Research Article A Comparison of Inflammatory and Oxidative Stress Markers in Adipose Tissue from Weight-Matched Obese Male and Female Mice

Transcription

1 Hindwi Pulishing Corportion Experimentl Dietes Reserh Volume 1, Artile ID , 8 pges doi:1.1155/1/ Reserh Artile A Comprison of Inflmmtory nd Oxidtive Stress Mrkers in Adipose Tissue from Weight-Mthed Oese Mle nd Femle Mie Kren J. Nikelson, 1 Kelly L. Stromsdorfer, 1 R. Tylor Pikering, 1 Tzu-Wen Liu, 1 Lur C. Ortinu, 1 Aileen F. Keting, nd Jmes W. Perfield II 1, 3 1 Deprtment of Nutrition nd Exerise Physiology, University of Missouri-Columi, 5 Willim Stringer Wing, Columi, MO 511, USA Deprtment of Animl Siene, Iow Stte University, Ames, IA 511, USA 3 Deprtment of Food Siene, University of Missouri-Columi, 5 Willim Stringer Wing, Columi, MO 511, USA Correspondene should e ddressed to Jmes W. Perfield II, perfieldj@missouri.edu Reeived 9 Ferury 1; Aepted 1 April 1 Ademi Editor: Pietro Glssetti Copyright 1 Kren J. Nikelson et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Expnsion of intr-dominl dipose tissue nd the ompnying inflmmtory response hs een put forwrd s unifying link etween oesity nd the development of hroni diseses. However, n pprent sexul dimorphism exists etween oesity nd hroni disese risk due to differenes in the distriution nd undne of dipose tissue. A rnge of experimentl protools hve een employed to demonstrte the role of estrogen in regulting helth enefits; however, most studies re onfounded y signifint differenes in ody weight nd diposity. Therefore, the purpose of this study ws to ompre weight-mthed oese mle nd femle mie to determine if the sex-dependent helth enefits remin when ody weight is similr. The development of oesity in femle mie reeiving high-ft diet ws delyed; however, susequent omprisons of weight-mthed oese mie reveled greter diposity in oese femle mie. Despite exess diposity nd enlrged dipoyte size, oese femles remined more gluose tolernt thn weight-mthed mle mie, nd this enefit ws ssoited with inresed expression of diponetin nd redutions in immune ell infiltrtion nd oxidtive stress in dipose tissue. Therefore, the protetive enefits of estrogen persist in the oese stte nd pper to improve the metoli phenotype of dipose tissue nd the individul. 1. Introdution Oesity is widely regrded s n independent risk ftor for rnge of hroni diseses inluding type dietes nd rdiovsulr disese [1, ]. Low-grde systemi inflmmtion hs een put forwrd s unifying link etween oesity nd the onset of these oesity-ssoited diseses [3 5]. Expnsion of intr-dominl dipose tissue is ssoited with inresed infiltrtion nd tivtion of immune ells, nd these events re signifint ontriutor to the systemi inflmmtion tht ours with oesity [, 7]. While n ext explntion for the umultion of immune ells in dipose tissue is unknown, one potentil ontriuting ftor iselevtedoxidtive stress [8, 9]. Therefore, deresing intrdominl oesity nd/or reduing dipose tissue oxidtive stress nd inflmmtion will positively influene hroni disese risk. Cler sex-sed differenes exist in dipose tissue distriution, inflmmtion, nd ultimtely the proility of developing hroni disese [1 1]. Speifilly, femles tend to hve higher ody ft ontent with the ft lolized suutneously while mles hve less totl ody ft nd their dipose tissue predomintes in the viserl region. Furthermore, niml studies hve demonstrted tht dietindued oesity nd insulin resistne our muh more rpidly in mle rodents s ompred to femles [13 15]. Estrogen is mjor ftor involved in this sexul dimorphism s it promotes suutneous ft umultion, hs ntiinflmmtory properties, nd is strong regultor of ppetite nd energy expenditure [1, 1, 1, 17]. To help eluidte

2 Experimentl Dietes Reserh the effets of estrogen on oesity, dipose tissue distriution, inflmmtion, nd insulin resistne studies hve utilized models of ovrietomy with or without repletion of estrogen nd/or ompred mle nd femle mie provided highft diet [13 15, 18]. While the outome mesures of these studies vried, they ll lerly demonstrte enefiil effet of estrogen. However, these studies were lso onfounded y ody weight differenes s intt femles or nimls reeiving estrogen were typilly smller nd hd smller dipose tissue depots. Therefore, the purpose of the urrent study ws to ompre weight-mthed oese mle nd femle mie to determine if the sex-dependent improvements in metoli helth our independent of differenes in ody weight. Following hroni exposure to high-ft diet, gluose tolerne test ws performed nd differenes in mrkers for inflmmtory nd oxidtive stress were ssessed in dipose tissue. Our dt demonstrte tht gluose tolerne remins improved in oese femle mie independent of differene in ody weight. Furthermore, despite inreses in totl diposity nd gondl dipoyte size, the oese femle mie displyed lower expression of mrkers for immune ells nd oxidtive stress whih re onsistent with n improved metoli phenotype.. Methods.1. Animls nd Animl Cre. The University of Missouri Animl Cre nd Use ommittee pproved ll proedures involving mie. Animls were mintined t ontrolled temperture ( C) nd 1-hour light: 1-hour drk yle. Six-to eight-week old mle nd femle C57BL/ mie were individully housed nd fed either how (Purin 51;.5 g/1 g ft) or high-ft diet (HFD; Reserh Diets D19; 35 g/1 g ft) for the durtion of the experiment. Body weight ws mesured weekly nd mie were kept on tretment until the verge ody weight of the HFD group ws 5 g. At this point, gluose tolerne testing nd tissue olletion were performed on the HFD group nd their gemthed how-fed ounterprts... Gluose Tolerne Testing. One the HFD-fed group rehed ody weight of 5 g; gluose tolerne test ws performed in oth HFD nd how-fed nimls. Following n overnight fst, seline lood smple ws tken from the til vein t time. Then n intrperitonel injetion of gluose (1 g/kg BW) ws dministered nd lood gluose onentrtions were determined using hndheld gluometer t 3,, 9, nd 1 minutes postinjetion. Gluose re under the urve (AUC) lultions were performed using GrphPd Prism softwre..3. Tissue Colletion. One week fter the gluose tolerne tests were performed, nimls were fsted 1 1 hours nd lood gluose ws mesured vi til nik. Animls were then euthnized y CO sphyxition followed y exsnguintion vi rdi punture. Plsm ws seprted y entrifugtion, liquoted, nd frozen for future nlysis. Gondl nd suutneous dipose tissues were exised, weighed, nd snp frozen for gene expression nlysis or fixed for histologil nlysis... Histologil Anlysis of Adipose Tissue. A portion of the gondl dipose tissue ws fixed in % prformldehyde, emedded in prffin, setioned, nd stined with hemtoxylin nd eosin (H&E). Digitl imges were quired with n Olympus BX51 light mirosope using n Olympus DP7 mer. Ded dipoytes were quntified y identifition of rown-like strutures (CLSs) within histologi setions of dipose tissue. The perentge of CLS present in gondl dipose tissue ws lulted nd used for omprison mong experimentl groups. Adipoyte volume ws lulted using the ross-setionl re otined from perimeter trings using Imge J softwre (Sun Mirosystems, Snt Clr, CA, USA)..5. Plsm Anlysis. An estrdiol EIA kit (Cymn Chemil Compny) ws used to determine fsting (overnight) plsm estrdiol onentrtions of femle mie... Rel-Time Quntittive PCR. Totl mrna ws extrted from dipose using RNesy lipid tissue kits with onolumn DNse digestion (Qigen). Purity nd onentrtion were determined with Nnodrop 1 spetrophotometer (Thermo Sientifi). 1 μg of RNA ws used to synthesize DNA with reverse trnsriptse polymerse hin retion kit (Applied Biosystems) nd diluted to 1 ng/μl. Expression of mrna ws determined using SYBR green qrt-pcr on n Applied Biosystems StepOne Plus RT-PCR system. Fold differene for gene expression ws lulted using ΔΔCT using the endogenous ontrol gene RPS Sttistil Anlysis. Tretment differenes were nlyzed y one-wy nlysis of vrine (ANOVA) with min effet signifine set t P <.5. Signifint min effets were followed y Tukey s multiple omprison test. Vlues re reported s men ± stndrd error. 3. Results 3.1. Onset of Oesity Is Delyed in Femle Mie Reeiving High-Ft Diet. Mle nd femle C57BL/ J mie reeived either low-ft how diet or HFD. The HFD indued more rpid ody weight gin in mle mie whih hieved the trget ody weight of 5 g in 1 weeks, while femle mie required n dditionl 17 weeks of HFD to reh the sme ody weight (Tle 1). This experimentl design ensured tht mie would e studied t ody weight ssoited with estlished oesity nd insulin resistne [] nd tht ody weight would not e onfounding vrile. However, due to the delyed ody weight gin in femle HFD mie ge did emerge s n unounted for vrile. Interestingly, despite hving similr ody weights, the diposity of the femle HFD mie ws greter thn the mle HFD group s oth gondl nd suutneous dipose tissue msses were signifintly inresed (Tle 1). Fsting lood gluose

3 Experimentl Dietes Reserh 3 Tle 1: Chrteristis of mle nd femle C57BL/ mie reeiving stndrd rodent how or high-ft diet (HFD). 1 Mle how Mle HFD Femle how Femle HFD Body weight (g) 8.8 ± ± 1..8 ±.5.5 ±. Age (weeks) Gondl AT 3 (g).51 ±.8.5 ±.17. ± ±.35 Suutneous AT (g).3 ±.1.9 ±..7 ±. 3.3 ±.8 Blood gluose (mg/dl) 99 ± 8 1 ± ± 8 11 ± 5 Plsm estrdiol (pg/ml) 9.5 ± ±.35 1 Dt re presented s mens ± SE; mens with different supersripts differ P<.5; n = 5 8. Age t whih the HFD-fed group rehed ody weight of 5 g nd metoli testing nd tissue olletion ourred. 3 AT; dipose tissue. onentrtions were elevted y oesity in the mle mie ut not the femle mie. Oesity lso did not lter plsm estrdiol onentrtions in the femle mie (Tle 1). 3.. Oese Femle Mie Hve Improved Gluose Tolerne s Compred to Weight-Mthed Oese Mle Mie. When the verge ody weight of HFD-fed mle nd femle mie rehed 5 g, n intrperitonel gluose tolerne test ws performed on tht experimentl group nd their gemthed how-fed ounterprts. Gluose re under the urve lultions reveled etter gluose tolerne in the femle HFD group s ompred to the mle HFD mie (Figure 1). This improvement is espeilly noteworthy sine there ws no differene in ody weight etween these two groups nd lso, the HFD femles hd greter totl ft mss (Tle 1). Further, despite the ge differene, gluose tolerne ws similr etween mle how nd femle how mie (Figure 1) Immune Cell Infiltrtion nd Oxidtive Stress Mrkers Are Redued in the Adipose Tissue of Oese Femle Mie. In order to etter understnd the oserved gluose tolerne differenes, we hrterized mrkers of inflmmtion nd oxidtive stress in gondl dipose tissue. In the urrent study, gondl dipose tissue mss ws greter in femle HFD mie s ompred to the mle HFD group (Tle 1). Consistent with n inresed mss of dipose tissue, reltive mrna expression of leptin in gondl dipose tissue ws elevted in oth mle HFD nd femle HFD groups with no differene etween genders oserved (Figure ). Interestingly, gondl dipose tissue mrna expression of diponetin ws redued in mle HFD mie ut remined unhnged in femle HFD mie (Figure ). Adiponetin expression is often orrelted with smller dipoyte size. Therefore, gondl dipoyte size ws quntified, nd surprisingly femle HFD mie hd lrger verge dipoyte size s ompred to mle HFD mie (Figure 3). This pprent disonnet etween dipoyte size nd diponetin expression my help explin the improvements in gluose tolerne. Inreses in dipoyte size lso re orrelted with n inresed presene of immune ells in dipose tissue [, 19]. Crown-like strutures (CLSs) re lusters of proinflmmtory immune ells tht lolize to ded dipoytes within dipose tissue []. Consistent with previous reports [, 1], gondl dipose tissue from the mle HFD group ontined elevted numers of CLS (Figure 3). Interestingly, the presene of CLS in the gondl dipose tissue of femle HFD mie ws redued y greter thn 5% when ompred to the mle HFD group (Figure 3). In support of this oserved redution, mrna expression of the mrophge mrkers F8 nd CD11 were lso deresed in femle HFD ompred to mle HFD mie(figure ). However, there ws no differene in the reltive expression of the inflmmtory ytokines IL- nd TNF-α or the hemokine MCP-1 (Figure ). Oesity used redution in the reltive mrna expression of enos in mle HFD gondl dipose tissue while there ws no hnge in the femle HFD group (Figure ). Furthermore, the oxidtive stress mrkers HO-1, pphox, nd prdx1 were inresed in mle HFD gondl dipose tissue, nd this inrese ws ttenuted in gondl dipose tissue from femle HFD mie (Figure ). Importntly, when gondl dipose tissue from mle how nd femle how mie ws ompred, dipoyte size ws smller in femle mie, ut there were no differenes in ny of the other vriles tht were mesured (Figures, 3, nd ). Overll, dipoyte size nd dipose tissue msses were greter in weight-mthed femle HFD mie s ompred to mle HFD mie. However, femle HFD mie hd redued immune ell infiltrtion nd oxidtive stress, nd this my e due, in prt, to inresed diponetin expression. Suutneous dipose tissue hs een reported to e metolilly different thn gondl dipose tissue [] nd therefore the gene expression profile of this tissue ws lso investigted. Suutneous dipose tissue mrna expression of CD11 nd F8 ws inresed in mle HFD mie s ompred to the femle HFD group (Figure 5). Oesity lso used n inrese in MCP-1 expression in this tissue; however, the inrese ws greter in the mle HFD nimls s ompred to the femle HFD mie. The expression of the inflmmtory ytokines IL- nd TNFα ws inresed in oth mle HFD nd femle HFD mie (Figure 5). Similr to the results reported for gondl dipose tissue, mrna expression of oxidtive stress mrkers in suutneous dipose tissue ws elevted in mle HFD mie s ompred to the femle HFD group (Figure 5). In ontrst to the gondl dipose tissue, we did not oserve ny differenes in diponetin expression in suutneous dipose tissue (dt not shown). No ppreile differenes in suutneous mrna expression for ny of the genes investigted were oserved etween mle how nd femle how groups (Figure 5).

4 Experimentl Dietes Reserh Blood gluose (mg/dl) Gluose AUC (mg/dl 1 min 1) Time (min) 3 1 Mle how Mle HFD () Femle how Femle HFD Mle how Mle HFD Femle how Femle HFD () Figure 1: Oese femle mie (femle HFD) hve improved gluose tolerne when ompred to weight-mthed oese mle mie (mle HFD). Mle nd femle C57BL/ mie were fed either stndrd rodent how or high-ft diet (HFD) from weeks of ge until the HFD-fed group hieved ody weight of 5 g. At tht time, gluose tolerne test ws performed in oth how- nd HFD-fed mles (1 weeks old) or femles (38 weeks old) nd lood gluose hnge over time plotted (). Corresponding lood gluose re under the urve (AUC) ws lulted (), dt re reported s men ± SE nd mens with different supersripts differ y P<.5. n = 5 8 per group. Reltive mrna expression Reltive mrna expression Mle how Mle HFD Adiponetin () () Leptin Femle how Femle HFD Figure : Adiponetin mrna expression in gondl dipose tissue is redued y oesity in mle mie ut not weight-mthed oese femle mie. Mle nd femle C57BL/ mie were fed either stndrd rodent how or high-ft diet (HFD) from weeks of ge until the HFD-fed group hieved ody weight of 5 g. At tht time, oth how- nd HFD-fed mles (1 weeks old) or femles (38 weeks old) were srified nd qrt PCR performed on gondl dipose tissue. Reltive mrna expression of diponetin ws redued y oesity in mle mie, while oesity hd no effet on diponetin expression in femle mie (). Consistent with n oese phenotype, mrna expression of leptin ws elevted in oth mle HFD nd femle HFD mie (). Dt re reported s men ± SE; n = 5 8 per group; mens with different supersripts differ y P<.5.. Disussion While numerous studies hve demonstrted the enefits of estrogen in oesity prevention nd hroni disese risk mngement [13 17, 3], our study is novel in tht weightmthed oese mle nd femle mie were evluted to determine if endogenous estrogen provides helth enefits in the oese stte tht re independent of ody weight differenes. As oserved from the irulting estrdiol levels, these femle mie hd not entered ovrin senesene. Postmenopusl mie hve een shown to disply more severe oese phenotype reltive to their yling, ge-mthed ontrols [], further supporting tht estrdiol provides protetion ginst HFD-indued oesity nd lterntions in gluose metolism. Consistent with previous reports [13 15], we oserved tht mle mie were more suseptile to HFD-indued oesity s ompred to the femle HFD group. Therefore, we reognize tht ge is potentil onfounding vrile tht ws not ounted for in the urrent study design s it took 17 weeks longer for the femle HFD group to reh the trget ody weight of 5 g. This trget ody weight ws seleted euse it hs een shown to e period of estlished oesity, dipose tissue inflmmtion, nd insulin resistneinmlemie[]. Regrdless of the differene in ge, oese femle mie hd improved gluose lerne during gluose tolerne test s ompred to weight-mthed oese mles. This improvement in gluose metolism ws supported y the oservtion tht oesity used n inrese in fsting lood gluose onentrtions in mles ut not femles when ompred to their how-fed littermtes. These dt demonstrte tht hroni exposure to HFD n indue n oese phenotype in femle mie; however, the development

5 Experimentl Dietes Reserh 5 Chow HFD Mle Femle () Crown-like strutures (%) 1 8 Mle HFD Femle HFD Cell size (μm 1) d Mle how Mle HFD Femle how Femle HFD () () Figure 3: Oese femle mie hve lrger dipoytes nd redued prevlene of rown-like strutures in gondl dipose tissue when ompred to weight-mthed oese mle mie. Mle nd femle C57BL/ mie were fed either stndrd rodent how or high-ft diet (HFD) from weeks of ge until the HFD-fed group hieved ody weight of 5 g. At tht time, oth how- nd HFD-fed mles (1 weeks old) or femles (38 weeks old) were srified nd histologil nlysis ws performed on gondl dipose tissue. Representtive H&E stins of gondl dipose tissue from eh of the four tretment groups re presented in pnel (). Setions were used to quntify the presene of rown-like struture () nd to lulte verge dipoyte re (). Dt re reported s men ± SE; n = 5 8 per group; mens with different supersripts differ y P<.5; P<.5; r = 1 μm. of insulin resistne in these nimls is not s severe s tht oserved in weight-mthed oese mle mie. In ddition, dvned ge is ssoited with the development of insulin resistne [5] suggesting the improvement in gluose tolerne my hve een greter if the signifint differene in ge did not exist etween the two groups. This differene in ge my explin why gluose tolerne ws similr etween the mle how nd femle how-groups while others hve reported differenes in gluose tolerne testing etween how-fed mle nd femle mie []. Given the strong orreltion etween metoli dysfuntion in dipose tissue nd impired gluose metolism; we exmined the dipose tissue from the four experimentl groups in n ttempt to etter understnd the oserved improvement in gluose tolerne in oese femles. The experimentl design preluded ody weight differenes etween mle HFD nd femle HFD groups; however, ody ft ontent of the femle HFD mie ws greter due to inresed gondl nd suutneous dipose tissue mss. This inrese in mss ws ssoited with inresed dipoyte size in the gondl dipose tissue nd is ontrry to our oservtion in how-fed nimls nd the reports of others studying len nimls []. Both inresed intr-dominl ft mss nd dipoyte size hve een ssoited with insulin resistne in mle mie due to n inresed infiltrtion nd tivtion of proinflmmtory immune ells in the dipose tissue [, 7, 19, 7, 8]. Here we oserve n pprent disonnet where femle mie hve inresed dipose tissue mss nd lrger dipoytes ut gluose tolerne is improved. When immune ell infiltrtion nd tivtion were ssessed, we oserved redution in the pperne of immune ells nd the formtion of CLS in femle HFD gondl dipose tissue. This orrelted with redutions in mrna expression of CD11 nd F8 lthough expression of proinflmmtory

6 Experimentl Dietes Reserh Reltive mrna expression Reltive mrna expression CD11 F8 IL- MCP-1 () TNF-α enos inos HO-1 pphox Prdx1 Mle how Mle HFD () Femle how Femle HFD Figure : Reltive mrna expression of mrkers for immune ell infiltrtion nd oxidtive stress is deresed in gondl dipose tissue isolted from oese femle mie s ompred to oese mle mie. Mle nd femle C57BL/ mie were fed either stndrd rodent how or high-ft diet (HFD) from weeks of ge until the HFD-fed group hieved ody weight of 5 g. At tht time, oth how- nd HFD-fed mles (1 weeks old) or femles (38 weeks old) were srified nd qrt PCR ws performed on gondl dipose tissue. Reltive mrna expression of mrkers for immune ell infiltrtion nd inflmmtion () s well s oxidtive stress () ws determined. Dt re reported s men ± SE; n = 5 8 per group; mens with different supersripts differ y P<.5. IL-: interleukin-; MCP-1: monoyte hemottrtnt protein-1; TNF-α: tumor nerosis ftor-lph; enos: endothelil nitri oxide synthse; inos: induile nitri oxide synthse; HO- 1: heme oxygense-1; pphox: NADPH suunit pphox; Prdx1: peroxiredoxin-1. ytokines ws not different etween mle HFD nd femle HFD groups, suggesting tht ftor other thn these ytokines ws potentilly responsile for the improvement in gluose tolerne. We then mesured mrna expression of the dipokines leptin nd diponetin in gondl dipose tissue. Consistent with previous studies [9], oesity used n inrese in leptin expression in oth HFD-fed groups. Interestingly, oesity used redution in diponetin expression in the mle HFD group, ut expression ws unhnged with oesity in the femle HFD group. These dt re not surprising s femles typilly hve higher diponetin levels thn mles Reltive mrna expression Reltive mrna expression CD11 F8 IL- MCP-1 () TNF-α enos inos HO-1 pphox Prdx1 Mle how Mle HFD () Femle how Femle HFD Figure 5: Reltive mrna expression of mrkers for immune ell infiltrtion nd oxidtive stress is ltered in suutneous dipose tissue isolted from oese femle mie s ompred to oese mle mie. Mle nd femle C57BL/ mie were fed either stndrd rodent how or high-ft diet (HFD) from weeks of ge until the HFD-fed group hieved ody weight of 5 g. At tht time, oth how- nd HFD-fed mles (1 weeks old) or femles (38 weeks old) were srified nd qrt PCR ws performed on suutneous dipose tissue. Reltive mrna expression of mrkers for immune ell infiltrtion nd inflmmtion () s well s oxidtive stress () ws determined. Dt re reported s men ± SE; n = 5 8 per group; mens with different supersripts differ y P<.5. IL-: interleukin-; MCP-1: monoyte hemottrtnt protein-1; TNF-α: tumor nerosis ftor-lph; enos: endothelil nitri oxide synthse; inos: induile nitri oxide synthse; HO- 1: heme oxygense-1; pphox: NADPH suunit pphox; Prdx1: peroxiredoxin-1. [3, 31] nd while this ppers to our independent of estrdiol levels, diponetin is ssoited with improved insulin sensitivity nd suggests potentil mehnism for our oserved improvement in the femles. However, dditionl studies investigting irulting onentrtions of diponetin re required to more fully explore this possiility. Endothelil dysfuntion nd oxidtive stress re dditionl stressors tht n influene gluose metolism nd were ssessed in dipose tissue. Redued mrna expression of enos nd elevtions in HO-1, pphox, nd prdx1 in the dipose tissue of mle HFD mie is inditive of oxidtive stress within the tissue [3 35]. Altertions in the profile of these genes were not s severe in the femle HFD dipose tissue reveling possile redution in oxidtive stress nd nother potentil mehnism y whih estrogen my hve provided enefiil effet. Nominl differenes

7 Experimentl Dietes Reserh 7 existed etween mle how nd femle how dipose tissue nd therefore it is unlikely tht ny of our oservtions in the oese ohorts were influened y seline differenes. Overll, our study demonstrtes tht if provided enough time, hroni exposure to hyperlori diet will indue n oese phenotype in femle mie tht is hrterized y exess dominl diposity nd enlrged dipoytes s ompred to weight-mthed oese mle mie. However, despite eing n older niml, femle mie mintined prtil protetion from the detrimentl effets of oesity s demonstrted y improved gluose tolerne testing. Furthermore, immune ell infiltrtion nd oxidtive stress were redued in the dipose tissue of oese femle mie, nd these hnges were ssoited with inresed diponetin expression. It is likely tht omintion of these ftors is responsile for the oserved improvement in gluose tolerne. While oesity did not lter irulting levels of estrogen we reognize tht estrogen my not e the only ftor influening the improved phenotype oserved in the femle HFD group. However, our dt re onsistent with studies tht hve more refully mnipulted irulting levels of estrogen. Future studies in weight-mthed oese femles will e required to extend nd verify these initil findings. Referenes [1] F. Asi, B. W. Brown Jr., C. Lmendol, T. MLughlin, nd G. M. Reven, Reltionship etween oesity, insulin resistne, nd oronry hert disese risk, Journlofthe Amerin College of Crdiology, vol., no. 5, pp ,. [] K. M. Flegl, B. I. Grurd, D. F. Willimson, nd M. H. Gil, Cuse-speifi exess deths ssoited with underweight, overweight, nd oesity, Journl of the Amerin Medil Assoition, vol. 98, no. 17, pp. 8 37, 7. [3] C. de Lu nd J. M. Olefsky, Inflmmtion nd insulin resistne, FEBS Letters, vol. 58, no. 1, pp , 8. [] A. W. Ferrnte Jr., Oesity-indued inflmmtion: metoli dilogue in the lnguge of inflmmtion, Journl of Internl Mediine, vol., no., pp. 8 1, 7. [5] G. S. Hotmisligil, Inflmmtion nd metoli disorders, Nture, vol., no. 711, pp. 8 87,. [] K. J. Strissel, Z. Stnhev, H. Miyoshi et l., Adipoyte deth, dipose tissue remodeling, nd oesity omplitions, Dietes, vol. 5, no. 1, pp , 7. [7] C. N. Lumeng, S. M. DeYoung, J. L. Bodzin, nd A. R. Sltiel, Inresed inflmmtory properties of dipose tissue mrophges reruited during diet-indued oesity, Dietes, vol. 5, no. 1, pp. 1 3, 7. [8] M. F. Gregor nd G. S. Hotmisligil, Themti review series: dipoyte iology. Adipoyte stress: the endoplsmi retiulum nd metoli disese, Journl of Lipid Reserh, vol. 8, no. 9, pp , 7. [9] S. Furukw, T. Fujit, M. Shimukuro et l., Inresed oxidtive stress in oesity nd its impt on metoli syndrome, Journl of Clinil Investigtion, vol. 11, no. 1, pp ,. [1] L. M. Brown, L. Gent, K. Dvis, nd D. J. Clegg, Metoli impt of sex hormones on oesity, Brin Reserh, vol. 135, pp , 1. [11] J. M. Bruun, C. B. Nielsen, S. B. Pedersen, A. Flyvjerg, nd B. Rihelsen, Estrogen redues pro-inflmmtory ytokines in rodent dipose tissue: studies in vivo nd in vitro, Hormone nd Metoli Reserh, vol. 35, no. 3, pp. 1 1, 3. [1] M. R. Meyer, D. J. Clegg, E. R. Prossnitz, nd M. Brton, Oesity, insulin resistne nd dietes: sex differenes nd role of oestrogen reeptors, At Physiologi, vol. 3, no. 1, pp. 59 9, 11. [13] R. E. Stuins, K. Njjr, V. B. Holom, J. Hong, nd N. P. Nunez, Oestrogen lters dipoyte iology nd protets femle mie from dipoyte inflmmtion nd insulin resistne, Dietes, Oesity nd Metolism, vol. 1, no. 1, pp. 58, 1. [1] R. E. Stuins, V. B. Holom, J. Hong, nd N. P. Nunez, Estrogenmodultes dominl diposity nd protets femle mie from oesity nd impired gluose tolerne, Europen Journl of Nutrition. In press. [15] K. L. Grove, S. K. Fried, A. S. Greenerg, X. Q. Xio, nd D. J. Clegg, A mirorry nlysis of sexul dimorphism of dipose tissues in high-ft-diet-indued oese mie, Interntionl Journl of Oesity, vol. 3, no., pp , 1. [1] D. J. Clegg, L. M. Brown, S. C. Woods, nd S. C. Benoit, Gondl hormones determine sensitivity to entrl leptin nd insulin, Dietes, vol. 55, no., pp ,. [17] N. H. Rogers, J. W. Perfield, K. J. Strissel, M. S. Oin, nd A. S. Greenerg, Redued energy expenditure nd inresed inflmmtion re erly events in the development of ovrietomy-indued oesity, Endorinology, vol. 15, no. 5, pp , 9. [18] Y. Kmei, M. Suzuki, H. Miyzki et l., Ovrietomy in mie dereses lipid metolism-relted gene expression in dipose tissue nd skeletl musle with inresed ody ft, Journl of Nutritionl Siene nd Vitminology, vol. 51, no., pp , 5. [19] S. P. Weiserg, D. MCnn, M. Desi, M. Rosenum, R. L. Leiel, nd A. W. Ferrnte Jr., Oesity is ssoited with mrophge umultion in dipose tissue, Journl of Clinil Investigtion, vol. 11, no. 1, pp , 3. [] S. Cinti, G. Mithell, G. Brtelli et l., Adipoyte deth defines mrophge loliztion nd funtion in dipose tissue of oese mie nd humns, Journl of Lipid Reserh, vol., no. 11, pp , 5. [1] J. W. Perfield II, Y. Lee, G. I. Shulmn et l., Tumor progression lous (TPL) regultes oesity-ssoited inflmmtion nd insulin resistne, Dietes, vol., no., pp , 11. [] T. T. Trn, Y. Ymmoto, S. Gest, nd C. R. Khn, Benefiil effets of suutneous ft trnsplnttion on metolism, Cell Metolism, vol. 7, no. 5, pp. 1, 8. [3] T. M. D Eon, S. C. Souz, M. Aronovitz, M. S. Oin, S. K. Fried, nd A. S. Greenerg, Estrogen regultion of diposity nd fuel prtitioning: evidene of genomi nd non-genomi regultion of lipogeni nd oxidtive pthwys, The Journl of Biologil Chemistry, vol. 8, no. 3, pp , 5. [] M. J. Romero-Aleshire, M. K. Dimond-Stni, A. H. Hsty, P. B. Hoyer, nd H. L. Brooks, Loss of ovrin funtion in the VCD mouse-model of menopuse leds to insulin resistne nd rpid progression into the metoli syndrome, Amerin Journl of Physiology, vol. 97, no. 3, pp. R587 R59, 9. [5] L. Lin, P. K. Sh, X. M et l., Altion of ghrelin reeptor redues diposity nd improves insulin sensitivity during ging y regulting ft metolism in white nd rown dipose tissues, Aging Cell, vol. 1, no., pp , 11.

8 8 Experimentl Dietes Reserh [] Y.Motel,J.Bouher,T.T.Trn,ndC.R.Khn, Sexnd depot differenes in dipoyte insulin sensitivity nd gluose metolism, Dietes, vol. 58, no., pp , 9. [7] K. J. Strissel, J. DeFuri, M. E. Shul, G. Bennett, A. S. Greenerg, nd M. S. Oin, T-ell reruitment nd Th1 polriztion in dipose tissue during diet-indued oesity in C57BL/ mie, Oesity, vol. 18, no. 1, pp , 1. [8] C. N. Lumeng, J. L. Bodzin, nd A. R. Sltiel, Oesity indues phenotypi swith in dipose tissue mrophge polriztion, Journl of Clinil Investigtion, vol. 117, no. 1, pp , 7. [9] R. S. Ahim nd S. Y. Osei, Adipokines in oesity, Frontiers of Hormone Reserh, vol. 3, pp , 8. [3] H. Nishizw, L. Shimomur, K. Kishid et l., Androgens derese plsm diponetin, n insulin-sensitizing dipoyte-derived protein, Dietes, vol. 51, no. 9, pp ,. [31] F. Bäkhed, H. Ding, T. Wng et l., The gut miroiot s n environmentl ftor tht regultes ft storge, Proeedings of the Ntionl Ademy of Sienes of the United Sttes of Ameri, vol. 11, no., pp ,. [3] P. Hnd, S. Ttey, N. O. Rizzo et l., Redued vsulr nitri oxide-gmp signling ontriutes to dipose tissue inflmmtion during high-ft feeding, Arterioslerosis, Thromosis, nd Vsulr Biology, vol. 31, no. 1, pp , 11. [33] A. Georgesu, D. Popov, A. Constntin et l., Dysfuntion of humn suutneous ft rterioles in oesity lone or oesity ssoited with type dietes, Clinil Siene, vol. 1, no. 1, pp. 3 7, 11. [3] J. M. Curtis, P. A. Grimsrud, W. S. Wright et l., Downregultion of dipose glutthione S-tnsferse A leds to inresed protein ronyltion, oxidtive stress, nd mitohondril dysfuntion, Dietes, vol. 59, no. 5, pp , 1. [35] D. Pitoo, F. Zrdi, E. Di Stsio et l., Oxidtive stress, nitri oxide, nd dietes, Review of Dieti Studies, vol. 7, no. 1, pp. 15 5, 1.

9 MEDIATORS of INFLAMMATION The Sientifi World Journl Hindwi Pulishing Corportion Gstroenterology Reserh nd Prtie Hindwi Pulishing Corportion Journl of Hindwi Pulishing Corportion Dietes Reserh Hindwi Pulishing Corportion Hindwi Pulishing Corportion Interntionl Journl of Journl of Endorinology Immunology Reserh Hindwi Pulishing Corportion Disese Mrkers Hindwi Pulishing Corportion Sumit your mnusripts t BioMed Reserh Interntionl PPAR Reserh Hindwi Pulishing Corportion Hindwi Pulishing Corportion Journl of Oesity Journl of Ophthlmology Hindwi Pulishing Corportion Evidene-Bsed Complementry nd Alterntive Mediine Stem Cells Interntionl Hindwi Pulishing Corportion Hindwi Pulishing Corportion Journl of Onology Hindwi Pulishing Corportion Hindwi Pulishing Corportion Prkinson s Disese Computtionl nd Mthemtil Methods in Mediine Hindwi Pulishing Corportion AIDS Behviourl Neurology Hindwi Pulishing Corportion Reserh nd Tretment Hindwi Pulishing Corportion Hindwi Pulishing Corportion Oxidtive Mediine nd Cellulr Longevity Hindwi Pulishing Corportion