courtney Gerger2 Paul Lee1

Lynn Weber2 and Joseph Fomusi Ndisang1

1 Department of Physiology University of Saskatchewan College of Medicine 107 Wiggins Road Saskatoon SK Canada S7N 5E52Department of Veterinary Biomedical Sciences University of Saskatchewan 52 Campus Drive Saskatoon SK Canada S7N 5E5

Correspondence should be addressed to Joseph Fomusi Ndisang josephndisangusaskca

Received 2 October 2013 Accepted 12 November 2013

Academic Editor Sharad Rastogi

Copyright copy 2013 Shuchita Tiwari et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Although many obese individuals are normoglycemic and asymptomatic of cardiometabolic complications this apparent healthystate may be a misnomer Since heart failure is a major cause of mortality in obesity we investigated the effects of heme-oxygenase(HO) on heart failure and cardiometabolic complications in obese normoglycemic Zucker-fatty rats (ZFs) Treatment with theHO-inducer hemin reduced markers of heart failure such as osteopontin and osteoprotegerin abated left-ventricular (LV) hyper-trophyfibrosis extracellular matrixprofibrotic proteins including collagen IV fibronectin TGF-1205731 and reduced cardiac lesionsFurthermore hemin suppressed inflammation by abating macrophage chemoattractant protein-1 macrophage-inflammatoryprotein-1 alpha TNF-120572 IL-6 and IL-1120573 but enhanced adiponectin atrial-natriuretic peptide (ANP) HO activity insulin sensitivityand glucose metabolism Correspondingly hemin improved several hemodynamicechocardiographic parameters including LV-diastolic wall thickness LV-systolic wall thickness mean-arterial pressure arterial-systolic pressure arterial-diastolic pressure LV-developed pressure +dPdt and cardiac output Contrarily the HO-inhibitor stannous mesoporphyrin nullified the hemin effectexacerbating inflammatoryoxidative insults and aggravated insulin resistance (HOMA-index) We conclude that perturbations ininsulin signaling and cardiac function may be forerunners to overt hyperglycemia and heart failure in obesity Importantly heminimproves cardiac function by suppressing markers of heart failure LV hypertrophy cardiac lesions extracellular matrixprofibroticproteins and inflammatoryoxidative mediators while concomitantly enhancing the HO-adiponectin-ANP axis

1 Introduction

The recent escalation of obesity in every segment of the pop-ulation including children adolescences and adults poses agreat health challenge of considerable socioeconomic burden[1 2] The impact on healthcare systems may become unsus-tainable given the numerous chronic diseases such as type-2 diabetes dyslipidemia hypertension and other relatedcardiometabolic complications associated with obesity [1ndash3]Cardiac complications including heart failure are among themajor causes ofmortality in obese individuals Obesity causeslipotoxicity and adipose tissue dysfunction with excessiveproduction of adipokines like tumor necrosis factor-120572 (TNF-120572) and interleukin 6 (IL-6) IL-1120573 all of which are implicated

in heart failure and related cardiometabolic complications [45] However obesity may not always translate into increasedrisk for these comorbidities [6] Some obese individualsdubbed ldquometabolically healthyrdquo are protected against obesity-related metabolic diseases These ldquometabolically healthyrdquoobese individuals are insulin sensitive with normal lipidmetabolism and cardiac function similar to healthy leanindividuals which is in stark contrast to ldquometabolicallyunhealthyrdquo obese individuals with high risk of developingcardiometabolic complications [6] However the apparentstate of good health in ldquometabolically healthyrdquo obese sub-phenotype may be a misnomer because the development ofseveral characteristics of metabolic syndrome is now beingobserved in many adults who previously manifested the

2 Oxidative Medicine and Cellular Longevity

healthy obese phenotype [7] suggesting that individuals witha healthy obese phenotype may not remain healthy for theirentire lives Several parameters including environmental andbehavioral factors may modify obesity subphenotypes andthe transition from healthy to unhealthy Whether healthyobese individuals can maintain insulin sensitivity duringthe entire life or whether healthy obesity simply representsdelayed onset of obesity related cardiometabolic complica-tions has to be clarified

In obesity excessive oxidative stress intense inflamma-tory activity insulin resistance deregulated lipidmetabolismaltered glucose metabolism and impaired mitochondrialbiogenesis are among the pathophysiological driving forcesthat precede the early stages of cardiac dysfunction Manycardiac complications have the common denominator ofelevated inflammation due to the infiltration of macrophageM1 phenotype [8] Generally macrophages exhibit two dif-ferent forms dubbed ldquoclassicalrdquo or M1 phenotype and ldquoalter-nativerdquo or M2 phenotype [8] and each phenotype expressesdistinct patterns of surface receptors when responding todifferent stimuli TheM1 phenotype stimulates inflammationwhile the M2 phenotype blunts inflammation [8] Duringmacrophage infiltration the M1 phenotype is stimulated bydifferent chemokines including macrophage inflammatoryprotein-1 alpha ((MIP-1120572) chemokine (C-C motif) ligand-3(CCL3)) and macrophage chemoattractant protein-1 ((MCP-1) [9] chemokine (C-C motif ligand-2 (CCL2)) [9] Theactivation of the macrophage M1 phenotype is generallyassociated with elevated levels of proinflammatory cytokinesincluding TNF-120572 IL-6 and IL-1120573 [10ndash12] Moreover thelevels of macrophage M1 phenotype MCP-1 TNF-120572 IL-6 and IL-1120573 are elevated in obesity and insulin resistance[9 10 13] and these factors play a major pathophysiolog-ical role in heart failure [4] In obesity markers of heartfailure such as osteopontin [14] and osteoprotegerin [15]are elevated [16 17] Similarly the levels of extracellularmatrixprofibrotic proteins like transforming growth factorbeta (TGF-120573) collagen and fibronectin are elevated in obe-sity [18]Therefore in obesity elevated chemokines cytokinesand increased macrophage-M1 infiltration would act inconcert with elevated extracellular matrixprofibrotic andheart-failure proteins to exacerbate cardiac tissue destruc-tion and compromise heart function Thus novel strategiescapable of selectively suppressingmacrophageM1 phenotypeproinflammatory cytokineschemokines and extracellularmatrixprofibrotic proteins are needed

In many pathophysiological conditions various stress-response immune-regulatory proteins including heme oxy-genase (HO-1) are activated as an innate defense mechanism[19ndash22] However the pathophysiological activation of HO-1 may only result in a transient or marginal increase of HOactivity that falls below the threshold necessary to activatethe downstream signaling components through which theHO system elicits its cytoprotective effects so a robust andsurmountable increase of HO activity with HO inducerslike hemin may be needed for cardioprotection [23ndash27]Generally HO is composed of two main isoforms (HO-1or inducible) and (HO-2 or constitutive) which are largelyresponsible for the antioxidant and anti-inflammatory effects

of HO [28] We recently reported the cardioprotective effectsof the HO system in Zucker diabetic fatty rats (ZDFs) [13] amodel characterized by obesity insulin resistance and overthyperglycemia However because ZDFs are hyperglycemictheir pathophysiological profile is not reflective of individualsdubbed ldquometabolically healthyrdquo a subtype of obesity char-acterized by normoglycemia [7] Given that the incidenceof cardiometabolic complications is increasing in manyadults who previously manifested the metabolically healthyobese phenotype [7] novel studies with animal models thatclosely mimic the pathophysiological profile of metabolicallyhealthy obese subtype are needed Therefore this study willinvestigate the effects of the HO system on cardiometaboliccomplications in Zucker fatty rats (ZFs) an obese modelwith normoglycemia and cardiometabolic complications [29]that closely mimic the pathophysiological profile of metabol-ically healthy obese individuals with normoglycemia and anapparent state of good health Although the HO system iscytoprotective its effects on cardiomyopathy in ZFs remainto be elucidated

Since dysfunctional insulin signaling obesity elevatedinflammation and cardiac hypertrophy are forerunners toheart failure this study will also investigate the multifacetedmechanisms by which theHO system preserves cardiac func-tion in ZFs Whether an upregulated HO system by hemin iscapable of modulating macrophage polarization towards theM2 phenotype that blunts inflammation while suppressingthe proinflammatory M1 phenotype will be assessed Asmacrophage infiltration is stimulated by chemokines likeMIP-1120572 and MCP-1 [9] and the effects of the HO system onthese chemokines in ZFs have not been reported this studywill also determine left-ventricular MIP-1120572 and MCP-1 andcorrelate changes of these chemokines to the expression ofthe proinflammatory macrophage-M1 phenotype in the leftventricle of ZFs Similarly the effect of hemin therapy onimportant markers of heart failure such as osteopontin [13]and osteoprotegerin [14] will be investigated Importantly nostudy has reported the levels of expression of osteopontinand osteoprotegerin in myocardial tissue of ZFs Thereforethis study will unveil the multifaceted mechanisms by whichhemin therapy improves cardiac function and insulin signal-ing in obesity

2 Materials and Methods

21 Animals Treatment Groups and Biochemical ParametersOur experimental protocol was in conformity with theGuide for Care and Use of Laboratory Animals stipulatedby the Canadian Council on Animal Care and the NationalInstitutes of Health (NIH Publication no 85-23 revised 1996)and was approved by University of Saskatchewan AnimalEthics Committee Male ZFs (12 weeks old) and sexage-matched Zucker lean (ZL) rats were purchased from CharlesRiver Laboratories (Willington MA USA) The animalswere housed at 21∘C with 12-hour lightdark cycles fed withstandard laboratory chow and had access to drinking waterad libitum

Oxidative Medicine and Cellular Longevity 3

The HO-inducer hemin (30mgkg ip Sigma St LouisMO USA) and HO-blocker stannous-mesoporphyrin((SnMP) 2mg100 g body weight ip) were purchased fromPorphyrin Products (Logan UT USA) and prepared as wepreviously reported and administered biweekly for 8 weeks[13 30 31] At 16 weeks of age the animals were randomlyassigned to the following experimental groups (119899 = 6 pergroup) (A) controls (ZF and ZL) (B) hemin-treated ZF andZL (C) ZF+hemin+SnMP and (D) ZF+vehicle dissolvinghemin and SnMP

During the treatment period body weight and glucosewere monitored on a weekly routine Body weight was mea-sured using a digital balance (ModelMettler PE1600MettlerInstruments Corporation Greifensee Zurich Switzerland)At the end of the 8-week treatment period the animalswere 24 weeks of age A day prior to killing the animalswere fasted in metabolic cages for 24 hr urine collectionand weighed Systolic blood pressure was determined bynoninvasive tail-cuff method (Model 29-SSP Harvard Appa-ratus Montreal Canada) while a Millar Mikro-Tip ultra-miniature tip sensor pressure transducer catheter (ModelSPR-407 Harvard Apparatus MontrealCanada) for invasivehemodynamic parameters In addition a Vevo 660 high fre-quency ultrasound machine (VisualSonics Markham ONCanada) equippedwith B-mode imaging was used for echocar-diographic measurements as we previously reported [13]After anaesthetizing the animals with pentobarbital sodium(50mgkg ip) blood was collected by cardiac punctureand the heart was cleaned and weighed with an analyticalbalance (Precisa Instruments Ltd Dietikon Switzerland) aswe previously reported [32] The atria were removed fromthe heart and the right ventricle free wall separated from theleft ventricle including the septum as we previously reported[32]

Left-ventricular HO activity was evaluated spectropho-tometrically as we previously reported [30 32] ELISAkits were used for HO-1 (Stressgen-Assay Design AnnArborMI USA) adiponectin (Phenix Pharmaceuticals IncBurlingame CA USA) TNF-120572 IL-6 and IL-1120573 (Immuno-Biological Laboratories Co Ltd Gunma Japan) MIP-1120572and MCP-1 (OmniKine Assay Biotechnology CompanyInc Sunnyvale CA USA) [33 34] while EIA kits for8-isoprostane ANP ET-1 cGMP and kits for cholesteroland triglyceride were purchased from Cayman (CaymanChemical Ann Arbor MI USA) following the manufac-turersrsquo instructions as we reported [13 30 32] Intraperi-toneal glucose tolerance test (IPGTT) andhomeostasismodelassessment of insulin resistance (HOMA-IR) were done as wepreviously reported [30]

22 Histological Morphological and ImmunohistochemicalAnalyses of Left Ventricle Histological and morphometricanalyses were done as we previously described [35] Sectionsof 5 120583mwere cut and stained with hematoxylin and eosin forhistological analysis Massonrsquos Trichrome staining detected

left-ventricular collagen deposition Morphometrical evalu-ation of left-ventricular longitudinal myocyte thickness wasdone by randomly measuring 30 cardiac muscle fibers fromeach experimental group by a blinded researcher using amicroscope (Aperio Scan ScopeModel CS Aperio Technolo-gies Inc Vista CA USA) and analyzed using Aperio ImageScope V1120780 software (Aperio e-Pathology SolutionVista CA USA) Morphologic assessment of collagen depo-sition in left-ventricular sections was accessed using AperioImageScope (Aperio Technologies Inc Vista CA USA)Each left-ventricular section was magnified at 200x and 20random snaps were taken per slide (20 times 6 = 120 images pergroup) subsequently scored semiquantitatively by a blindedresearcher as we previously reported [13 35]

Immunohistochemistry was done as we previouslyreported [36] Sections of 5 120583m of left-ventricular tissuewere treated with bovine serum albumin in phosphate-buffered saline to block nonspecific staining and incubatedovernight with ED1 (1 500 dilution sc-59103 Santa CruzBiotechnology CA USA) The sections were later treatedwith with goat anti-mouse IgG for 30min (1 200 dilutionJackson Immuno-Research Laboratories Inc ME USA)Immunohistochemical staining was done using the standardavidin-biotin complex method with the chromagen 331015840-diaminobenzidine (DAB) at the final detection step Sectionsof heart tissuewere scanned using virtualmicroscope (AperioScan Scope Model CS Aperio Technology Inc Vista CAUSA) Quantitative assessment of ED1 was done by a blindedresearcher who randomly examined 20ndash22 fields of each left-ventricular section magnified at 200x Macrophages whichwere positively stained with ED1 (brown from immune-stained sections) were quantified by manually counting theED1-stained cells around the blood vessels and interstitialspaces of myocardium

23 Western Immunoblotting Left-ventricular tissue washomogenized as previously reported [13 30ndash32 35] Pri-mary antibodies ((Santa Cruz Biotechnology Santa CruzCA USA) ED-1 (CD68) (sc-59103) ED-2 (CD163) (sc-58956) CD-14 (sc-9150) CD-206 (sc-48758) CD-36 (sc-9154) osteopontin (sc-21742) osteoprotegerin (sc-11383)PI3K (sc67306) IRS-1 (sc-559) collagen IV (sc-11360)fibronectin (sc-18825) TGF-120573123 (sc7892)) and GLUT4 (ab654 Abcam Inc Cambridge MA USA) were used Densit-ometric analysis was done with UN-SCAN-IT software (SilkScientific Inc Orem UT USA) G6PDH antibody (A9521Sigma St Louis MO USA) was used as a control to ascertainequivalent loading

24 Statistical Analysis All data are expressed as means plusmnSEM from at least four independent experiments unless oth-erwise stated Statistical analyses were done using two-wayANOVA using Statistical Analysis System (SAS) softwareVersion 93 (SAS Institute Inc Cary NC USA) and Studentrsquos119905-test Group differences at the level of 119875 lt 005 wereconsidered statistically significant


0

7

14

21

28

35

42

Left-

vent

ricul

ar H

O-1

conc

entr

atio

n (n

gm

L)

lowast

dagger

ZL control ZL +hemin

ZF control ZF +hemin

ZF + hemin+ SnMP

(a)

lowast

dagger daggerLeft-

vent

ricul

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O ac

tivity

(nm

ol b

iliru

bin

mg

prot

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hr)

0

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16



ZF + hemin+ SnMP

(b)

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5

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Left-

vent

ricul

ar cG

MP

(pm

olm

g pr

otei

n)

daggerdagger

lowast



ZF + hemin+ SnMP

(c)

Figure 1 Effects of the HO inducer hemin and the HO inhibitor SnMP onHO-1 HO activity and cGMP in the left ventricle of ZLs and ZFs(a) Hemin enhanced HO-1 whereas SnMP nullified the effects of hemin (b) Hemin increased HO activity while SnMP abolished the hemineffect (c) Hemin enhanced cGMP which however was abolished by SnMP Bars represent means plusmn SEM 119899 = 6 rats per group (lowast119875 lt 001versus ZL control dagger119875 lt 005 versus Z control 119875 lt 001 versus ZF+hemin+SnMP or ZF control)

3 Results

31 Hemin Therapy Upregulates the HO System to ImproveCardiac Function To investigate the mechanisms underly-ing the improvement of cardiac function in obese insulin-resistant ZFs we measured the concentration of HO-1 andHO activity In ZF-control rats the basal level of HO-1concentration and HO activity was significantly lower thanthat of ZL control (Figures 1(a) and 1(b)) However heminadministration increased HO-1 and HO activity in ZF by 84-and 113-fold respectively The enhanced HO activity wouldincrease endogenous carbon monoxide that would in turnstimulate cGMP [30 32] Both cGMP and carbon monox-ide are known to enhance insulin signaling and glucosemetabolism [37] Accordingly we detected a 34-fold increaseof cGMP in hemin-treated animals (Figure 1(c)) In contrastthe coadministration of the HO blocker SnMP and the HOinducer and hemin abolished the hemin-induced increaseof HO-1 and HO activity with corresponding reduction ofcGMP levels (Figure 1(c)) Hemin therapy also enhancedHO-1 HO activity and cGMP levels in ZL-control rats(Figures 1(a) 1(b) and 1(c)) In hemin-treated ZLs HO-1 HO activity and cGMP were enhanced by 31- 28- and

24-fold respectively as compared to 84- 113- and 34-fold respectively in hemin-treated ZFs suggesting greaterselectivity of hemin to the HO system in unhealthy ZFscharacterized obesity insulin resistance and cardiomyopathy[29]

Since cardiac hypertrophy is amongst the forerunnersto heart failure we investigated the effects of hemin oncardiac hypertrophy Our results indicate that hemin therapysignificantly reduced cardiac hypertrophy in ZF whereas thecoadministration of hemin and SnMP nullified the effect(Table 1) Echocardiography was used to further assess left-ventricular hypertrophy Our hemodynamic data obtainedduring catheterization of the left side of hearts from ZFsshowed association between elevated myocardial hyper-trophic response and obesity Asignificant 2-fold increasein left-ventricular free wall thickness an important indexof cardiac hypertrophy [38] was observed during dias-tole and systole and interestingly these were abated byhemin by 333 and 156 respectively (Table 2) Otherhemodynamic deficiencies in ZFs including abnormalitiesin left-ventricular end-diastolic volume left-ventricular end-systolic volume stroke volume and cardiac output whichwere reduced by 175 16 83 and 77 respectively


Table 1 Effect of hemin and stannous mesoporphyrin (SnMP) on physiological and biochemical variables in Zucker fatty (ZF) and Zuckerlean (ZL) rats

ParametersAnimal groups

ZL control ZL + hemin ZF control ZF + hemin ZF + hemin +SnMP ZF + vehicle

Body weight (g) 4729 plusmn 97 4455 plusmn 116dagger 7468 plusmn 215sect 6859 plusmn 147sect 6914 plusmn 152$ 7028 plusmn 246Fasting glucose(mmoL) 72 plusmn 06 65 plusmn 03lowast 82 plusmn 05sect 69 plusmn 04lowast 85 plusmn 04lowast 81 plusmn 04

Heart weight (g) 15 plusmn 003 11 plusmn 002dagger 34 plusmn 006sect 20 plusmn 004sect 30 plusmn 003$ 31 plusmn 004Cardiachypertrophy(gKg body weight)

31 plusmn 007 25 plusmn 006lowast 46 plusmn 016sect 29 plusmn 008lowast 44 plusmn 017lowast 44 plusmn 009

dagger

119875 lt 005 versus ZL $119875 lt 005 versus ZF sect119875 lt 005 versus ZL control lowast119875 lt 005 versus ZF-control or ZL-control

Table 2 Effect of hemin therapy on hemodynamic and echocardiographic parameters

ParametersExperimental groups 119875 value

Effect of heminon ZFZF ZL ZF + hemin ZF

versus ZLZF versusZF + hemin

Arterial systolic pressure(mmHg) 153 plusmn 52 124 plusmn 34 134 plusmn 63lowast 0001 0018 Reduced by 124

Arterial diastolic pressure(mmHg) 109 plusmn 32 92 plusmn 25 96 plusmn 47lowast 0003 0024 Reduced by 119

Mean arterial pressure(mmHg) 123 plusmn 38 102 plusmn 27 109 plusmn 52lowast 0002 0022 Reduced by 114

Total peripheral resistances(mmHgsdotminmL) 17 plusmn 01 14 plusmn 01 14 plusmn 01 0060 0080 Reduced by 176

LV developed pressure(mmHg) 161 plusmn 41 136 plusmn 80 140 plusmn 58lowast 0012 0030 Reduced by 130

+dpdt(max) (mmHgsec) 3634 plusmn 127 3050 plusmn 200 3050 plusmn 169lowast 0027 0027 Reduced by 161

Heart rate (beatsmin) 328 plusmn 92 331 plusmn 72 289 plusmn 142lowast 0814 0020 Reduced by 119LV diastolic wall thickness(mm) 27 plusmn 01 17 plusmn 01 18 plusmn 01lowast 00001 00001 Reduced by 333

LV systolic wall thickness(mm) 32 plusmn 01 21 plusmn 01 27 plusmn 01lowast 00001 0007 Reduced by 156

LV end diastolic volume(mL) 033 plusmn 001 040 plusmn 004 038 plusmn 002 0075 0169 Increased by 152

LV end systolic volume(mL) 011 plusmn 001 016 plusmn 001 014 plusmn 001 0001 0089 Increased by 273

Stroke volume (mL) 022 plusmn 001 024 plusmn 002 025 plusmn 002 0564 0311 Increased by 136Cardiac output (mLmin) 728 plusmn 36 789 plusmn 90 818 plusmn 98 0519 0412 Increased by 124Values for each echocardiography and hemodynamic endpoint were averaged for each rat and the mean values used in statistical analyses with 119899 number ofrats Differences among treatment groups were compared using 1-way ANOVA followed by Fisherrsquos Least Square Difference (LSD) posteriori tests Differencesof 119875 lt 005were considered statistically significant Values are means plusmn SE 119899 = 6 per group lowast119875 lt 005 versus control ZF rats 119875 lt 005 versus control ZL ratsLV left ventricle +dpdt

(max) maximal rate of increase in left ventricular pressure

(Table 2) were increased by hemin therapy by 152 273136 and 124 respectively Hemin therapy also improvedcardiac hemodynamics by lowering arterial-systolic pressurearterial-diastolic pressure mean-arterial pressure and total-peripheral resistance by 124 114 and 122 176respectively with corresponding reduction of +dpdt (themaximal rate of increase in left-ventricular pressure) left-ventricular developed pressure and heart rate

Treatment with hemin and SnMP caused loss of bodyweight in ZL controls and ZFs which however did notexceed 9 (Table 1) The loss of weight may not be due totoxicity as we recently showed that several indices of toxi-city including plasma gamma-glutamyltransferase aspartate

aminotransferase and alanine aminotransferase were withinnormal range [30] AlthoughZFs had normoglycemia heminand SnMP affected blood glucose In hemin-treated animalsthere was a slight but significant reduction of glycemiawhereas in SnMP-treated animals a slight increase wasobserved (Table 1) Similarly hemin therapy slightly reducedglycemia in ZL controls The vehicle dissolving hemin andSnMP had no effect on the measured parameters

32 Hemin Therapy Abates MCP-1 MIP-1120572 TNF-120572 Endo-thelin-1 and 8-Isoprostane but Enhanced ANP in ZFs Since8-isoprostane stimulates ET-1 [39] and both ET-1 and 8-isoprostane are involved in the oxidative destruction of tissue


0

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ZF + hemin+ SnMP

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-1 (p

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L)

(a)


lowast

0

20

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ZF + hemin+ SnMP

Urin

ary

8-iso

pros

tane

(ng

24 h

rs)

(b)

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lowastlowast

0

300

600

900



ZF + hemin+ SnMP

Left-

vent

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ar A

NP

(ng

mg

prot

ein)

(c)

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lowast

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ZF + hemin+ SnMP

Left-

vent

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IP-1120572

(pg

mg

prot

ein)

(d)

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lowast

00

09

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ZF + hemin+ SnMP

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vent

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IP-1120572

(ng

mg

prot

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(e)

lowast


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120



ZF + hemin+ SnMP

Left-

vent

ricul

ar T

NF-120572

(pg

mL)

(f)

Figure 2 Effects of theHO inducer hemin and theHO inhibitor SnMP on endothelin-1 ANPMCP-1MIP1-120572 and TNF-120572 in left-ventriculartissue from ZLs and ZFs Hemin therapy (a) reduced endothelin-1 (b) attenuated 8-isoprostane (c) increased ANP (d) suppressed MCP-1(d) abated MIP-1120572 and (f) reduced TNF-120572 whereas SnMP abolished the hemin effects Bars represent means plusmn SEM 119899 = 6 rats per group(lowast119875 lt 005 lowastlowast119875 lt 001 versus ZL control dagger119875 lt 005 daggerdagger119875 lt 001 versus ZL control 119875 lt 001 versus ZF+hemin+SnMP or ZF control)

we measured ET-1 and 8-isoprostane ET-1 in untreatedZFs was markedly elevated as compared to ZL controls(Figure 2(a)) but was significantly abated by hemin In con-trast the coadministration of hemin and the HO blockerSnMP annulled the effect of hemin (Figure 2(a)) Becauseelevated oxidative stress is linked to impaired insulin-signaling and cardiac dysfunction we measured urinary 8-isoprostane an important marker of oxidative stress [40]

In ZFs the basal levels of 8-isoprostane were significantlyelevated (Figure 2(b)) but were reduced by hemin whereascotreatment of hemin with SnMP nullified the effects Giventhat ET-1 and ANP are known to interact reciprocally [41]we investigated whether the hemin-dependent suppressionof ET-1 (Figure 2(a)) would be associated with a parallelpotentiation of ANP In ZFs the basal ANP levels weremarkedly depressed by 17-fold (Figure 2(c)) but interestingly


were robustly enhanced by hemin by 33-fold In contrast thecoadministration of hemin with SnMP abolished the effectsof hemin

We also investigated the effects of hemin on MIP-1120572 andMCP-1 since these chemokines trigger macrophage infiltra-tion [9] In ZFs the basal MCP-1 levels were significantlyincreased by 46-fold (Figure 2(d)) but were attenuated byhemin by 28-fold whereas the coadministration with SnMPnullified the effects of hemin (Figure 2(d)) Although hemintherapy greatly attenuated MCP-1 by 64 in ZF howevercomparable levels as observed in the ZL controls were notreinstated Hemin therapy was also effective in suppressingMIP-1120572 (Figure 2(e)) In ZFs the basal MIP-1120572 levels weresignificantly elevated by 49-fold but were reduced by heminby 35-fold whereas the cotreatment of hemin with SnMPnullified the effects (Figure 2(e)) Since TNF-120572 is implicatedin macrophage infiltration [9] we also assessed the effectsof hemin on TNF-120572 In ZFs the basal levels of TNF-120572were elevated by 35-fold but were significantly attenuatedby hemin by 26-fold (Figure 2(f)) whereas cotreatment withSnMP abolished the effect of hemin

Hemin therapy also affected ET-1 8-isoprostane ANPMCP-1 and MIP-1120572 in ZL controls although the magnitudeof effect was smaller than that in ZFs (Figure 2)

33 Hemin Selectively Abated the Proinflammatory Macro-phage M1 Phenotype but Enhanced the Anti-Inflammatory M2Phenotype in the Left Ventricle of ZFs After having observedthe hemin-dependent reduction of cytokineschemokinesimplicated in macrophage infiltration such as MIP-1120572 MCP-1 and TNF-120572 we investigated whether the suppressionof these chemokinescytokines in the left ventricle of ZFswould be accompanied by the selective attenuation of theproinflammatory macrophage M1 phenotype using a specificmarker such as ED1 [42] to quantify the expression of theproinflammatoryM1 phenotype in left-ventricular tissue andother markers for the assessment of anti-inflammatory M2phenotype including ED2 [42] CD14 [43 44] CD206 [8]and CD36 [45 46]

Our Western immunoblotting and relative densitometryrevealed that the basal expression of the proinflammatorymacrophageM1phenotypemarker ED1 inZFswasmarkedlyelevated by 48-fold as compared to ZL controls (Figure 3(a))but was significantly reduced by hemin by 35-fold althoughcontrol levels were not attained On the other hand thebasal expression of several markers of the anti-inflammatorymacrophage M2 phenotype including ED2 CD206 CD36and CD14 was significantly depressed in ZFs by 21- 57- 36-and 29-fold respectively (Figures 3(b) 3(c) 3(d) and 3(e))Interestingly hemin therapy greatly enhanced the depressedED2 CD206 CD36 and CD14 in ZFs by 38- 41- 23- and26-fold respectively suggesting that a novel mechanism bywhich hemin therapy blunts inflammation is by selectivelymodulating the polarization of macrophage toward the M2phenotype that dampens inflammation

34 Hemin Therapy Suppressed Macrophage Infiltration inthe Left Ventricle of ZFs Following the observation from

our Western blot experiment that hemin therapy reducedleft-ventricular ED-1 a marker of macrophage infiltrationwe use the ED-1 antibody to further confirm macrophageinfiltration in the left ventricle by immunohistochemistry(Figure 4(a)) Our results reveal that left-ventricular sec-tions from ZL-control rats were almost devoid of the darkbrown ED1 positive staining However in untreated ZF-control rats a greater number of ED1-positively stained darkbrown cells were observed indicating increased macrophageinfiltration Interestingly in hemin-treated ZFs there wasa marked reduction in the number of dark brown-stainedmacrophages suggesting reduction of macrophage infiltra-tion Correspondingly hemin therapy significantly reducedthe quantitative ED1 score (Figure 4(b))

35 HeminTherapy Enhanced Insulin Signaling but SuppressedExtracellular Matrix and Profibrotic Proteins Implicated inCardiac Injury Since visceral adiposity and elevated inflam-mation impair insulin signaling [47] we investigated theeffects of hemin therapy on the expression of important com-ponents of the insulin signal transduction pathway includingIRS-1 PI3K and GLUT4 In in ZFs the basal expression ofIRS-1 PI3K and GLUT4 was significantly reduced by 112-25- and 23-fold as compared to the ZL control (Figures 5(a)5(b) and 5(c)) but was enhanced by hemin by 57- 401- and19-fold respectively

To further confirm the antihypertrophic effect of hemintherapy we measured collagen IV an important proteinimplicated in cardiac hypertrophy and fibrosis [35] In ZFsthe basal expression of left-ventricular collagen IV wassignificantly elevated by 69-fold but was abated by heminby 28-fold (Figure 5(d)) Given that excessive deposition ofextracellular matrixprofibrotic proteins and inflammationdue to macrophage infiltration are cardinal pathophysiolog-ical events implicated in cardiac insult [47 48] while atrialnatriuretic peptide (ANP) and adiponectin are known tosuppress fibrosis caused by the deposition of extracellularmatrix [49 50] we investigated whether the concomitantpotentiation of ANP adiponectin and the HO system byhemin would abate TGF-120573 In ZFs the basal expressionof TGF-120573 was significantly elevated by 46-fold but wasmarkedly attenuated by hemin by 34-fold (Figure 5(e)) SinceTGF-120573 mobilizes the extracellular matrix by stimulatingfibronectin and collagen to cause fibrosis and cardiac injury[49 51] we also measured the expression of fibronectin InZF rats the basal expression of fibronectin was increased by75-fold butwasmarkedly attenuated by hemin therapy by 45-fold (Figure 5(f))

36 Hemin Improved Glucose Tolerance Enhanced the Insulin-Sensitizing Protein Adiponectin but Abated Insulin Resis-tance After having observed the hemin-induced potenti-ation of insulin signalling to further confirm the role ofhemin therapy on glucosemetabolism we assessed the effectsof hemin on glucose tolerance insulin resistance and theinsulin-sensitizing protein adiponectin in ZFs an obesemodel with elevated inflammation Since inflammation dueto macrophage infiltration is implicated in insulin resistance


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ricul

arED

1G

6PD

H (

)

37 kD

110 kD

G6PDH

ED1

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

130 kD

G6PDH

ED2

0

10

20

30

40

50

Left-

vent

ricul

arED

2G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

20

40

60

80

Left-

vent

ricul

arCD

206

G6P

DH

()

37 kD

170 kD

G6PDH

CD206

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

0

20

40

60

80

Left-

vent

ricul

arCD

36G

6PD

H (

)

lowast

37 kD

90 kD

G6PDH

CD36

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

15

30

45

60

Left-

vent

ricul

arCD

14G

6PD

H (

)37 kD

130 kD

G6PDH

CD14

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

Figure 3 Effects of hemin on ED1 ED2 CD206 CD36 and CD14 in left-ventricular tissue from ZLs and ZFs Hemin therapy (a) abatedED1 but (b) enhanced ED2 (c) increased CD206 (d) enhanced CD36 and (e) increased CD14 in ZFs Bars represent means plusmn SEM 119899 = 4rats per group (lowast119875 lt 001 versus all groups 119875 lt 001 versus all groups)

and cardiomyopathy [47 48] and ZFs are characterized byinsulin resistance [29] we investigated whether the hemin-dependent suppression of macrophage infiltration would beaccompanied by improved glucose metabolism In untreatedZFs IPGTT analysis showed marked increase in glycemiaas compared to ZL controls and hemin-treated ZFs at alltime points tested (Figure 6(a)) suggesting improved glucosetolerance in hemin-treated ZFs Although ZFs were hyper-insulinemic with elevated basal glycemia when challengedwith a bolus injection of glucose only to a meagre glucose-stimulated insulin release was observed (Figure 6(b)) sug-gesting glucose intolerance On the other hand glucosechallenge to ZL controls and hemin-treated ZFs greatlystimulated insulin release (Figure 6(b)) suggesting improvedglucose tolerance Hemin also reduced the elevated insulinresistance HOMA-IR in ZFs (Figure 6(c)) whereas coad-ministration with SnMP reversed the effects of hemin(Figure 6(c))

We also investigated the effects of hemin therapy onadiponectin an anti-inflammatory insulin sensitizing andcardioprotective protein [52 53] Interestingly hemin therapysignificantly enhanced the depressed basal adiponectin levelsin ZFs whereas treatment with SnMP abolished and furtherreduced the depressed levels of adiponectin (Figure 6(d))Hemin therapy also reduced HOMA-IR index in ZL controlsand enhanced adiponectin although the effectwas less intenseas compared to ZFs

37 Hemin Therapy Suppressed Left-Ventricular Fibrosis Car-diomyocyte Hypertrophy and Longitudinal Cardiac MyofibrilThickness in ZFs Histological and morphometric analysesusingMassonrsquos trichrome andhematoxylin and eosin stainingwere done to further confirm the cardioprotection by heminCardiomyocytes appeared as dark reddish while extracellular


Left-ventricular ED-1 (macrophage infiltration)

ZL control ZF control ZF + hemin

(a)

0

2

4

6

8


ED1

cells

per

fiel

d in

left

vent

ricle

(b)

Figure 4 (a) Representative photomicrographs of cross-sections of the left-ventricle showing macrophage infiltration (ED1-positive cellsstained dark brown) (magnification times200) (b) Quantitative analyses of macrophage infiltration per field indicating that in ZFs the number ofED1-positive dark-brown cells (macrophage infiltration) was markedly elevated as compared to ZL control but interestingly was significantlyattenuated by hemin therapy Bars represent means plusmn SEM 119899 = 6 rats per group (119875 lt 001 versus all groups)

matrix such as collagen stained blue (Figure 7(a)) Left-ventricular sections from ZL controls appeared morpholog-ically normal with scanty interstitial collagen deposition Incontrast left-ventricular images from ZFs showed moderate-to-severe fibrosis with scarring of cardiomyocytes and inter-stitial and perivascular collagen depositions (Figure 7(a))Interestingly hemin therapy attenuated the severity of scar-ring and intestinal and perivascular collagen deposition evi-denced by reduced extracellular and perivascular blue stain-ing (Figure 7(a)) Correspondingly semiquantitative analysisshowed that hemin therapy significantly abated the elevatedcollagen deposition and perivascular fibrosis in ZFs reinstat-ing comparable levels to ZL control (Figure 7(b))

Hemin therapy was also effective against cardiomyocytehypertrophy (Figure 7(c)) In ZFs cardiomyocytes wereenlargedwith increscent nuclei and the innermyofibril spaceswere decreased as compared to normal cardiomyocytes inZL controls (Figure 7(c)) In ZFs the longitudinal cardiacmyofibril thickness was 37 higher than that of ZL controls(Figure 7(d)) but was reduced by 27 in hemin-treatedZFs Although ZL-control values were not reinstated hemin

increased intermyofibril spaces in ZFs close to the levelsobserved in ZL controls (Figure 7)

38 Hemin Therapy Suppressed the Elevated Expression ofMarkers of Heart Failure in the Left Ventricle of ZFs Tofurther confirm the cardioprotective effects of an upregulatedHO system we investigated the effects of hemin therapy onimportant markers of heart failure such as osteopontin [13]and osteoprotegerin [14] Since left-ventricular hypertrophyis associated with heart failure [54] we determined whetherthe hemin-dependent suppression of left-ventricular hyper-trophy in ZFs would be accompanied by the reduction ofmarkers of heart failure Our results indicate that in ZFs thebasal expression levels of osteopontin and osteoprotegerinwere significantly elevated by 46- and 71-fold respectivelyas compared to ZL controls (Figures 8(a) and 8(b)) Inter-estingly treatment with hemin attenuated the expressionsof osteopontin and osteoprotegerin by 35- and 33-foldrespectively (Figures 8(a) and 8(b))


37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)

Figure 5 Effects of hemin on the expression of important proteins of the insulin signal transduction pathway such as IRS-1 PI3K GLUT4and the expression of profibroticextracellular matrix proteins including collagen IV TGF-120573 and fibronectin in left-ventricular tissue fromZLs and ZFs Representative Western immunoblotting and relative densitometry of the expressed proteins normalized by G6PHD indicatesthat hemin therapy significantly (a) enhanced IRS-1 (b) increased PI3K (c) upregulated GLUT4 but (d) abated collagen IV (e) reducedTGF-120573 and (f) suppressed fibronectin in ZFs Bars represent means plusmn SEM 119899 = 4 rats per group (lowast119875 lt 001 versus all groups 119875 lt 001versus all groups)

4 Discussion

Thepresent study indicates that themultifacetedmechanismsby which hemin therapy improves cardiomyopathy in obesityinclude (i) the suppression of visceral adiposity (ii) thereduction of macrophage M1 phenotype (iii) the attenuationof markers of heart failure (iv) the reduction of extracel-lular matrixprofibrotic proteins and (v) the ameliorationof insulin resistance with corresponding enhancement ofglucose metabolism In ZFs excessive visceral adiposityincreased macrophage infiltration and the elevated levels of8-isoprostane MIP-1120572 MCP-1 TNF-120572 IL-6 IL-1120573 and ET-1proteins of heart failure and extracellular-matrix depositionare among the complexmolecular processes that characterizethe intricate relationship between inflammation oxidative

stress cardiac fibrosis and the progressive development ofinsulin resistance and cardiomyopathy [5 47ndash49 55ndash57]Importantly the present study unveils that hemin therapyselectively enhances the anti-inflammatory macrophage M2phenotype in left-ventricular tissue of ZFs while concomi-tantly abating the proinflammatory M1 phenotype sug-gesting that a novel mechanism by which hemin therapysuppresses cardiac inflammation in obesity is by selec-tively favoring the polarization of macrophage towards theM2 phenotype that ablates inflammation Correspondinglyhemin therapy abated several chemokines and cytokines thatpromote macrophage infiltration including MIP-1120572 MCP-1TNF-120572 IL-6 and IL-1120573 [9ndash11] Interestingly the suppressionof visceral adiposity and inflammation in hemin-treatedZFs was accompanied by reduced insulin resistance and


0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger

ZF + heminZF control

ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger

ZF + heminZL controlZF control

(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)

ZL control ZF control ZF + hemin+ SnMP

ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)

ZL control ZF control ZF +hemin

ZF + hemin+ SnMP

ZL + hemin

(d)

Figure 6 Effects of hemin on glucose tolerance insulin resistance (HOMA-IR index) and adiponectin Hemin therapy (a) improved glucosetolerance (IPGTT) (b) increased glucose-stimulated insulin release (c) reduced insulin resistance and (d) increased adiponectin Barsrepresent means plusmn SEM 119899 = 6 rats per group (lowast119875 lt 005 lowastlowast119875 lt 001 versus ZL control dagger119875 lt 001 versus ZL control 119875 lt 001 versusZF+hemin+SnMP or ZF control)

improved glucose intolerance and the potentiation of impor-tant components of the insulin signal transduction pathwayincluding IRS-1 PI3K and GLUT4 which in addition tothe hemin-dependent enhancement of adiponectin an anti-inflammatory insulin sensitizing and cardioprotective pro-tein [52 53] may account for improved glucose metabolismin obese conditions

Hemin therapy also reduced LV hypertrophy cardiacfibrosis and cardiomyocyte longitudinal muscle-fiber thick-ness a pathophysiological feature of cardiomyocyte hyper-trophy [35] with a corresponding suppression of markersof heart failure such as osteopontin and osteoprotegerin[14 15] as well as the reduction of extracellularmatrix proteinlike TGF-120573 fibronectin and collagen which are implicatedin cardiac hypertrophy and fibrosis [49 57] Since TGF-120573mobilizes the extracellular matrix by stimulating fibronectinand collagen causing tissue damage and hypertrophy [4957] the concomitant reduction of TGF-120573 fibronectin and

collagen IV in ZFs may account for reduced cardiac lesionsAnother mechanism by which the HO system suppressesextracellular matrix and profibrotic agents like TGF-120573 andET-1 may be due to the HO-dependent potentiation of ANPa substance known to suppress extracellular matrix [41 58]Generally ANP and ET-1 have opposing effects [59] Forexample ANP reduces fibrosis by inhibiting TGF-1205731 andfibronectin [41] while ET-1 acts in conjunction with TGF-1205731 to stimulate the synthesis of fibronectin [58] SimilarlyANP suppresses inflammation to reduce insulin resistance[59] while ET-1 stimulates inflammatoryoxidative insultscausing insulin resistance [60] On the other hand ANPstimulates the production of adiponectin [61] a protein withinsulin-sensitizing and anti-inflammatory effects [52] Theeffects of ANP are largely mediated by cGMP [62] andadiponectin is also known to enhance cGMP [63] MoreoverANP and the HO system have mutual stimulatory effectsAccordingly ANP enhances HO [64 65] and similarly



200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)

Figure 7 Effect of hemin on histological lesions in the left ventricle of ZLs and ZFs (a) Representative Masonrsquos trichrome-stained imagesrevealing severe cardiac muscle scaring and collagen deposition in ZFs (b) Semiquantitative evaluation showed that hemin therapy reducedcollagen deposition in ZFs (c) Representative hematoxylin and eosin-stained images revealing severe longitudinal muscle-fiber thickness inZFs (d) Quantitative evaluation showed that hemin reduced longitudinal muscle-fiber thickness plusmn SEM 119899 = 6 rats per group (lowast119875 lt 005lowastlowast119875 lt 001 versus all groups)


0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)

Figure 8 Effect of hemin on markers of heart failure such as osteopontin and osteoprotogerin in the left ventricle of ZLs and ZFsRepresentative Western immunoblotting and relative densitometry of the expressed proteins normalized by G6PHD indicates that hemintherapy significantly (a) abated osteopontin and (b) reduced osteoprotogerin in ZFs Bars represent means plusmn SEM 119899 = 4 rats per group(lowast119875 lt 001 versus all groups)

the HO system has been shown to upregulate ANP andadiponectin [23 66] Therefore the synergistic potentiationof the HO-adiponectin-ANP axis and insulin signaling withcorresponding ablation of extracellular matrixheart failureproteins the reduction of oxidative stress and inflammationmediators such as macrophage M1 phenotype MIP-1120572 andMCP-1 TNF-120572 IL-6 IL-1120573 ET-1 and 8-isoprostane areamong themultifacetedmechanisms bywhich hemin therapyimproved cardiac function Thus novel strategies capable ofpotentiating the HO-adiponectin-ANP axis would improvecardiomyopathy and insulin signaling in obesity

Cardiomyocyte hypertrophy and myocardial fibrosis areearly microscopic changes in heart failure Subsequentlymacroscopic alterations including increased left-ventricularwall thickness diastolicsystolic dysfunction and impairedcardiac hemodynamics become evident Interestingly hemintherapy modulated several hemodynamic and echocardio-graphic parameters to improve cardiac function [67] Theseinclude the reduction of left-ventricular diastolic wall thick-ness left-ventricular systolic wall thickness mean-arterialpressure arterial-systolic pressure arterial-diastolic pres-sure left-ventricular developed pressure +dPdt and total-peripheral resistance with corresponding enhancement ofstroke volume and cardiac out and thus improved cardiacfunction in hemin-treated ZFsHemin therapy also enhancedthe HO system cGMP adiponectin and ANP abated 8-isoprostane MCP-1 MIP-1120572 TNF-120572 IL-6 IL-1120573 and ET-1and loweredHOMA-IR index in ZL-control rats although themagnitude was smaller as compared to ZFs with depressedHO activity The reasons for this selective effect of HOare not fully understood However it is possible that asZL controls are healthy animals with normalfunctional

insulin-signalling the HO system may be more stable ascompared to ZFs which have deregulated HO system withdepressed HO-1 and HO activity Importantly the selectivityof the HO system in diseased conditions could be exploredagainst the comorbidity of insulin-resistant diabetes andobesity

Although we recently reported the cardioprotectiveeffects of the HO system in ZDFs [13] a model character-ized by obesity insulin resistance and overt hyperglycemiapathophysiological profile of ZDF is not reflective of themetabolically healthy individuals who are characterized byobesity and normoglycemia [7] In contrast ZFs closelymimic the pathophysiological profile ofmetabolically healthyobese individuals with normoglycemia so our findings maybe applicable to this subtype of obese individuals Moreoverwith the rising incidence of cardiometabolic complicationsin many adults who previously manifested the metabolicallyhealthy obese phenotype [7] novel studies with animalmodels that closely mimic the pathophysiological profile ofmetabolically healthy obese subtype are needed Thereforestudying the effects of the HO system on ZFs may offernew perspective in the pathophysiology of cardiometaboliccomplications and especially the progressive deterioration ofcardiac function which may eventually lead to heart failuregiven the elevated levels of proteins of heart failure detectedin untreated ZFs

Collectively our study unveils the beneficial effect ofupregulating the HO system in the comorbidity of obesityand insulin resistance and suggests that the suppression ofoxidative mediators macrophage-M1-phenotype infiltrationand extracellular matrixremodeling proteins are among themultifaceted mechanisms by which the HO system main-tains and enhances insulin signaling and counteract diabetic


cardiomyopathy These data suggest that although ZFs arenormoglycemic perturbations of insulin signaling and car-diac functionmay be forerunners to overt hyperglycemia andheart failure in obesity

5 Conclusion

The novelty of our study includes (i) the hemin-inducedselective enhancement of the anti-inflammatory M2 pheno-type in left-ventricular tissue of ZFs and parallel reduction ofthe proinflammatory macrophage M1 phenotype and MIP-1120572 a chemokine implicated in macrophage infiltration (ii)the hemin-dependent suppression of heart failure proteinssuch as osteopontin and osteoprotegerin (iii) the suppressionof inflammatory cytokines in ZFs and (iv) the hemin-induced reduction of insulin resistance and improvementof cardiac function in ZFs Since we recently reportedthat the HO system suppressed pericardial adiposity in amodel characterized by obesity insulin resistance and overthyperglycemia [13] and the present study indicates thathemin therapy abates cardiac inflammation in obesity aninterorgan crosstalk of inflammatory mediators between themyocardium and pericardial adipose tissue can be envis-aged in the pathophysiology of diabetic cardiomyopathyImportantly the concomitant modulation of macrophagepolarization in left ventricles towards the anti-inflammatoryM2 phenotype alongside the parallel reduction of proinflam-matory cytokines and chemokines implicated in macrophageinfiltration and tissue destructionmay be indicative of a puta-tive interorgan crosstalk and the movement of inflammatorymediators from the pericardial fat to the myocardium orvice versa and this may be particularly important in theprogressive development of cardiomyopathy insulin resis-tance and related cardiometabolic complications Althoughthis linkage has to be established this study would setthe stage for further exploration of the putative interorgancommunication between pericardial fat and the heart

Conflict of Interests

The authors have no conflict of interests that could beperceived as prejudicing the impartiality of the researchreported

Acknowledgment

This work was supported by a grant from the Heart amp StrokeFoundation of Saskatchewan Canada to Joseph FomusiNdisang

References

[1] V S Malik W C Willett and F B Hu ldquoGlobal obesitytrends risk factors and policy implicationsrdquo Nature ReviewsEndocrinology vol 9 no 1 pp 13ndash27 2013

[2] J C Han D A Lawlor and S Y Kimm ldquoChildhood obesityrdquoThe Lancet vol 375 no 9727 pp 1737ndash1748 2010

[3] S Tiwari and J F Ndisang ldquoThe role of obesity incardiomyopa-thy and nephropathyrdquo Current Pharmaceutical Design In press

[4] B Mittendorfer and L R Peterson ldquoCardiovascular conse-quences of obesity and targets for treatmentrdquo Drug DiscoveryToday vol 5 no 1 pp 53ndash61 2008

[5] J F Ndisang and R Wang ldquoNovel therapeutic strategies forimpaired endothelium-dependent vascular relaxationrdquo ExpertOpinion on Therapeutic Patents vol 12 no 8 pp 1237ndash12472002

[6] M Bluher ldquoAre there still healthy obese patientsrdquo CurrentOpinion in Endocrinology Diabetes and Obesity vol 19 no 5pp 341ndash346 2012

[7] C den Engelsen K J Gorter P L Salome and G E RuttenldquoDevelopment of metabolic syndrome components in adultswith a healthy obese phenotype a three year follow-uprdquoObesityvol 21 no 5 pp 1025ndash1030 2013

[8] Y Hirata M Tabata H Kurobe et al ldquoCoronary atheroscle-rosis is associated with macrophage polarization in epicardialadipose tissuerdquo Journal of the American College of Cardiologyvol 58 no 3 pp 248ndash255 2011

[9] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[10] S Lallukka K Sevastianova J Perttila et al ldquoAdipose tissue isinflamed in NAFLD due to obesity but not in NAFLD due togenetic variation in PNPLA3rdquo Diabetologia vol 56 no 4 pp886ndash892 2013

[11] M Neumeier S Bauer H Bruhl et al ldquoAdiponectin stimulatesrelease of CCL2 -3 -4 and -5 while the surface abundanceof CCR2 and -5 is simultaneously reduced in primary humanmonocytesrdquo Cytokine vol 56 no 3 pp 573ndash580 2011

[12] J F Ndisang ldquoRole of heme oxygenase in inflammationinsulin-signalling diabetes and obesityrdquo Mediators of Inflam-mation vol 2010 Article ID 359732 18 pages 2010

[13] A Jadhav S Tiwari P Lee and J F Ndisang ldquoThe hemeoxygenase system selectively enhances the anti-inflammatorymacrophage-m2 phenotype reduces pericardial adiposity andameliorated cardiac injury in diabetic cardiomyopathy inzucker diabetic Fatty ratsrdquo Journal of Pharmacology and Exper-imental Therapeutics vol 345 no 2 pp 239ndash249 2013

[14] M Rosenberg C Zugck M Nelles et al ldquoOsteopontin a newprognostic biomarker in patients with chronic heart failurerdquoCirculation vol 1 no 1 pp 43ndash49 2008

[15] T Ueland C P Dahl J Kjekshus et al ldquoOsteoprotegerinpredicts progression of chronic heart failure results fromCORONArdquo Circulation vol 4 no 2 pp 145ndash152 2011

[16] M Zeyda K Gollinger J Todoric et al ldquoOsteopontin is anactivator of human adipose tissue macrophages and directlyaffects adipocyte functionrdquo Endocrinology vol 152 no 6 pp2219ndash2227 2011

[17] E S Oh E-J Rhee KWOh et al ldquoCirculating osteoprotegerinlevels are associated with age waist-to-hip ratio serum totalcholesterol and low-density lipoprotein cholesterol levels inhealthy Korean womenrdquo Metabolism vol 54 no 1 pp 49ndash542005

[18] M Keophiphath V Achard C Henegar C Rouault KClement and D L Lacasa ldquoMacrophage-secreted factorspromote a profibrotic phenotype in human preadipocytesrdquoMolecular Endocrinology vol 23 no 1 pp 11ndash24 2009

[19] H J Duckers M Boehm A L True et al ldquoHeme oxygenase-1protects against vascular constriction and proliferationrdquoNatureMedicine vol 7 no 6 pp 693ndash698 2001

[20] M Mishra and J F Ndisang ldquoA critical and comprehensiveinsight on heme oxygenase and related products including


carbonmonoxide bilirubin biliverdin and ferritin in type-1 andtype-2 diabetes rdquo Current Pharmaceutical Design In press

[21] S Tiwari and J F Ndisang ldquoThe Heme oxygenase system andtype-1 diabetesrdquo Current Pharmaceutical Design In press

[22] S Tiwari and J F Ndisang ldquoHeme oxygenase system andhypertension a comprehensive insightrdquo Current Pharmaceuti-cal Design In press

[23] J F Ndisang N Lane N Syed and A Jadhav ldquoUp-regulatingthe heme oxygenase system with hemin improves insulinsensitivity and glucose metabolism in adult spontaneouslyhypertensive ratsrdquo Endocrinology vol 151 no 2 pp 549ndash5602010

[24] J F Ndisang ldquoThe Heme oxygenase system selectively mod-ulates proteins implicated in metabolism oxidative stress andinflammation in spontaneously hypertensive ratsrdquo CurrentPharmaceutical Design In press

[25] J F Ndisang and A Jadhav ldquoUpregulating the Heme oxygenasesystem suppresses left ventricular hypertrophy in adult spon-taneously hypertensive rats for 3 monthsrdquo Journal of CardiacFailure vol 15 no 7 pp 616ndash628 2009

[26] J F Ndisang N Lane and A Jadhav ldquoUpregulation of theheme oxygenase system ameliorates postprandial and fastinghyperglycemia in type 2 diabetesrdquo The American Journal ofPhysiology vol 296 no 5 pp E1029ndashE1041 2009

[27] J F Ndisang and R Wang ldquoAge-related alterations in solubleguanylyl cyclase and cGMP pathway in spontaneously hyper-tensive ratsrdquo Journal of Hypertension vol 21 no 6 pp 1117ndash11242003

[28] J F Ndisang H E N Tabien and RWang ldquoCarbon monoxideand hypertensionrdquo Journal of Hypertension vol 22 no 6 pp1057ndash1074 2004

[29] I G Poornima P Parikh and R P Shannon ldquoDiabetic car-diomyopathy the search for a unifying hypothesisrdquo CirculationResearch vol 98 no 5 pp 596ndash605 2006

[30] J F Ndisang N Lane and A Jadhav ldquoThe heme oxygenasesystem abates hyperglycemia in zucker diabetic fatty rats bypotentiating insulin-sensitizing pathwaysrdquo Endocrinology vol150 no 5 pp 2098ndash2108 2009

[31] J F Ndisang and A Jadhav ldquoHemin therapy suppresses inflam-mation and retroperitoneal adipocyte hypertrophy to improveglucose metabolism in obese rats co-morbid with insulinresistant type-2 diabetesrdquoDiabetes Obesity andMetabolism vol15 no 11 pp 1029ndash1039 2013

[32] A Jadhav E Torlakovic and J F Ndisang ldquoInteraction amonghemeoxygenase nuclear factor-120581B and transcription activatingfactors in cardiac hypertrophy in hypertensionrdquo Hypertensionvol 52 no 5 pp 910ndash917 2008

[33] J F Ndisang and M Mishra ldquoThe Heme oxygenase systemselectively suppresses the proinflammatory macrophage M1phenotype and potentiates insulin signaling in spontaneouslyhypertensive ratsrdquo The American Journal of Hypertension vol26 no 9 pp 1123ndash1131 2013

[34] J F Ndisang and A Jadhav ldquoHemin therapy improves kidneyfunction in male streptozotocin-induced diabetic rats roleof the heme oxygenaseatrial-natriuretic peptideadiponectinaxisrdquo Endocrinology In press

[35] A Jadhav and J F Ndisang ldquoTreatment with heme arginatealleviates adipose tissue inflammation and improves insulinsensitivity and glucose metabolism in a rat model of Humanprimary aldosteronismrdquo Free Radical Biology andMedicine vol53 no 12 pp 2277ndash2286 2012

[36] A Jadhav E Torlakovic and J F Ndisang ldquoHemin therapyattenuates kidney injury in deoxycorticosterone acetate-salthypertensive ratsrdquoTheAmerican Journal of Physiology vol 296no 3 pp F521ndashF534 2009

[37] HMosen A Salehi P Alm et al ldquoDefective glucose-stimulatedinsulin release in the diabetic Goto-Kakizaki (GK) rat coincideswith reduced activity of the islet carbon monoxide signalingpathwayrdquo Endocrinology vol 146 no 3 pp 1553ndash1558 2005

[38] M LMuiesanM Salvetti CMonteduro et al ldquoLeft ventricularconcentric geometry during treatment adversely affects cardio-vascular prognosis in hypertensive patientsrdquo Hypertension vol43 no 4 pp 731ndash738 2004

[39] M Fukunaga T Yura and K F Badr ldquoStimulatory effect of8-Epi-PGF(2120572) an F2-isoprostane on endothelin-1 releaserdquoJournal of Cardiovascular Pharmacology vol 26 supplement 3pp S51ndashS52 1995

[40] N Delanty M P Reilly D Pratico et al ldquo8-epi PGF(2120572) gen-eration during coronary reperfusion a potential quantitativemarker of oxidant stress in vivordquo Circulation vol 95 no 11 pp2492ndash2499 1997

[41] M Piechota M Banach R Irzmanski et al ldquoN-terminal brainnatriuretic propeptide levels correlatewith procalcitonin andC-reactive protein levels in septic patientsrdquoCellular andMolecularBiology Letters vol 12 no 2 pp 162ndash175 2007

[42] K Aki A Shimizu Y Masuda et al ldquoANG II receptor blockadeenhances anti-inflammatory macrophages in anti-glomerularbasement membrane glomerulonephritisrdquo The American Jour-nal of Physiology vol 298 no 4 pp F870ndashF882 2010

[43] A Anzai T Anzai S Nagai et al ldquoRegulatory role of dendriticcells in postinfarction healing and left ventricular remodelingrdquoCirculation vol 125 no 10 pp 1234ndash1245 2012

[44] M Heusinkveld P J de vos van Steenwijk R Goedemans et alldquoM2 macrophages induced by prostaglandin E2 and IL-6 fromcervical carcinoma are switched to activated M1 macrophagesby CD4+ Th1 cellsrdquo Journal of Immunology vol 187 no 3 pp1157ndash1165 2011

[45] A Bhattacharjee S Pal G M Feldman and M K CathcartldquoHck is a key regulator of gene expression in alternativelyactivated human monocytesrdquo Journal of Biological Chemistryvol 286 no 42 pp 36709ndash36723 2011

[46] S Gibbings N D Elkins H Fitzgerald et al ldquoXanthineoxidoreductase promotes the inflammatory state of mononu-clear phagocytes through effects on chemokine expressionperoxisome proliferator-activated receptor-120574 sumoylation andHIF-1120572rdquo Journal of Biological Chemistry vol 286 no 2 pp 961ndash975 2011

[47] G A Rosito J M Massaro U Hoffmann et al ldquoPericardialfat visceral abdominal fat cardiovascular disease risk factorsand vascular calcification in a community-based sample theframingham heart studyrdquo Circulation vol 117 no 5 pp 605ndash613 2008

[48] H Sell C Habich and J Eckel ldquoAdaptive immunity in obesityand insulin resistancerdquo Nature Reviews vol 8 no 12 pp 709ndash716 2012

[49] P Li D Wang J Lucas et al ldquoAtrial natriuretic peptide inhibitstransforming growth factor 120573-induced Smad signaling andmyofibroblast transformation in mouse cardiac fibroblastsrdquoCirculation Research vol 102 no 2 pp 185ndash192 2008

[50] F Fang L Liu Y Yang et al ldquoThe adipokine adiponectinhas potent anti-fibrotic effects mediated via adenosinemonophosphate-activated protein kinase novel target for


fibrosis therapyrdquo Arthritis Research and Therapy vol 14 no 5article R229 2012

[51] M Dobaczewski C Gonzalez-Quesada and N G Frangogian-nis ldquoThe extracellular matrix as a modulator of the inflamma-tory and reparative response following myocardial infarctionrdquoJournal of Molecular and Cellular Cardiology vol 48 no 3 pp504ndash511 2010

[52] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo Journal of Biolog-ical Chemistry vol 284 no 38 pp 25569ndash25575 2009

[53] K Robinson J Prins and B Venkatesh ldquoClinical reviewadiponectin biology and its role in inflammation and criticalillnessrdquo Critical Care vol 15 no 2 article 221 2011

[54] T A McKinsey and E N Olson ldquoToward transcriptionaltherapies for the failing heart chemical screens to modulategenesrdquo Journal of Clinical Investigation vol 115 no 3 pp 538ndash546 2005

[55] M Konishi S Sugiyama Y Sato et al ldquoPericardial fat inflam-mation correlates with coronary artery diseaserdquo Atherosclerosisvol 213 no 2 pp 649ndash655 2010

[56] M O Al Chekakie C C Welles R Metoyer et al ldquoPericardialfat is independently associated with human atrial fibrillationrdquoJournal of the American College of Cardiology vol 56 no 10 pp784ndash788 2010

[57] J Ren M Yang G Qi et al ldquoProinflammatory protein CARD9is essential for infiltration of monocytic fibroblast precursorsand cardiac fibrosis caused by angiotensin II infusionrdquo TheAmerican Journal of Hypertension vol 24 no 6 pp 701ndash7072011

[58] X Shi-Wen L Kennedy E A Renzoni et al ldquoEndothelin is adownstream mediator of profibrotic responses to transforminggrowth factor 120573 in human lung fibroblastsrdquo Arthritis andRheumatism vol 56 no 12 pp 4189ndash4194 2007

[59] C Moro E Klimcakova K Lolmede et al ldquoAtrial natriureticpeptide inhibits the production of adipokines and cytokineslinked to inflammation and insulin resistance in human sub-cutaneous adipose tissuerdquoDiabetologia vol 50 no 5 pp 1038ndash1047 2007

[60] Y Chien Y-H Lai C F Kwok and L-T Ho ldquoEndothelin-1suppresses long-chain fatty acid uptake and glucose uptake viadistinct mechanisms in 3T3-L1 adipocytesrdquo Obesity vol 19 no1 pp 6ndash12 2011

[61] O Tsukamoto M Fujita M Kato et al ldquoNatriuretic peptidesenhance the production of adiponectin in human adipocytesand in patients with chronic heart failurerdquo Journal of theAmerican College of Cardiology vol 53 no 22 pp 2070ndash20772009

[62] K N Pandey ldquoBiology of natriuretic peptides and their recep-torsrdquo Peptides vol 26 no 6 pp 901ndash932 2005

[63] R Riba B Patel A Aburima and K M Naseem ldquoGlobu-lar adiponectin increases cGMP formation in blood plateletsindependently of nitric oxiderdquo Journal of Thrombosis andHaemostasis vol 6 no 12 pp 2121ndash2131 2008

[64] A K Kiemer N Bildner N C Weber and A M VollmarldquoCharacterization of heme oxygenase 1 (heat shock protein 32)induction by atrial natriuretic peptide in human endothelialcellsrdquo Endocrinology vol 144 no 3 pp 802ndash812 2003

[65] T Polte A Hemmerle G Berndt N Grosser A Abate andH Schroder ldquoAtrial natriuretic peptide reduces cyclosporintoxicity in renal cells role of cGMP and heme oxygenase-1rdquo FreeRadical Biology and Medicine vol 32 no 1 pp 56ndash63 2002

[66] J F Ndisang and A Jadhav ldquoHeme arginate therapy enhancedadiponectin and atrial natriuretic peptide but abatedendothelin-1 with attenuation of kidney histopathologicallesions in mineralocorticoid-induced hypertensionrdquo Journal ofPharmacology and Experimental Therapeutics vol 334 no 1pp 87ndash98 2010

[67] J Liu C S Fox D A Hickson et al ldquoPericardial fat andechocardiographic measures of cardiac abnormalities the jack-son heart studyrdquo Diabetes Care vol 34 no 2 pp 341ndash346 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


healthy obese phenotype [7] suggesting that individuals witha healthy obese phenotype may not remain healthy for theirentire lives Several parameters including environmental andbehavioral factors may modify obesity subphenotypes andthe transition from healthy to unhealthy Whether healthyobese individuals can maintain insulin sensitivity duringthe entire life or whether healthy obesity simply representsdelayed onset of obesity related cardiometabolic complica-tions has to be clarified

In obesity excessive oxidative stress intense inflamma-tory activity insulin resistance deregulated lipidmetabolismaltered glucose metabolism and impaired mitochondrialbiogenesis are among the pathophysiological driving forcesthat precede the early stages of cardiac dysfunction Manycardiac complications have the common denominator ofelevated inflammation due to the infiltration of macrophageM1 phenotype [8] Generally macrophages exhibit two dif-ferent forms dubbed ldquoclassicalrdquo or M1 phenotype and ldquoalter-nativerdquo or M2 phenotype [8] and each phenotype expressesdistinct patterns of surface receptors when responding todifferent stimuli TheM1 phenotype stimulates inflammationwhile the M2 phenotype blunts inflammation [8] Duringmacrophage infiltration the M1 phenotype is stimulated bydifferent chemokines including macrophage inflammatoryprotein-1 alpha ((MIP-1120572) chemokine (C-C motif) ligand-3(CCL3)) and macrophage chemoattractant protein-1 ((MCP-1) [9] chemokine (C-C motif ligand-2 (CCL2)) [9] Theactivation of the macrophage M1 phenotype is generallyassociated with elevated levels of proinflammatory cytokinesincluding TNF-120572 IL-6 and IL-1120573 [10ndash12] Moreover thelevels of macrophage M1 phenotype MCP-1 TNF-120572 IL-6 and IL-1120573 are elevated in obesity and insulin resistance[9 10 13] and these factors play a major pathophysiolog-ical role in heart failure [4] In obesity markers of heartfailure such as osteopontin [14] and osteoprotegerin [15]are elevated [16 17] Similarly the levels of extracellularmatrixprofibrotic proteins like transforming growth factorbeta (TGF-120573) collagen and fibronectin are elevated in obe-sity [18]Therefore in obesity elevated chemokines cytokinesand increased macrophage-M1 infiltration would act inconcert with elevated extracellular matrixprofibrotic andheart-failure proteins to exacerbate cardiac tissue destruc-tion and compromise heart function Thus novel strategiescapable of selectively suppressingmacrophageM1 phenotypeproinflammatory cytokineschemokines and extracellularmatrixprofibrotic proteins are needed

In many pathophysiological conditions various stress-response immune-regulatory proteins including heme oxy-genase (HO-1) are activated as an innate defense mechanism[19ndash22] However the pathophysiological activation of HO-1 may only result in a transient or marginal increase of HOactivity that falls below the threshold necessary to activatethe downstream signaling components through which theHO system elicits its cytoprotective effects so a robust andsurmountable increase of HO activity with HO inducerslike hemin may be needed for cardioprotection [23ndash27]Generally HO is composed of two main isoforms (HO-1or inducible) and (HO-2 or constitutive) which are largelyresponsible for the antioxidant and anti-inflammatory effects

of HO [28] We recently reported the cardioprotective effectsof the HO system in Zucker diabetic fatty rats (ZDFs) [13] amodel characterized by obesity insulin resistance and overthyperglycemia However because ZDFs are hyperglycemictheir pathophysiological profile is not reflective of individualsdubbed ldquometabolically healthyrdquo a subtype of obesity char-acterized by normoglycemia [7] Given that the incidenceof cardiometabolic complications is increasing in manyadults who previously manifested the metabolically healthyobese phenotype [7] novel studies with animal models thatclosely mimic the pathophysiological profile of metabolicallyhealthy obese subtype are needed Therefore this study willinvestigate the effects of the HO system on cardiometaboliccomplications in Zucker fatty rats (ZFs) an obese modelwith normoglycemia and cardiometabolic complications [29]that closely mimic the pathophysiological profile of metabol-ically healthy obese individuals with normoglycemia and anapparent state of good health Although the HO system iscytoprotective its effects on cardiomyopathy in ZFs remainto be elucidated

Since dysfunctional insulin signaling obesity elevatedinflammation and cardiac hypertrophy are forerunners toheart failure this study will also investigate the multifacetedmechanisms by which theHO system preserves cardiac func-tion in ZFs Whether an upregulated HO system by hemin iscapable of modulating macrophage polarization towards theM2 phenotype that blunts inflammation while suppressingthe proinflammatory M1 phenotype will be assessed Asmacrophage infiltration is stimulated by chemokines likeMIP-1120572 and MCP-1 [9] and the effects of the HO system onthese chemokines in ZFs have not been reported this studywill also determine left-ventricular MIP-1120572 and MCP-1 andcorrelate changes of these chemokines to the expression ofthe proinflammatory macrophage-M1 phenotype in the leftventricle of ZFs Similarly the effect of hemin therapy onimportant markers of heart failure such as osteopontin [13]and osteoprotegerin [14] will be investigated Importantly nostudy has reported the levels of expression of osteopontinand osteoprotegerin in myocardial tissue of ZFs Thereforethis study will unveil the multifaceted mechanisms by whichhemin therapy improves cardiac function and insulin signal-ing in obesity

2 Materials and Methods

21 Animals Treatment Groups and Biochemical ParametersOur experimental protocol was in conformity with theGuide for Care and Use of Laboratory Animals stipulatedby the Canadian Council on Animal Care and the NationalInstitutes of Health (NIH Publication no 85-23 revised 1996)and was approved by University of Saskatchewan AnimalEthics Committee Male ZFs (12 weeks old) and sexage-matched Zucker lean (ZL) rats were purchased from CharlesRiver Laboratories (Willington MA USA) The animalswere housed at 21∘C with 12-hour lightdark cycles fed withstandard laboratory chow and had access to drinking waterad libitum











0

7

14

21

28

35

42

Left-

vent

ricul

ar H

O-1

conc

entr

atio

n (n

gm

L)

lowast

dagger



ZF + hemin+ SnMP

(a)

lowast

dagger daggerLeft-

vent

ricul

ar H

O ac

tivity

(nm

ol b

iliru

bin

mg

prot

ein

hr)

0

4

8

12

16



ZF + hemin+ SnMP

(b)

0

5

10

15

20

25

Left-

vent

ricul

ar cG

MP

(pm

olm

g pr

otei

n)

daggerdagger

lowast



ZF + hemin+ SnMP

(c)


3 Results











dagger























0

2

4

6


lowastlowast



ZF + hemin+ SnMP

Left-

vent

ricul

ar en

doth

elin

-1 (p

gm

L)

(a)


lowast

0

20

40

60

80

100



ZF + hemin+ SnMP

Urin

ary

8-iso

pros

tane

(ng

24 h

rs)

(b)

daggerdaggerdagger

lowastlowast

0

300

600

900



ZF + hemin+ SnMP

Left-

vent

ricul

ar A

NP

(ng

mg

prot

ein)

(c)

daggerdagger

daggerdagger

lowast

0

4

8

12



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(pg

mg

prot

ein)

(d)

daggerdagger

daggerdagger

lowast

00

09

18

27



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(ng

mg

prot

ein)

(e)

lowast


0

20

40

60

80

100

120



ZF + hemin+ SnMP

Left-

vent

ricul

ar T

NF-120572

(pg

mL)

(f)
















0

8

16

24

32

40

Left-

vent

ricul

arED

1G

6PD

H (

)

37 kD

110 kD

G6PDH

ED1

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

130 kD

G6PDH

ED2

0

10

20

30

40

50

Left-

vent

ricul

arED

2G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

20

40

60

80

Left-

vent

ricul

arCD

206

G6P

DH

()

37 kD

170 kD

G6PDH

CD206

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

0

20

40

60

80

Left-

vent

ricul

arCD

36G

6PD

H (

)

lowast

37 kD

90 kD

G6PDH

CD36

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

15

30

45

60

Left-

vent

ricul

arCD

14G

6PD

H (

)37 kD

130 kD

G6PDH

CD14

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)








(a)

0

2

4

6

8


ED1

cells

per

fiel

d in

left

vent

ricle

(b)







37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)


4 Discussion




0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























0

7

14

21

28

35

42

Left-

vent

ricul

ar H

O-1

conc

entr

atio

n (n

gm

L)

lowast

dagger



ZF + hemin+ SnMP

(a)

lowast

dagger daggerLeft-

vent

ricul

ar H

O ac

tivity

(nm

ol b

iliru

bin

mg

prot

ein

hr)

0

4

8

12

16



ZF + hemin+ SnMP

(b)

0

5

10

15

20

25

Left-

vent

ricul

ar cG

MP

(pm

olm

g pr

otei

n)

daggerdagger

lowast



ZF + hemin+ SnMP

(c)


3 Results











dagger























0

2

4

6


lowastlowast



ZF + hemin+ SnMP

Left-

vent

ricul

ar en

doth

elin

-1 (p

gm

L)

(a)


lowast

0

20

40

60

80

100



ZF + hemin+ SnMP

Urin

ary

8-iso

pros

tane

(ng

24 h

rs)

(b)

daggerdaggerdagger

lowastlowast

0

300

600

900



ZF + hemin+ SnMP

Left-

vent

ricul

ar A

NP

(ng

mg

prot

ein)

(c)

daggerdagger

daggerdagger

lowast

0

4

8

12



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(pg

mg

prot

ein)

(d)

daggerdagger

daggerdagger

lowast

00

09

18

27



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(ng

mg

prot

ein)

(e)

lowast


0

20

40

60

80

100

120



ZF + hemin+ SnMP

Left-

vent

ricul

ar T

NF-120572

(pg

mL)

(f)
















0

8

16

24

32

40

Left-

vent

ricul

arED

1G

6PD

H (

)

37 kD

110 kD

G6PDH

ED1

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

130 kD

G6PDH

ED2

0

10

20

30

40

50

Left-

vent

ricul

arED

2G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

20

40

60

80

Left-

vent

ricul

arCD

206

G6P

DH

()

37 kD

170 kD

G6PDH

CD206

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

0

20

40

60

80

Left-

vent

ricul

arCD

36G

6PD

H (

)

lowast

37 kD

90 kD

G6PDH

CD36

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

15

30

45

60

Left-

vent

ricul

arCD

14G

6PD

H (

)37 kD

130 kD

G6PDH

CD14

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)








(a)

0

2

4

6

8


ED1

cells

per

fiel

d in

left

vent

ricle

(b)







37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)


4 Discussion




0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























dagger























0

2

4

6


lowastlowast



ZF + hemin+ SnMP

Left-

vent

ricul

ar en

doth

elin

-1 (p

gm

L)

(a)


lowast

0

20

40

60

80

100



ZF + hemin+ SnMP

Urin

ary

8-iso

pros

tane

(ng

24 h

rs)

(b)

daggerdaggerdagger

lowastlowast

0

300

600

900



ZF + hemin+ SnMP

Left-

vent

ricul

ar A

NP

(ng

mg

prot

ein)

(c)

daggerdagger

daggerdagger

lowast

0

4

8

12



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(pg

mg

prot

ein)

(d)

daggerdagger

daggerdagger

lowast

00

09

18

27



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(ng

mg

prot

ein)

(e)

lowast


0

20

40

60

80

100

120



ZF + hemin+ SnMP

Left-

vent

ricul

ar T

NF-120572

(pg

mL)

(f)
















0

8

16

24

32

40

Left-

vent

ricul

arED

1G

6PD

H (

)

37 kD

110 kD

G6PDH

ED1

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

130 kD

G6PDH

ED2

0

10

20

30

40

50

Left-

vent

ricul

arED

2G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

20

40

60

80

Left-

vent

ricul

arCD

206

G6P

DH

()

37 kD

170 kD

G6PDH

CD206

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

0

20

40

60

80

Left-

vent

ricul

arCD

36G

6PD

H (

)

lowast

37 kD

90 kD

G6PDH

CD36

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

15

30

45

60

Left-

vent

ricul

arCD

14G

6PD

H (

)37 kD

130 kD

G6PDH

CD14

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)








(a)

0

2

4

6

8


ED1

cells

per

fiel

d in

left

vent

ricle

(b)







37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)


4 Discussion




0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6


lowastlowast



ZF + hemin+ SnMP

Left-

vent

ricul

ar en

doth

elin

-1 (p

gm

L)

(a)


lowast

0

20

40

60

80

100



ZF + hemin+ SnMP

Urin

ary

8-iso

pros

tane

(ng

24 h

rs)

(b)

daggerdaggerdagger

lowastlowast

0

300

600

900



ZF + hemin+ SnMP

Left-

vent

ricul

ar A

NP

(ng

mg

prot

ein)

(c)

daggerdagger

daggerdagger

lowast

0

4

8

12



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(pg

mg

prot

ein)

(d)

daggerdagger

daggerdagger

lowast

00

09

18

27



ZF + hemin+ SnMP

Left-

vent

ricul

ar M

IP-1120572

(ng

mg

prot

ein)

(e)

lowast


0

20

40

60

80

100

120



ZF + hemin+ SnMP

Left-

vent

ricul

ar T

NF-120572

(pg

mL)

(f)
















0

8

16

24

32

40

Left-

vent

ricul

arED

1G

6PD

H (

)

37 kD

110 kD

G6PDH

ED1

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

130 kD

G6PDH

ED2

0

10

20

30

40

50

Left-

vent

ricul

arED

2G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

20

40

60

80

Left-

vent

ricul

arCD

206

G6P

DH

()

37 kD

170 kD

G6PDH

CD206

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

0

20

40

60

80

Left-

vent

ricul

arCD

36G

6PD

H (

)

lowast

37 kD

90 kD

G6PDH

CD36

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

15

30

45

60

Left-

vent

ricul

arCD

14G

6PD

H (

)37 kD

130 kD

G6PDH

CD14

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)








(a)

0

2

4

6

8


ED1

cells

per

fiel

d in

left

vent

ricle

(b)







37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)


4 Discussion




0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























0

8

16

24

32

40

Left-

vent

ricul

arED

1G

6PD

H (

)

37 kD

110 kD

G6PDH

ED1

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

130 kD

G6PDH

ED2

0

10

20

30

40

50

Left-

vent

ricul

arED

2G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

20

40

60

80

Left-

vent

ricul

arCD

206

G6P

DH

()

37 kD

170 kD

G6PDH

CD206

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

0

20

40

60

80

Left-

vent

ricul

arCD

36G

6PD

H (

)

lowast

37 kD

90 kD

G6PDH

CD36

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

15

30

45

60

Left-

vent

ricul

arCD

14G

6PD

H (

)37 kD

130 kD

G6PDH

CD14

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)








(a)

0

2

4

6

8


ED1

cells

per

fiel

d in

left

vent

ricle

(b)







37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)


4 Discussion




0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(a)

0

2

4

6

8


ED1

cells

per

fiel

d in

left

vent

ricle

(b)







37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)


4 Discussion




0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















37 kD

185 kD

G6PDH

IRS1

0

20

40

60

80

Left-

vent

ricul

arIR

S1G

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(a)

37 kD

190 kD

G6PDH

PI3K

0

15

30

45

60

Left-

vent

ricul

arPI

3KG

6PD

H (

)

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(b)

0

8

16

24

32

40

Left-

vent

ricul

arG

LUT4

G6P

DH

()

37 kD

50 kD

G6PDH

GLUT4

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(c)

37 kD

190 kD

G6PDH

Collagen IV

0

15

30

45

60

Left-

vent

ricul

arco

llage

n-IV

G6P

DH

()

lowast

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(d)

0

8

16

24

32

Left-

vent

ricul

arTG

F-120573

G6P

DH

()

37 kD

13 kD

G6PDH

TGF-120573

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(e)

0

25

50

75

Left-

vent

ricul

arfib

rone

ctin

G6P

DH

()

233 kD

37 kDG6PDH

Fibronectin

ZLcontrol

ZFcontrol

ZF +hemin

ZLcontrol

ZFcontrol

ZF +hemin

(f)


4 Discussion




0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

7

14

21

28

dagger

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

IPGTT

dagger

dagger

dagger


ZL control

(a)

2

4

6

8

10

Time (min)0 30 60 90 120

Plas

ma i

nsul

in (m

gL)

daggerdagger

daggerdagger

dagger


(b)

0

50

100

150dagger

dagger

lowast

HO

MA-

insu

lin re

sista

nce

(mg

dLlowast

mU

mL)


ZL + hemin

ZF + hemin

(c)

0

5

10

15

20

25

daggerdagger

lowastlowast

Plas

ma a

dipo

nect

in (120583

gm

L)


ZF + hemin+ SnMP

ZL + hemin

(d)







200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















200x 200x 200x

(a)C

olla

gen

scor

e


lowastlowast25

20

15

10

05

00

(b)


200x 200x 200x

(c)

Myo

cyte

s thi

ckne

ss (120583

m) lowast


30

20

10

0

(d)



0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

12

24

36

48

60

lowast

Left-

vent

ricul

aros

teop

ontin

G6P

DH

()

37 kD

55 kD

G6PDH

Osteopontin



(a)

0

15

30

45

60

75

lowast

Left-

vent

ricul

aros

teop

rote

gerin

G6P

DH

()

37 kD

60 kD

G6PDH

Osteoprotegerin



(b)









5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article The Risk of Heart Failure and...

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