Hydrodynamic Delivery of mil10 Gene Protects Mice From High-fat Diet-induced Obesity and Glucose Intolerance

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1 originl rtile The Amerin Soiety of Gene & Cell Therpy Hydrodynmi Delivery of mil Gene Protets Mie From High-ft Diet-indued Oesity nd Gluose Intolerne Mingming Go, Chuno Zhng, Yongjie M, Le Bu, Linn Yn nd Dexi Liu Deprtment of Phrmeutil nd Biomedil Sienes, College of Phrmy, University of Georgi, Athens, Georgi, USA High-ft diet () indued oesity is ssoited with low-grde inflmmtion, insulin resistne (IR), nd gluose intolerne. The ojetive of this study is to ssess the effet of interleukin (IL), n nti-inflmmtory ytokine, on loking -indued oesity nd oesityssoited metoli disorders y hydrodynmi delivery of IL-ontining plsmid. Animls fed regulr how or reeived two injetions (one on dy nd the other on dy ) of plsmids ontining green fluoresene protein (GFP) or mouse IL (mil) gene. Blood onentrtion of mil rehed ~ ng/ml on dy 7 in nimls reeiving mil plsmid DNA. The trnsfetion effiieny of liver ells ws the sme in nimls fed regulr how or. No differene ws seen in nimls on regulr how when injeted with plsmids ontining either or mil gene. Overexpression of mil prevented weight gin of nimls on. Intrperitonel gluose tolerne test (IPGTT) nd insulin tolerne tests (ITT) showed tht mil mintined insulin sensitivity nd prevented gluose intolerne. The mehnisti study revels tht mil suppressed mrophge infiltrtion nd redued the development of rownlike strutures in dipose tissue (AT). Colletively, these results suggest tht mintining higher level of IL through gene trnsfer ould e n effetive strtegy in preventing diet-indued oesity. Reeived 7 Novemer ; epted My ; dvne online pulition 8 June. doi:.8/mt.. INTRODUCTION The inidene of oesity hs inresed drstilly in reent dedes. Aording to the Centers for Disese Control, more thn % dults in the United Sttes re overweight, ~% of whom re linilly oese (ody mss index: > kg/m ). Oesity inreses the risk of numer of helth onditions inluding IR, hypertension, type dietes, ftty liver disese, theroslerosis, degenertive disorders, irwy disese, nd some ners. This luster of pthologies hs lso strted to emerge in hildren t young ge, phenomenon tht ws inoneivle only few dedes go. There re urrently few drugs ville in lini for the prevention or tretment of oesity-ssoited diseses. The sfety issues nd side effets re still outstnding onerns with most of these drugs. Therefore, there is n urgent need for new pprohes to ddress oesity nd oesity-ssoited omplitions. Although extensive studies hve een onduted, the ext underlying mehnism for pthogenesis of oesity nd oesityssoited metoli syndrome remins ontroversil.,7 Reent studies showed tht hroni inflmmtion plyed ritil roles in the development of oesity, espeilly in its omplitions inluding ftty liver nd gluose intolerne. 8,9 Chroni inflmmtion is usully ssoited with n imlne etween proinflmmtory nd nti-inflmmtory ytokines. Interleukin (IL) is n nti-inflmmtory ytokine tht ttenutes the inflmmtory proesses indued y tumor nerosis ftor-α (TNFα), IL, nd IL while upregulting the relese of IL- reeptor ntgonist., IL is negtively orrelted with ody mss index, perentge ft mss, nd fsting gluose levels. Low levels of IL re ssoited with oesity, metoli syndrome nd type dietes. For exmple, Mnigrsso et l. showed tht ndroid oesity ws ssoited with onomitnt redution of IL. Srpelli et l. reported tht vrints of the IL promoter gene re ssoited with oesity nd IR in Itlin sujets. Blüher et l. lso demonstrted signifint derese in IL in prllel with the impirment of gluose tolerne. Reently, Hong et l. showed tht IL protein dministrtion n prevent diet-indued IR in mouse skeletl musle. An lterntive nd more promising pproh in hieving enefiil effets from IL is to overexpress it using gene delivery. In this study, we investigted the effets of hydrodynmi delivery of mouse IL (mil) gene on energy homeostsis in mie fed high-ft diet (). First, we onfirmed tht hydrodynmi injetion with mil gene inresed lood mil level in mie fed regulr how or with similr effiieny. Next, we evluted the effets of mil overexpression on nimls y performing ody omposition nlysis, gluose nd insulin tolerne tests (ITT), quntittive PCR, nd histohemistry of different orgns. Our dt suggest tht hydrodynmi trnsfer of mil gene n e regrded s potentil linil strtegy for prevention of oesity nd its relted diseses. Correspondene: Dexi Liu, Deprtment of Phrmeutil nd Biomedil Sienes, College of Phrmy, University of Georgi, Phrmy South, West Green Street, Athens, Georgi, USA. E-mil: dliu@ug.edu 8 vol. no., 8 8 ot.

2 The Amerin Soiety of Gene & Cell Therpy Hydrodynmi Delivery of mil Gene RESULTS Effiieny of hydrodynmi gene delivery is not ffeted y the feeding diet nd genes delivered To onfirm tht nimls fed different diet re eqully sensitive to hydrodynmi gene delivery, we performed hydrodynmi injetion with plsmids ontining either mil or green fluoresent protein () gene in mie fed regulr how or. Hydrodynmi injetion did not use pprent morphologil hnges in the mouse liver, nd ontrol mie injeted with gene showed strong signls of green fluoresene (Figure ). Both sprtte minotrnsferse nd lnine minotrnsferse levels were in norml rnge 7 dys fter injetion (Figure,), onfirming tht nd mil gene expression did not use liver dmge. The mrna level of mil ws ~-fold higher thn tht of ontrol nimls injeted with (Figure d). As expeted, mie injeted with mil gene showed signifintly higher level of lood mil ompred with ontrol (Figure e). These results demonstrte tht diet does not ffet the delivery effiieny of hydrodynmi proedure nd suffiient level of mil gene expression ws hieved in the liver resulting in elevted protein levels of mil in the lood (Figure d,e). Time ourse (Supplementry Figure S) shows tht irulting mil level in lood rehes pek level (~ ng/ml) dys fter hydrodynmi injetion, delines with time, nd rehes stedy level mil mil Fluoresene H&E 8 7 AST (U/l) ALT (U/l) mil mil mil mil d Reltive mrna level of mil 8 8 e mil (pg/ml) mil mil mil mil Figure Impt of diet on hydrodynmi gene delivery effiieny. Animls fed regulr how or high-ft diet () were hydrodynmilly injeted with plsmids ontining either green fluoresent protein () or mouse interleukin (mil) gene nd killed 7 dys lter. () Representtive imges of hemtoxylin nd eosin (H&E) stining nd green fluoresene of the mouse livers. () Blood onentrtion of sprtte minotrnsferse. () Blood onentrtion of lnine minotrnsferse. (d) mrna level of mil in mouse liver. (e) Blood onentrtion of mil. Vlues in ( e) represent verge ± SD (n = ). P <. ompred with how-fed -injeted mie, P <. ompred with -fed -injeted mie. Moleulr Therpy vol. no. ot. 8

3 Hydrodynmi Delivery of mil Gene The Amerin Soiety of Gene & Cell Therpy (~ ng/ml) in dys whih stys t the sme level until the end of experiment on dy. mil gene trnsfer loked -indued weight gin without signifint impt on len mss nd verge food intke Two plsmid injetions were performed on nimls when fed speifi diet, one on dy nd the other on dy. Compred with ontrol nimls injeted with gene, mie injeted with mil gene showed irulting mil onentrtion t ~ ng/ ml t the end of 7-week experiment (Figure ), onfirming tht two injetions were enough to mintin effetive lood onentrtions of mil. Hydrodynmi gene trnsfer of mil gene ompletely loked -indued ody weight gin (Figure ). Animls fed for 7 weeks showed n ~% inrese in ody weight when gene ws injeted while only ~% ws seen in those injeted with mil gene (Figure ). The ody omposition nlysis showed tht injetion of the mil gene hd no signifint impt on len mss, onfirming tht the differene in ody weight gin ws primrily due to n inrese in ft mss (Figure ). Although mie injeted with mil gene showed slightly lower umultive food intke (Figure d, insert) ompred with ontrol mie, oth groups hd similr verge food intke over 7 weeks of feeding (Figure d). The lulted lori intke of nimls fed regulr how or injeted with or mil gene ws. ±.,.9 ±.7, nd.7 ±.7 kl/ mouse/dy, respetively. Injetion of mil gene loked hypertrophy in dipoytes To investigte the impt of mil gene expression on morphologil hnge in AT, we onduted tissue setion nd hemtoxylin nd eosin (H&E) stining. Compred with ontrol mie fed regulr how, mie fed nd injeted with gene showed hypertrophi dipoytes in epididyml white AT (EWAT) nd inguinl WAT, nd lso hd more ft umultion in rown AT s evidened y the inresed mount of vuoles in H&E stining (Figure ). Injetion of the mil gene loked dipoyte hypertrophy in these tissues (Figure ). In mie injeted with the gene, 7-week feeding inresed the size of dipoyte y ~.8- nd.9-fold in EWAT nd inguinl WAT, respetively (Figure,). Agin, mil gene injetion ompletely loked the enlrgement of the dipoytes (Figure,). Injetion of mil gene prevented etopi ft umultion in liver Beuse oesity nd hypertrophy in dipoytes my use etopi ft umultion in the liver, we disseted the liver nd performed histohemil exmintion. Neither onsumption nor mil gene injetion used n pprent hnge in liver pperne (Figure ). In mie injeted with the gene, onsumption used lrge mount of ft umultion in the liver, s evidened y vuoles in H&E stining nd red dots in Oil-red O stining (Figure ). Injetion of mil gene prevented indued etopi ft umultion in the liver. Hepti triglyeride mil (pg/ml) Body omposition (g) + mil Ft mss Len mss Body weight hnge (% originl) d Food intke (g/mouse/dy) + mil Aumultive food intke (g/mouse) Time (weeks) Δ =. g + mil + mil + mil Figure Effet of hydrodynmi delivery of mouse interleukin (mil) gene on ody weight, ody omposition, nd food intke. () Blood onentrtion of mil in mie t the end of 7-week feeding with seleted diet. () Body weight gin over 7-week feeding period. Arrows indite the time of hydrodynmi injetion of mil plsmids. () Body omposition. (d) Food intke. Insert in d represents umultive food intke per mouse. Vlues represent verge ± SD (n = ). P <. ompred with -fed -injeted mie, P <. ompred with how-fed -injeted mie. GFP, green fluoresent protein;, high-ft diet. 8 vol. no. ot.

4 The Amerin Soiety of Gene & Cell Therpy Hydrodynmi Delivery of mil Gene + mil BAT IWAT EWAT Are of epididyml white dipoyte ( μm ) mil Are of inguinl white dipoyte ( μm ) mil Figure Hydrodynmi delivery of mouse interleukin (mil) gene prevented hypertrophy in dipoytes. () Representtive imges from hemtoxylin nd eosin stining of EWAT, IWAT, nd BAT. Arrows indite rown-like strutures. () Averge size of dipoytes in EWAT. () Averge size of dipoytes in IWAT. Vlues in nd represent verge ± SD of totl numer of dipoytes seen in five tissue slies ( dipoytes per slie). P <. ompred with how-fed -injeted mie, P <. ompred with -fed -injeted mie. BAT, rown dipose tissue; EWAT, epididyml white dipose tissue; GFP, green fluoresent protein;, high-ft diet; IWAT, inguinl white dipose tissue. determintion showed similr results (Figure ), further onfirming the mil effet on loking hepti umultion of ft. Blood sprtte minotrnsferse nd lnine minotrnsferse levels suggest tht neither feeding nor mil gene trnsfer used liver dmge(figure,d). Injetion of mil gene mintined gluose homeostsis Beuse oesity nd etopi ft umultion re usully ssoited with gluose intolerne, we onduted intrperitonel gluose tolerne test (IPGTT) nd ITT to evlute gluose homeostsis. feeding for 7 weeks used gluose intolerne in mie injeted with gene, s evidened y elevted lood gluose during IPGTT (Figure ); mil gene injetion signifintly ttenuted gluose exursion nd mintined gluose tolerne (Figure ). Are under the urve lultion showed similr results (Figure ). Next, we performed ITT to estimte IR. Results of ITT showed feeding used severe IR in mie injeted with the gene (Figure ). Injetion of the mil gene mintined insulin sensitivity in mie fed (Figure ). Clultion of homeostsis model ssessment-ir (HOMA-IR), whih reflets the degree of IR, showed similr results (Figure d). Injetion of mil gene prevented hyperinsulinemi without signifint influene in the pnreti islet To further onfirm -indued IR in mie fed, we mesured lood insulin level. Consistent with ove results, feeding indued hyperinsulinemi in mie injeted with ut not with the mil gene (Figure ). To investigte the influene of mil gene injetion in the islet, we performed histohemil exmintion inluding H&E stining nd immunohistohemistry (IHC) stining ginst insulin. H&E stining showed tht neither feeding nor mil gene injetion used hypertrophy in islet (Figure ). Islet size mesurements showed similr results (Figure ). Next, we deteted insulin levels in islets using IHC stining nd found there is no differene in islet insulin ontent in nimls with or without mil gene delivery (Figure d). Injetion of mil gene prevented -indued GLUT derese in ATs -indued oesity nd dipoyte hypertrophy re usully ssoited with GLUT derese in AT. To ssess -indued GLUT hnge, we onduted IHC stining using ATs inluding EWAT, inguinl WAT, nd rown AT. In mie injeted with the Moleulr Therpy vol. no. ot. 8

5 Hydrodynmi Delivery of mil Gene The Amerin Soiety of Gene & Cell Therpy + mil Oil-red O H&E Piture d TG in liver (mg/g tissue) AST (U/l) 8 7 ALT (U/l) + mil + mil + mil Figure Hydrodynmi delivery of mouse interleukin (mil) gene loked etopi ft umultion in liver. () Representtive imges of liver from hemtoxylin nd eosin (H&E) nd Oil-red O stining. () Liver triglyeride level. () Blood onentrtion of sprtte minotrnsferse nd (d) lood onentrtion of lnine minotrnsferse in mie t the end of 7-week feeding period. Vlues in ( d) represent verge ± SD (n = ). P <. ompred with how-fed -injeted mie, P <. ompred with -fed -injeted mie. GFP, green fluoresent protein;, high-ft diet; TG, triglyeride. Blood gluose (mg/dl) AUC of IPGTT ( mg/dl minute) 7 Time (minutes) + mil 9 d Blood gluose (% of initil vlue) HOMA-IR Time (minutes) + mil 9. + mil + mil Figure Hydrodynmi delivery of mouse interleukin (mil) gene mintined gluose tolerne in mie fed. () Profiles of lood gluose onentrtion s funtion of time upon intrperitonel injetion of gluose. () Are under the urve of IPGTT. () Profiles of gluose onentrtion (perentge of initil vlue) s funtion of time upon intrperitonel injetion of insulin. (d) Results of HOMA-IR nlysis for insulin resistne. Vlues represent verge ± SD (n = ). P <. ompred with how-fed -injeted mie, P <. ompred with -fed -injeted mie. GFP, green fluoresent protein;, high-ft diet; HOMA-IR, homeostsis model ssessment-insulin resistne; IPGTT, intrperitonel gluose tolerne test. 8 vol. no. ot.

6 The Amerin Soiety of Gene & Cell Therpy Hydrodynmi Delivery of mil Gene + mil Insulin (ng/ml) d + mil Are of islet ( μm ) + mil Merge DAPI Insulin + mil Figure Hydrodynmi delivery of mouse interleukin (mil) gene prevented hyperinsulinemi without signifint influenes in islets. () Blood insulin levels. () Representtive imges from the hemtoxylin nd eosin (H&E) stining of the pnres. Arrows indite islets. () Averge size of the islet. (d) Representtive imges of immunohistohemistry stining ginst insulin in islets. Vlues in represent verge ± SD (n = ). P <. ompred with how-fed -injeted mie, P <. ompred with -fed -injeted mie. DAPI,,-dimidino--phenylindole; GFP, green fluoresent protein;, high-ft diet. gene, 7-week feeding mrkedly deresed GLUT in ll three types of ATs (Figure 7). Two injetions of mil gene prevented -indued GLUT derese. Dt from western lot nlysis (Figure 7) show tht 7-week feeding deresed GLUT level y ~% in EWAT nd y ~8% in rown AT ut not in musle, nd hydrodynmi trnsfer of mil gene prevented the derese. Quntittive PCR nlysis t mrna level revels the sme trend in WAT (Figure 7). Injetion of mil gene suppressed mrophge infiltrtion nd loked development of rown-like strutures in AT To investigte the impt of mil gene delivery on the development of rown-like struture in EWAT, we onduted IHC stining ginst f/8, whih is moleulr mrker for mrophges. feeding for 7 weeks gretly inresed the mount of rown-like strutures in EWAT of mie injeted with ut not mil gene (Figure 8). Quntittive nlysis showed similr results (Figure 8). Quntifition of F/8 positive mrophges revels the verge level t ~.,.7, nd.8 mrophges per mm for how-fed mie, -fed ontrol mie, nd -fed mil-treted mie, respetively. We lso exmined mrophge infiltrtion y ssessing the mrna level of mrophge mrker genes. Results in Figure 8,d lerly show tht in mie injeted with the gene, feeding mrkedly inresed the expression of f/8 nd d8 y ~.- nd.-fold, respetively. Injetion of the mil gene loked elevtion of these genes. Similr onlusion ws otined with nlysis of dditionl mrker genes for mrophges (d, d), dendriti ells (d, d8, d) nd T regultory ells (d, d9, d, foxp; Supplementry Figure S). Injetion of mil gene modulted gene expression in WAT nd suppressed -indued elevtions of proinflmmtory ytokines The hypertrophy of WAT nd infiltrtion of mrophges were ssoited with signifint inrese in mrna levels of leptin, mp, tnfα, interferon-γ (ifnγ), ilβ, nd il, nd injetion of the mil gene ompletely loked these inreses (Supplementry Figure S,). In ontrst, 7-week feeding slightly ut signifintly redued the trnsription of glut (Figure 7) nd diponetin (Supplementry Figure S) while injetion of the mil gene prevented this redution. The mrna level of spse gene ws elevted in -fed ontrol nimls nd mil expression loked the elevtion (Supplementry Figure S). In ddition, we determined irulting levels of set of proinflmmtory ytokines inluding TNFα, IFNγ, ILβ, nd IL (Supplementry Figure S). -fed ontrol nimls showed higher levels of proinflmmtory ytokines nd injetion of the mil gene loked the elevtion. Moleulr Therpy vol. no. ot. 87

7 Hydrodynmi Delivery of mil Gene The Amerin Soiety of Gene & Cell Therpy + mil IWAT EWAT BAT GLUT GAPDH GLUT GAPDH GLUT GAPDH + mil BAT EWAT Musle Reltive expression of glut mil Figure 7 Hydrodymi delivery of mouse interleukin (mil) gene mintined GLUT level in dipose tissues. () Representtive imges of immunohistohemistry stining ginst GLUT in BAT, EWAT, nd IWAT. () GLUT protein levels in BAT, EWAT, nd musle of mie. Tissue smples were olleted t the end of the 7-week feeding period, homogenized, loded t ~ μg protein per lne, nd seprted y % SDS-PAGE. The ntiody ginst GLUT (#; Am) ws diluted t :, for this determintion. () Reltive mrna levels of glut in EWAT. Vlues in represent verge ± SD (n = ). P <. ompred with how-fed -injeted mie, P <. ompred with -fed -injeted mie. BAT, rown dipose tissue; EWAT, epididyml white dipose tissue; GFP, green fluoresent protein;, high-ft diet; IWAT, inguinl white dipose tissue. DISCUSSION In this study, we demonstrted tht hydrodynmi delivery of the mil gene proteted mie from -indued oesity nd gluose intolerne (Figures nd ). Injetion of the mil gene loked hypertrophy in dipoytes nd lso prevented etopi ft umultion in the liver (Figures nd ). The elevted expression of mil y hydrodynmi gene delivery mintined GLUT gene expression in AT nd loked IR (Figures nd 7). Mehnisti studies showed tht mil gene injetion suppressed mrophge infiltrtion, redued the development of rown-like strutures in AT nd loked the elevtions of irulting proinflmmtory ytokines (Figure 8 nd Supplementry Figure S). Oesity is hrterized y low-grde inflmmtion whose moleulr origin is unknown. As typil nti-inflmmtory ytokine, IL is importnt in modulting inflmmtion nd regulting metoli homeostsis. Previous studies hve shown tht IL is involved in the development of oesity nd gluose intolerne, nd exogenous dministrtion of IL protein llevited diet-indued IR in mouse skeletl musle. A more reent study y Ogur et l. reported tht deno-ssoited virusmedited delivery of IL gene generted enefiil effet on oesity-ssoited glomerulopthy in Zuker ftty (Zuker-f/ f) rts, nd tht the slightly elevted IL mrkedly deresed lood onentrtions of triglyeride, holesterol s well s glyosylted hemogloin. Consistent with these studies, our dt showed tht trnsfer of the mil gene into mie fed exerted enefiil effets in metoli homeostsis. These results suggest tht high IL level in lood is ple of exerting enefiil effets on metoli homeostsis through suppressing the -indued hroni inflmmtion. While the preventive effet of IL on -indued oesity is ler, dditionl studies re needed to exmine the therpeuti tivity of IL for tretment of oesity. Etopi ft umultion in the liver is typil feture of diet-indued oesity. Previous studies hve shown tht redution in IL expression is involved in this proess. Using IL knok-out mie, den Boer et l. demonstrted endogenous IL proteted mie ginst -indued hepti stetosis. 7 Studies y Cintr et l. nd Morri et l. showed tht IL is protetive ftor ginst hepti inflmmtion nd IR, nd its expression is prtilly ontrolled y prolifertor-tivted reeptor gmm otivtor-α. 8,9 Hshem et l. proposed tht the rtio of IL to TNF-α should e onsidered s preditive iomrker for nonloholi ftty liver disese. A more reent study y Gotoh et l. demonstrted tht spleen-derived IL my help to protet mie ginst the development of non-loholi ftty pnres disese. In line with these studies, our dt lerly show tht mil gene delivery prevents -indued etopi lipid umultion in the 88 vol. no. ot.

8 The Amerin Soiety of Gene & Cell Therpy Hydrodynmi Delivery of mil Gene + mil * * * * * d Crown-like struture per mm + mil Reltive expression of f/ mil Reltive expression of d mil Figure 8 Hydrodynmi delivery of mouse interleukin (mil) gene suppressed mrophge infiltrtion nd loked the development of rown-like strutures in dipose tissue. () Representtive imges of immunohistohemistry stining ginst f/8 in EWAT. Asterisks (*) indite rown-like strutures. () Quntifition of rown-like strutures in EWAT. () Reltive mrna level of f/8. (d) Reltive mrna level of d8. Vlues in ( d) represent verge ± SD (n = ). P <. ompred with how-fed -injeted mie, P <. ompred with -fed -injeted mie. EWAT, epididyml white dipose tissue; GFP, green fluoresent protein;, high-ft diet. liver s evidened y H&E stining, Oil-red O stining, nd liver triglyeride determintion (Figure ). Etopi ft umultion nd hroni inflmmtion ply ritil role in development of IR. Centrl nd peripherl IR inreses gluoneogenesis nd dereses lood gluose sorption, leding to gluose intolerne. Mny studies hve shown tht low pity of IL prodution is tightly ssoited with IR nd gluose intolerne.,9, Cintr et l. showed tht knokdown of IL y neutrlizing nti-il ntiody or n ntisense oligonuleotide inresed hepti expression of the inflmmtory ytokines nd deresed insulin signling in mouse liver. 8 Moreover, Hong et l. reported tht exogenous IL protein dministrtion llevited diet-indued IR in mouse skeletl musle. Consistent with these studies, our results show tht mil overexpression mintined mrna nd protein levels of GLUT in the ATs (Figure 7), loked IR, nd prevented gluose intolerne in -fed mie (Figure ), nd these effets were independent of the funtion of IL in pnres sine no pprent hnge ws oserved in pnreti tissue (Figure ). Oesity nd hypertrophy of dipoytes re ssoited with n inrese in poptosis of dipoyte nd ell deth. Murno et l. showed tht ded dipoytes, deteted s rown-like strutures, were prevlent in viserl ft depots of genetilly modified oese mie. Another typil feture of oesity is mrophge infiltrtion to WAT. Although still ontroversil, the infiltrted mrophges seem to funtion s svenger to remove ded dipoytes. Unfortuntely, ftty ids relesed from these dipoytes n tivte toll-like reeptor in mrophges nd initite trnsription of multiple proinflmmtory ytokines, leding to hroni inflmmtion. Reent studies y Heno-Meji et l. nd Stienstr et l. showed tht initition of inflmmtion plys ritil roles in the pthogenesis of oesity nd its ssoited diseses inluding ftty liver nd IR., Their studies lerly show tht loking the sde of inflmmtion uses mie to e resistnt to diet-indued oesity nd ftty liver. Moreover, Shi et l. reported tht mie lking toll-like reeptor re sustntilly proteted ginst systemi lipid infusion-indued IR nd prtilly proteted ginst diet-indued IR. Consistent with these studies, our dt showed tht overexpression of mil suppressed mrophge infiltrtion nd redued development of rownlike strutures in AT (Figure 8), nd led to multiple enefiil onsequenes in metoli homeostsis (Figures, nd ). We elieve tht the enefiil effets of mil overexpression re t lest prtilly medited y its funtion in suppressing inflmmtion. Evidently, some other pthwys my lso e involved nd dditionl studies re still needed to further eluidte the underlying mehnism in detil. In summry, in this study, we demonstrte tht overexpression of mil using hydrodynmi gene delivery n protet mie from -indued ft umultion nd gluose intolerne. Our dt suggest tht trnsfer of IL gene ould e n effetive pproh in preventing -indued oesity, n urgent helth prolem tht ffets out % Amerins. Moleulr Therpy vol. no. ot. 89

9 Hydrodynmi Delivery of mil Gene The Amerin Soiety of Gene & Cell Therpy MATERIALS AND METHODS Mterils. The plive plsmid vetor ws purhsed from Mirus Bio (Mdison, WI). The mouse IL gene ws loned from omplementry DNA sequenes of mie nd inserted into the multiple loning sites of the plive vetor using restrition enzyme digestion. The onstruted plsmid ws onfirmed y DNA sequening. Plsmid ontining GFP gene ws onstruted nd onfirmed y using similr proedures. These plsmids were prepred y the method of esium hloride-ethidium romide grdient entrifugtion nd kept in sline t 8 C until use. The purity of the plsmid preprtions ws exmined y soreny t nd 8 nm nd % grose gel eletrophoresis. Animls nd tretments. The used in this study ws purhsed from Bio-Serv (Frenhtown, NJ; #F8) in whih % of totl lories re from ft, % from rohydrtes nd % from proteins. Mle C7BL/ mie (~7 g) purhsed from Chrles River Lortories (Wilmington, MA) were housed under stndrd ondition with -hour light-drk yle. The use of nimls ws omplint with relevnt poliies, nd the niml protool ws pproved y the Institutionl Animl Cre nd Use Committee of the University of Georgi (protool numer, A 7-Y-A). The proedure for hydrodynmi gene delivery in mie hs previously een reported. 7,8 Briefly, lrge volume of sline solution (equivlent to 9% ody weight) ontining plsmid DNA ws injeted into mouse til vein within 8 seonds. In the expression-onfirming experiment, mie were divided into four groups (n = for eh group), inluding two how-feeding groups nd two -feeding groups. For eh diet, one group of mie ws hydrodynmilly injeted with plsmid ontining gene nd the other with mil gene driven y lumin promoter in plive plsmid vetor. Mie were killed 7 dys fter injetion. Blood smples were olleted for determining lood levels of sprtte minotrnsferse, lnine minotrnsferse, nd mil. Liver smples were olleted for histohemil exmintion nd mil expression nlysis. In experiment designed to ssess the effet of mil gene trnsfer on niml growth, mie were divided into three groups (n = for eh group). Group one ws fed with regulr how nd hydrodynmilly injeted with gene. The other two groups of niml were fed, nd one group ws hydrodynmilly injeted with nd the other with mil gene ontining plsmids. Hydrodynmi gene delivery ws performed on dy nd dy when nimls were fed with seleted diet. Body weight nd food intke were mesured weekly. Body omposition nlysis using EhoMRI- (Eho Medil Systems, Houston, TX) ws onduted t the end of the experiment. Protein determintions of IL, TNFα, IFNγ, ILβ, nd IL. Blood levels of mil, TNFα, IFNγ, ILβ, nd IL were determined using ELISA kits from ebiosiene (Sn Diego, CA). For mil determintion, lood smples were olleted t predetermined time-point nd t the end of 7-week feeding with seleted diet. For TNFα, IFNγ, ILβ, nd IL determintions, ll of the lood smples were olleted t the end of 7-week feeding. Serum ws prepred from lood y entrifugtion t, rpm for minutes, nd kept frozen t 8 C until use. ELISA ws onduted using kit following protool provided y the mnufturer. Gene expression nlysis nd western lot. We purified totl mrna from the liver nd ATs using TRIZOL regent purhsed from Invitrogen (Crlsd, CA). Reverse trnsription-pcr ws performed using Supersript RT III enzyme kit (#7-) from Invitrogen. Quntittive nlysis of mrna ws onduted y using SYBR Green s detetion regent nd the ΔΔC t method for dt nlysis. 9 GAPDH mrna ws utilized s n internl referene nd primer sequenes re listed in Supplementry Tle S. Melting urve nlysis of ll rel-time PCR produt ws onduted nd showed single DNA duplex. For western lot, tissue smples were homogenized nd loded t ~ μg totl proteins per lne, nd seprted on % SDS-PAGE. GLUT ntiody (#; Am, Cmridge, MA) ws diluted t :,, nd the protein nds were quntified using Bio-Rd Quntity One -D nlysis softwre (BIO-RAD, Herules, CA). Evlution of gluose homeostsis. IPGTT nd ITT were performed in the lst week of niml growth experiment. Mie were fsted for 8 hours efore IPGTT nd intrperitonelly injeted with gluose in sline ( g/ kg). Blood ws olleted t predetermined time-points (,,, nd minutes) fter gluose injetion nd ws determined using gluose test strips nd gluose meters. For ITT, mie were fsted for 8 hours nd intrperitonelly injeted with insulin (Humulin:. U/kg) purhsed from Eli Lilly (Indinpolis, IN). Blood gluose level ws determined using the sme method s ove t,,, nd minutes fter insulin injetion. Insulin onentrtion in lood ws determined using ommeril kit (#--; Merodi Developing Dignostis, Winston Slem, NC). We lulted HOMA-IR using the following formul: HOMA-IR = (fsting insulin (ng/ml) fsting plsm gluose (mg/dl)/). Oil-red O stining. Animls were killed t desirle times nd orgns were immeditely olleted nd frozen in liquid nitrogen. The frozen setions were mde t 8 μm thikness nd fixed in % neutrl uffered formlin for minutes nd wshed with phosphte-uffered sline. Tissue slies were rinsed with % isopropnol for minutes efore eing stined with freshly prepred Oil-red O working solution (Eletron Mirosopy Sienes, Htfield, PA) for minutes. The setions were rinsed with % isopropnol gin for minutes efore eing ounterstined with hemotoxylin for minute nd exmined under n optil mirosope (ECLIPSE Ti; Nikon, Melville, NY). Liver triglyeride determintion. Liver triglyeride level ws mesured following previously reported method. In rief; frozen liver smples ( mg per smple) were homogenized in mixture solution of hloroform nd methnol (:) nd inuted overnight t C. The smples were entrifuged t, rpm for minutes t C. Superntnts were dried, re-dissolved in % Triton-X nd triglyeride onentrtion ws determined following the instrution of the ommeril kit (#TR; Thermo-Sientifi, Pittsurgh, PA). Immunofluoresene histohemil study. Tissue smples emedded in prffin were ut t μm thikness nd dried. The setions were immersed in mmol/l itrte uffer (ph.) nd proessed in thermostti wter th for ntigen retrievl. These setions were loked in % norml serum nd % ovine serum lumin in Tris-uffered sline for hours efore inution with primry ntiodies. The following primry ntiodies were used: ntiody for insulin (#9; Cell Signling, Boston, MA), ntiody for GLUT (#; Am), nd ntiody for F/8 (#M; Spring Biosienes, Plesnton, CA). A seondry ntiody onjugted with fluoresent dye (#; Cell Signling) ws employed for detetion. H&E stining. Tissues were fixed in % neutrl uffered formlin nd dehydrted ginst grdients of ethnol efore emedded into prffin. Tissue setions were ut t μm thikness nd dried t 7 C for hour efore inution in xylene, followed y stndrd H&E stining using ommeril kit (#; BBC Biohemil, Atlnt, GA). Size mesurement of dipoytes ws rried out using NIS-Elements imging pltform purhsed from Nikon Instruments (Melville, NY). Sttistis. The dt re expressed s mens ± SD for eh group nd sttistil signifine ws nlyzed using nlysis of vrine. A vlue of P <. ws onsidered signifint. SUPPLEMENTARY MATERIAL Figure S. Time ourse of mil gene expression. Figure S. Effet of mil gene expression on expression of mrker genes in white dipose tissue. Figure S. Effet of hydrodynmi delivery of mil gene on expression of seleted genes in white dipose tissue. Figure S. Effet of mil expression on lood onentrtion of proinflmmtory ytokines. Tle S. PCR primer sequenes. 8 vol. no. ot.

10 The Amerin Soiety of Gene & Cell Therpy Hydrodynmi Delivery of mil Gene ACKNOWLEDGMENTS The study ws supported in prt y grnts from Ntionl Institutes of Helth (ROEB77 nd ROHL989). We thnk Ryn Fugett for English editing. The uthors delred no onflit of interest. REFERENCES. Ynovski, SZ nd Ynovski, JA (). Oesity prevlene in the United Sttes up, down, or sidewys? N Engl J Med : Zimmet, P, Alerti, KG nd Shw, J (). Glol nd soietl implitions of the dietes epidemi. Nture : Cprio, S, Dniels, SR, Drewnowski, A, Kufmn, FR, Plinks, LA, Rosenloom, AL et l. (8). Influene of re, ethniity, nd ulture on hildhood oesity: implitions for prevention nd tretment: onsensus sttement of Shping Ameri s Helth nd the Oesity Soiety. Dietes Cre :.. Dietrih, MO nd Horvth, TL (). Limittions in nti-oesity drug development: the ritil role of hunger-promoting neurons. Nt Rev Drug Disov : Pi-Sunyer, FX (). Pthophysiology nd long-term mngement of the metoli syndrome. Oes Res (suppl.): 7S 8S. 7. 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