Methyl-β-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue*

Transcription

1 University of Nebrsk - Linoln DigitlCommons@University of Nebrsk - Linoln Publitions from USDA-ARS / UNL Fulty U.S. Deprtment of Agriulture: Agriulturl Reserh Servie, Linoln, Nebrsk 213 Methyl-β-yloextrin lters ipokine gene expression n gluose metolism in swine ipose tissue* T. G. Rmsy USDA-ARS, timothy.rmsy@rs.us.gov L. Blomberg USDA-ARS, lenn.blomberg@rs.us.gov T. J. Cpern USDA-ARS Follow this n itionl works t: Rmsy, T. G.; Blomberg, L.; n Cpern, T. J., "Methyl-β-yloextrin lters ipokine gene expression n gluose metolism in swine ipose tissue*" (213). Publitions from USDA-ARS / UNL Fulty This Artile is brought to you for free n open ess by the U.S. Deprtment of Agriulture: Agriulturl Reserh Servie, Linoln, Nebrsk t DigitlCommons@University of Nebrsk - Linoln. It hs been epte for inlusion in Publitions from USDA-ARS / UNL Fulty by n uthorize ministrtor of DigitlCommons@University of Nebrsk - Linoln.

2 Animl (213), 7:1, pp & The Animl Consortium 213. This is work of the U.S. Government n is not subjet to opyright protetion in the Unite Sttes. oi:1.117/s niml Methyl-b-yloextrin lters ipokine gene expression n gluose metolism in swine ipose tissue* T. G. Rmsy -, L. Blomberg n T. J. Cpern Animl Biosienes n Biotehnology Lortory, Animl n Nturl Resoures Institute, USDA/ARS, Beltsville Agriulturl Reserh Center, USDA, Beltsville MD 275, USA (Reeive 26 Otober 212; Aepte 25 April 213; First publishe online 19 July 213) This stuy ws esigne to etermine whether methyl-b-yloextrin () n substitute for lbumin in inubtion meium for neontl swine ipose tissue explnts, or whether ffets metolism n ytokine expression. Subutneous ipose tissue explnts (1 6 1 mg) were prepre from 21-y-ol pigs. Explnts were inubte in meium 199 supplemente with 25 mm HEPES, 1. nm insulin t 37C. The meium lso ontine bovine serum lbumin () or t %,.5%,.1%,.2% or.3%. Tissue explnts were trete with these mei for 1 h n then swithe to the sme bsl inubtion meium ontining.5%. Explnts were remove from bsl meium t 2 or h of inubtion, n rel-time PCR ws performe to ssess expression of tumor nerosis (TNF) n interleukin 6 (IL6), etyl CoA rboxylse (ACAC) n ftty i synthse (FASN). Alterntively, rtes of 1 C-gluose oxition n lipogenesis were monitore 6 insulin (1 nm), following tretment. Inubtion with h miniml effets on gene expression or ipose tissue metolism, only prouing oubling in TNF mrna unne ( P,.1). Tretment with inrese TNF mrna unne by eightfol ( P,.9), wheres IL6 gene expression inrese 1-fol ( P,.1) with suppression in ACAC n FASN expression ( P,.1). This ws prllele by inhibition of insulin-stimulte gluose oxition n lipogenesis ( P,.1). Aition of TNF ntiboy to the inubtion meium llevite this inhibition of insulin-stimulte gluose metolism by,3% ( P,.5). Keywors: ipose tissue, yloextrin, tumor nerosis ftor, lipogenesis Implitions Albumin is inlue in the inubtion meium for ipose tissue for multiple resons. However, lbumin hs reently been emonstrte to lter ytokine expression by humn n roent ipoytes. Cyloextrins hve been use s n lterntive to lbumin in vitro n in vivo. However, the present stuy emonstrtes tht methyl-b-yloextrin is not suitle s n lbumin substitute, s it reues insulin-sensitive gluose metolism through inution of tumor nerosis ftor. Introution The role of ipose-erive ytokines hs reently ome to the forefront beuse of their intertions with ipoyte metolism n ipose tissue funtion (Jobi et l., 2; Rsouli n Kern, 2). Reserh into the regultion of the * Mention of tre nme, proprietry prout or venor oes not onstitute gurntee or wrrnty of the prout by the U.S. Deprtment of Agriulture or imply its pprovl to the exlusion of other prouts or venors tht lso my be suitle. - E-mil: timothy.rmsy@rs.us.gov expression of these prrine ytokines in vitro typilly requires the use of lbumin in the meium to reue lysis of the ipoytes through intertion with glss or plsti ontiners; to bin ftty is relese by the ipoytes, n thus prevent feebk inhibition of metolism; to funtion s rrier for ftty is to improve solubility; or to bin vrious proteins introue into the meium to prevent those e proteins from bining to the vessel. Shlesinger et l. (26) emonstrte tht lbumin (or it ontminnts) n intert with the ipoyte to inue the expression of ipose-erive ytokines (ipokines), whih omplites the nlysis of ipokine regultion. Run et l. (23) reporte tht the proess of ipoyte isoltion (in the presene of lbumin) n lter ipokine expression within ipose tissue. Therefore, lterntives to lbumin my be neessry beuse of lbumin inution of ytokine expression, whih omplites nlysis of ipoyte-erive ytokines. b-yloextrin hs been use to bin ftty is uring the in vitro nlysis of ftty i oxition (Kto et l., 1993 n 199) n this woul suggest tht it my serve s substitute for lbumin in ipose tissue inubtion. 169

3 Cyloextrin inution of pig ipokines Methyl-b-yloextrin () hs been use to bin ftty is for in vivo infusion into growing swine (Wry-Chen et l., 21) without the report of ny eleterious effets when use t 2% onentrtion (w/v) in the infuste, wheres vrious porine n bovine lbumins t 5% onentrtion (w/v) inue nphylti responses when infuse into swine. The onlusion ws tht my serve s n lterntive rrier for ftty is uring infusions in swine. This implies tht i not inue ytokines uring the infusion n suggests tht oul be n lterntive to lbumin for inlusion in ipose inubtions uring the nlysis of ytokine gene expression. However, Le Ly et l. (21) emonstrte tht n lter gluose metolism n gene expression in rt ipoytes. Inubtion of isolte inguinl rt ipoytes with 1 mm oul reue insulin-stimulte gluose oxition by,5%. They postulte tht this ws beuse of the epletion of holesterol from the plsm membrne by s supplementtion of holesterol to the oul prelue the reution in insulin-stimulte gluose oxition. However, holesterol epletion lso inues gene expression within 3T3-L1 ipoytes (Le Ly et l., 21). Inubtion of ifferentite 3T3-L1 ells with omptin (n inhibitor of sterol synthesis), n use of lipoprotein-efiient serum in the meium resulte in 7% reution in holesterol ontent of the 3T3-L1 ipoytes fter ys. The mrna unne of tumor nerosis ftor (TNF) n interleukin 6 (IL6) ppere to be inrese pproximtely fivefol n threefol fter ys, lthough sttistil nlysis ws not performe. The use of s substitute for lbumin for in vitro nlysis of ipoyte funtion my be limite by the reporte tions of to lter ipoyte metolism n its potentil effet on the expression ipokines. The present stuy ws esigne to etermine whether lters gluose metolism n ipokine gene expression in ipose tissue explnts from neontl swine. Methoology Experimentl esign The first experiment ompre responses in ipokine gene expression, following inubtion with inresing onentrtions of bovine serum lbumin () or. Albumin n were use t (ontrol),.5%,.1%,.2% n.3% in the meium n prepre through seril ilution. Subutneous ipose tissue explnts (1 mg) erive from 21-y-ol pigs were inubte with these onentrtions of lbumin or for 1 h. All explnts were then rinse n blotte with subsequent trnsfer to bsl meium ontining.5% lbumin for 2 or h of inubtion. The ipose tissue explnts were then ollete for the nlysis of TNF n IL6 mrna unne, n subsequently etyl CoA rboxylse (ACAC) n ftty i synthse (FASN) gene expression. The effet of on ipose metolism ws exmine by mesuring 1 C-gluose oxition n inorportion into lipi oring to methos previously esribe (Rmsy, 23). Following.2% or.2% tretments for 1 h, tissue explnts were trnsferre to 25 ml Erlenmeyer flsks with meium ontining.5% 6 1 nm insulin n 1 C-gluose. Gluose metolism ws then monitore for 2 h with subsequent olletion of 1 CO 2 n extrtion of 1 C- lele lipis from the tissue explnts. Alterntively, - n -trete explnts were ple in bsl meium (meium 199, 25 mm HEPES, 1 mm gluose,.5%, 1 nm insulin) for 6 h n then trnsferre to Erlenmeyer flsks for 2-h 1 C-gluose inubtion ( totl of h post-/ tretment). This ws to llow omprison with our gene expression t from the -h inubtions esribe ove. The selete for use in this experiment Sigm #953 (low enotoxin,,.1 ng/mg; Sigm-Alrih, St. Louis, MO, USA) ws selete beuse low-enotoxin preprtions proue miniml ipokine responses in humn ipoytes, reltive to other types of preprtions (Shlesinger et l., 26). The ws ell ulture gre n lso purhse from Sigm-Alrih (#C555). The onentrtion rnge selete ws bse on the t of Le Ly et l. (22) tht emonstrte n,35% reution in gluose oxition by isolte ipoytes with.5% n 5% reution with,1.3% (1. mm) tretment. The ipokine genes selete for nlysis were TNF n IL6. This ws bse on preliminry experiments tht inite tht these two ipokines re the most highly expresse by porine ipose tissue n emonstrte the lrgest response to inubtion with mong number of ytokines n metoli genes (t not presente). Tumor nerosis ftor hs been the most hrterize of ll ipokines n hs key regultory roles in the expression of vriety of proteins (Cwthorn n Sethi, 2), wheres IL6 is serete by pig ipose tissue (Ajuwon n Spurlok, 25) n ultures of pig ipoytes (Husmn et l., 26). Aetyl CoA rboxylse n FASN re key regultory genes in the pthwy for lipogenesis (Lenhr, 211), n thus were selete s metoli mrkers. Tissue hnling Briefly, ipose tissue ws issete using sterile tehnique from 21-y-ol pigs (Yorkshire 3 Lnre 3 Poln Chin), n 5 3 to per experiment, with ifferent pigs use for eh experiment. Dorsl subutneous ipose tissue smples from between the seon n fourth thori vertebre were quire, following euthnsi by pentobrbitl soium injetion (2 mg/kg BW), oring to proeures pprove by the Institutionl Are Animl Use n Cre Committee. Aipose tissue strips were ple in Hnks buffer (37C, ph 7., Sigm-Alrih, St. Louis, MO, USA) n were then issete len of ny extrneous musle tissue n further seprte into 1 m 2 ubes. Aipose tissue explnts (1 6 1 mg) were prepre by sliing these tissue ubes with Stie Riggs mirotome in lminr flow hoo by proeures previously esribe (Rmsy n Rihrs, 2). Tissue slies were rinse twie with fresh Hnks 1691

4 Rmsy, Blomberg n Cpern buffer (37C, ph 7.), blotte free of exess liqui n weighe. Tissue slies were then trnsferre to 12-well tissue ulture pltes ontining 1 ml of meium 199 with 25 mm Hepes, 1. nm insulin n % to.3% or % to.3% t ph 7.. Triplite tissue slies were inubte with these mei in tissue ulture inubtor t 37C with 95% ir/5% CO 2 for 1 h. Porine insulin (Sigm-Alrih, St. Louis, MO, USA) ws solubilize in.1 N HCl. Iniviul hormone liquots were thwe for eh y of use n ilute in inubtion meium to the pproprite onentrtion. Following 1-h inubtion with or, tissue explnts were rinse three times with fresh meium 199 (37C), blotte n trnsferre to nother 12-well tissue ulture plte ontining bsl meium tht omprise meium 199, 25 mm HEPES, 1 nm insulin n.5%. Controls were ple in the sme meium but without. Prllel ultures were inubte for either 2 or h to evlute the effets of on gene expression. Following this inubtion perio, tissue explnts were rinse three times with meium 199 (37C), blotte n then frozen in liqui nitrogen n store t 2C for lter nlysis of gene expression. Gluose metolism Inubtion meium omprise Meium 199 n supplemente with 25 mm Hepes, 1 mm gluose,.5%, 1 nm insulin n.5 mci D-[U- 1 C]- gluose/ml (Morvek Biohemils, Bre, CA, USA). This meium (2 ml) ws ple in 25 ml silionize Erlenmeyer flsks. Following ition of riolele meium n explnts, flsks were gsse for 1 min with 95% ir, 5% rbon ioxie n then ppe with rubber stoppers ontining enter well. Following 2 h of inubtion,.5 ml 1 N H 2 SO ws injete into the meium to kill the metoli tivity of the tissue. After 1 minutes, methylbenzythonium hyroxie (Sigm- Alrih, St. Louis, MO, USA) ws injete into the enter well to permit pture of CO 2 for the next 3 min. Center wells were subsequently trnsferre to sintilltion vils ontining 1 ml of sintilltion oktil (Bio-sfe II, Reserh Prouts Interntionl Corp., Mount Prospet, IL, USA) for ounting. Tissue explnts were remove with foreps, blotte n trnsferre to srew ppe tubes ontining 5 ml of Dole s solution for lipi extrtion by the metho use by ecingolni (1972). Inorportion of 1 C into totl lipi n ftty is ws etermine following sponifition, oring to the methos of Azin n Mrtin (193). Eh metoli experiment ws repete three times using three ifferent nimls n using triplite explnts/tretment for eh pig. Dt were lulte s nmole gluose utilize/hour per 1 mg tissue, but were represente in the grphs s reltive to tissue inubte without or, ontrol whose vlue ws rbitrrily set to 1.. An itionl experiment ws onute to ssess the potentil role of TNF or IL6 serete by the explnts uring inubtion on the metolism of the ipose tissue. Antiboies to porine TNF (1 mg/ml) or IL6 (2.5 mg/ml) were inlue in the bsl n riotive inubtion mei uring the 2- n -h inubtions n ompre with mei without the ntiboies. The nti-pig mouse monolonl ntiboy to TNF n the nti-pig got polylonl ntiboy to IL6 were purhse from R&D Systems (Minnepolis, MN, USA). Antiboy onentrtions for use in the meium were bse on preliminry experiments to proue mximl response. Otherwise, the methoology ws ientil to tht esribe ove for mesuring gluose metolism. Rel-time PCR nlysis of gene expression Totl RNA ws isolte using Qigen RNesy spin olumns, oring to the mnufturer s protool (Qigen, Vleni, CA, USA). Integrity of RNA ws ssesse vi grose gel eletrophoresis n RNA onentrtion ws etermine spetrophotometrilly using A26 n A2 mesurements. All primer sets were esigne to spn n intron s previously esribe n utilize for rel-time PCR (Rmsy n Azin, 27; Rmsy n Cpern, 29; Rmsy et l., 21). The following primers were use for generting 1-bse mplion orresponing to portion of the TNF oing sequene: 5 -CCCCTCTGAAAAAGACACCA-3 (forwr), 5 -TCGAAGTGCAGTAGGCAGAA-3 (reverse). The primers for IL6 were use to rete 215 bse mplion: 5 -ATGGCAGAAAAAGACGGATG-3 (forwr), 5 -GTGGTGG CTTTGTCTGGATT-3 (reverse). Aetyl CoA rboxylse primers inlue 5 -CTCCAGGACAGCACAGATCA-3 (forwr), 5 -GCCGAAACATCTCTGGGATA-3 (reverse) to proue 17-bse mplion, wheres 21-bse mplion ws proue with the following primers for ftty i synthse: 5 -AACGTCCTGCTGAAGCCTAA-3 (forwr), 5 -CTCCTTG GAACCGTCTGTGT-3 (reverse). The 1S rrna ws use s reltive stnr for omprisons (QuntumRNA TM Universl 1S Internl Stnr; Ambion, In., Austin, TX, USA) in prllel retions. Therml yling n t quisition were performe with Bio-R icyler IQ system (Bio-R Lortories In., Herules, CA, USA). Reverse trnsription (RT) n rel-time PCR nlysis were performe in two-tube ssy s previously esribe (Rmsy n Cpern, 29). RT ws one using Supersript First-Strn Synthesis System for RT-PCR kit (Invitrogen, Crlsb, CA, USA). Mster mix ws me ontining rnom hexmers (5 ng/ml), 1 mm NTP mix, RNse-free H 2 O n RNA (1 mg/ml). The RNA mix ws nnele t 65C for 5 min. A seon mster mix ws prepre with 1X RT buffer, 25 mm MgCl 2,.1 mm ithiothreitol n 1. ml RNseOut. This seon mster mix ws e to the RNA mix n inubte t 25C for 2 min. Supersript II ws then e n inubte t 25C for 1 min, 2Cfor5min n 7C for 15 min. An liquot of RNse H (1. ml) ws then e n inubte t 37C for 2 min. Rel-time PCR ws rrie out using the IQ sybr green supermix kit (Bio-R Lortories In., Herules, CA, USA). A 2 ml retion mix ws me ontining 12.5 ml sybr green supermix, 1. ml forwr primer (1 mm), 1. ml reverse primer (1 mm) n 9.5 ml sterile wter. This retion mix ws e to eh well, followe by 1. ml RT prout (25 ml totl volume). Prmeters for ll retions were s 1692

5 Cyloextrin inution of pig ipokines follows: 1 yle t 95C for 15 min (PCR tivtion), followe by 3 yles, 9C for 15 s, 5C for 3 s, 72C for 3 s, with finl extension t 72C for min. Melting urve nlysis ws performe on ll rel-time PCR retions to onfirm speifiity n ientity of the rel-time PCR prouts. A nontemplte ontrol ws run for every ssy. Speifiity of rel-time PCR prouts ws further onfirme by grose gel eletrophoresis. Quntifition of gene expression At the en of the PCR, bseline n threshol rossing vlues (CT) for ll nlyze genes were lulte using the BioR softwre, n the CT vlues were exporte to Mirosoft Exel for nlysis. The reltive expression of the genes of interest, stnrize ginst the mount of 1S mrna, ws lulte using the DDCT metho (Winer et l., 1999; Livk n Shmittgen, 21). Vlues were lulte s the men 6 s.e.m. of uplite etermintions from triplite tissue smples erive from eh of four iniviul nimls for eh experiment. Dt re expresse reltive to vlues etermine for inubtions without or, ontrol whose vlue ws rbitrrily set t 1.. Sttistil nlysis Dt were nlyze by nlysis of vrine using SigmPlot 12 softwre (SPSS Siene, Chigo, IL, USA) to test for the effets of tretment with v. lbumin. Men seprtion ws nlyze using Stuent Newmn Keuls test. Mens were efine s being ifferent t P,.5. Results Tumor nerosis ftor mrna unne ws inrese 2 h fter tretment with.3% (P,.5; Figure 1), but not with lower onentrtions of (P..5). However, inubtion with.5% inrese TNF by,% (P,.1) reltive to bsl meium or.5%, when mesure 2 h fter exposure. The response by TNF gene expression to inrese to mximl eightfol with.2% in the tretment meium (P 5.9). Performing n -h inubtion fter or tretment before smple olletion resulte in no hnge in TNF mrna unne from the ontrol inubtion without or (P 5.23; Figure 1b). Interleukin 6 mrna unne ws unffete by tretment with t 2 h following tretment (P..5), unne (TNF/1S) %.1%.2%.3% b b unne (IL6/1S) %.1%.2%.3% b unne (ACAC/1S) %.1%.2%.3% unne (FASN/1S) %.1%.2%.3% unne (TNF/1S) unne (ACAC/1S) %.1%.2%.3%.5%.1%.2%.3% b b b b b unne (IL6/1S) unne (FASN/1S) %.1%.2%.3% b.5%.1%.2%.3% b b b Figure 1 Chnges in mrna unne in response to ifferent onentrtions of lbumin or with time. Tissue explnts (,1 mg) were expose to % (ontrol),.5%,.1%,.2% or.3% lbumin or in meium 199 with 25 mm Hepes n 1. nm insulin t 37C, ph 7. for 1 h. Explnts were then blotte n trnsferre to fresh meium omprising meium 199 with 25 mm Hepes n 1. nm insulin, ph 7. n inubte for either 2 or h t 37C. Aipose tissue explnts were then blotte n frozen in liqui N 2 for lter RNA extrtion n subsequent rel-time PCR for 1S n TNF mplions. () mrna unne for the genes of interest t 2 h fter tretment with or. (b) mrna unne for the genes of interest t h fter ening tretment with or. The reltive expression of the genes of interest, stnrize ginst the mount of 1S mrna, ws lulte using the DDCT metho. Dt re expresse s the perent of reltive unne of the gene of interest to 1S in the ontrol ipose explnts s lulte with the (% lbumin/% ). 5 low enotoxin (,1. ng/mg); 5 methyl-b-yloextrin. Mens not shring ommon supersript letter re ifferent (P,.5). 1693

6 Rmsy, Blomberg n Cpern Reltive Gluose Oxition Ins +Ins 2 Hr Hr Reltive Lipogenesis Ins +Ins b 2 Hr Hr Figure 2 Gluose metolism in response to tretment with lbumin or. 1 C-gluose oxition n inorportion into lipi ws performe, oring to proeures esribe in the Methoology. Following.2% or.2% tretments for 1 h, tissue explnts were trnsferre to 25 ml Erlenmeyer flsks with meium ontining.5% 6 1 nm insulin n.5 mci (U)- 1 C-gluose. Gluose metolism ws then monitore for 2 h with subsequent olletion of 1 CO 2 n extrtion of 1 C-lele lipis from the tissue explnts. Alterntively, - n -trete explnts were ple in bsl meium (meium 199, 25 mm HEPES, 1 mm gluose,.5%, 1 nm insulin) for 6 h before trnsferring to Erlenmeyer flsks for the 2-h 1 C-gluose inubtion ( totl of h fter tretment). () Gluose oxition t 2 n h. (b) Lipogenesis from gluose t 2 n h fter or tretment. Eh metoli experiment ws repete three times with explnts erive from three seprte pigs n using triplite explnts/tretment for eh pig. Dt re expresse s reltive % of metoli tivity of explnts inubte in meium without or. 5 low-enotoxin (,1. ng/mg); 5 methyl-byloextrin; Ins 5 1 nm porine insulin. Mens not shring ommon supersript letter re ifferent (P,.5). wheres n inrese in IL6 gene expression ws etete with.5% in the meium n IL6 expression inrese up to 25-fol with.2% in the tretment meium (P,.19; Figure 1). An -h inubtion following the initil 1 h tretment resulte in muh higher inrese in IL6 mrna unne thn the 2-h inubtion (Figure 1b). Tretment with.5% resulte in 36-fol inrese in IL6 mrna unne, wheres.2% proue 1-fol inrese in IL6 gene expression reltive to the ontrol inubte with bsl meium. The tretment gin h no etetle effet on IL6 mrna unne (P..5). Anlysis of the mrna unne for enzymes tht re rte limiting for lipogenesis emonstrte tht neither ACAC nor FASN were ffete within 2 h of exposure to or (P..5; Figure 1, respetively). Inubtion for h following removl of the or tretment resulte in eline in ACAC mrna unne with.1% or.5% in the meium (P,.1; Figure 1b). No further hnge in ACAC gene expression ourre with higher onentrtions of thn.1% (P..5); however inresing the onentrtion of to.2% le to further 7% suppression in ACAC mrna unne (P,.1). Similrly, tretment with.1% or higher onentrtions of subsequently reue FASN mrna unne (P,.; Figure 1b). Inubtion with 1 C-gluose for 2 h, following 1-h exposure to or, emonstrte tht neither tretment ffete bsl or insulin-stimulte gluose oxition t tht time point (Figure 2). However, inubtion for h (with inlusion of 1 C gluose uring the lst 2 h) following.2% or.2% tretment emonstrte tht oul limit the insulin response reltive to the tretment. Exposure of tissue explnts to insulin following tretment resulte in more thn fourfol inrese in gluose oxition, wheres tretment resulte in less thn threefol inrese in gluose oxition with insulin tretment (P,.9). Lipogeni rtes were lso ffete by tretment (Figure 2b). Insulin-stimulte gluose inorportion into ftty is by,1%, following exposure to.2% in Reltive Perent Stimultion H +I 2H +I H +I H +I Oxition * Lipogenesis Figure 3 Effet of ition of n nti-tnf monolonl ntiboy on insulinstimulte gluose metolism. 1 C-gluose oxition n inorportion into lipi ws performe, oring to proeures esribe in the Methoology. Following.2% or.2% tretments for 1 h, tissue explnts were trnsferre to 25 ml Erlenmeyer flsks with meium ontining.5% 1 1 nm insulin n.5 mci (U)- 1 C-gluose 6 1 mg nti-pig TNF monolonl ntiboy. Gluose metolism ws then monitore for 2 h with subsequent olletion of 1 CO 2 n extrtion of 1 C-lele lipis from the tissue explnts. Alterntively, - n trete explnts were ple in meium 199 ontining 25 mm HEPES, 1 mm gluose,.5%, 1. nm insulin, with or without 1 mg nti-pig TNF monolonl ntiboy for 6 h before trnsferring to Erlenmeyer flsks for the 2-h 1 C-gluose inubtion 6 1 mg nti-pig TNF monolonl ntiboy ( totl of h fter tretment). Dt re expresse s reltive % hnge in oxition or lipogenesis reltive to explnts inubte in meium without ntiboy. * 5 Different from explnts not trete with the nti-pig TNF ntiboy (n 5 3; P,.5). 5 low-enotoxin (,1. ng/mg); 5 methyl-b-yloextrin; I 5 1 nm porine insulin. the 2-h inubtion (P,.1), wheres inhibite the evelopment of n insulin response in ipose tissue explnts (P..5). The insulin response by -trete tissue ws muh greter t h thn t 2 h with pproximtely sixfol inrese in ftty i synthesis (P,.1). In ontrst, tretment resulte in limiting insulin stimultion of bsl gluose inorportion into ftty is to,15% (P,.1). Neither nor ffete bsl oxition or lipogenesis (P..5). Inubtion with 1 mg of monolonl TNF ntiboy i not lter insulin-stimulte gluose oxition or lipogenesis, following tretment with.2% t either 2 or h (Figure 3). * 169

7 Cyloextrin inution of pig ipokines There ws lso no effet of the ntiboy on the metolism of tissue explnts trete with.2% for 2 h. However, o-inubtion of the TNF ntiboy in the meium for h, following tretment with.2%, inrese the insulinstimulte gluose oxition by 26% (P,.5) n the lipogeni rte by 35% (P,.5). Tretment with n IL6 ntiboy h no effet on ny metoli prmeters (P..5; t not presente). Disussion Tretment of ipose tissue explnts with resulte in n ute inrese in TNF mrna unne. These experiments were purposefully performe with ipose tissue explnts to prelue onfouning the t with the use of isolte ipoytes, beuse the proess of ipoyte isoltion my lso inrese TNF expression (Run et l., 23). The observe inrese in TNF mrna unne ws in the sme rnge s reporte by Le Ly et l. (21), lthough the present results were obtine within 2 h of tretment n h issipte by h. This ws followe lter by lrge surge in IL6 gene expression, muh greter thn reporte in 3T3-L1 ipoytes trete with holesterol synthesis inhibitor (Le Ly et l., 21). This my prtilly be ounte for in the present stuy beuse mesurements were me within the first 2 h of tretment, wheres gene expression ws not nlyze until fter ys of tretment in the stuy with 3T3-L1 ells (Le Ly et l., 21). Tumor nerosis ftor n stimulte the expression of IL6 (Fsshuer et l., 23), whih my ount for some of the inrese in IL6 mrna unne, following the inrese in TNF gene expression. Conversely, IL6 hs been shown to inhibit TNF protein expression (Strkie et l., 23). An inrese in IL6 protein in ssoition with the rise in IL6 gene expression in the present stuy woul inhibit TNF gene trnsription n oul ontribute to the sene of ny effet of on TNF mrna unne h following tretment, but this requires further reserh. These hnges in ipokine gene expression were ssoite with reution in ACAC n FASN mrna unne within h of tretment with. Tumor nerosis ftor hs been emonstrte to inhibit ACAC trnsription in the 3A-5 ipoyte ell line (Ppe n Kim, 19) n the 3T3-2A ipoyte ell line (Doerrler et l., 199), wheres FASN gene expression ws emonstrte to be inhibite by TNF in the 3T3-2A ipoytes (Doerrler et l., 199). In ontrst, IL6 hs not been reporte to lter ACAC expression or FASN expression in ipose tissue. However, IL6 hs been emonstrte to reue lipogenesis in 3T3-L1 ipoytes in ssoition with reution in gluose trnsport (Lgthu et l., 23). The eline in ACAC n FASN mrna unne with tretment in the present stuy suggeste tht lipogenesis is ltere by tretment. This ws onfirme by mesuring 1 C-gluose inorportion into ftty is. The t inite tht only insulin stimulte, n not bsl lipogenesis, ws ffete by tretment. Previous reserh hs only ssesse n reporte n effet on gluose oxition by isolte ipoytes (Le Ly et l., 21), n this ws lso observe in the present stuy with tissue explnts. The knowlege mehnism of tion for is to reue holesterol ontent of the plsm membrne; uses the ollpse of veole in the plsm membrne by the removl of holesterol (Breen et l., 212). The role of veole my be quite importnt to the physiology n metolism of the ipoyte, s they n onstitute up to 5% of the plsm membrne surfe re of the ipoyte (Thorn et l., 23). The insulin reeptor n insulin reeptor substrte-i (IRS-1) re two signling proteins tht re lolize to the holesterol-rih veole in humn ipoytes (Krlsson et l., 2), wheres only the insulin reeptor is lolize in rt ipoytes (Prpl et l., 21). Tretment of humn ipoytes with b-yloextrin (1 mm) resulte in reution in membrne holesterol ontent n reution in gluose trnsport, lthough insulin reeptor phosphoryltion n IRS-1phosphoryltion were unffete (Krlsson et l., 2). Tretment of rt ipoytes with similr onentrtion of b-yloextrin resulte in reution in membrne holesterol ontent n reutions in gluose trnsport n IRS-1 phosphoryltion by insulin (Prpl et l., 21). These results re in greement with Le Ly et l. (21), emonstrting the evelopment of n insulin resistne following 1. mm tretment. The present stuy using pig ipose tissue explnts lso observe reution in insulin-stimulte gluose oxition n lipogenesis, following tretment with reltive to lbumin t h following the tretment. At 2 h fter tretment, only n effet on lipogenesis ws etete, lthough the insulin response by gluose oxition following tretment ws 2% less thn fter tretment (P..5). The results t h re intriguing, beuse this is the timefrme following the elevtion in TNF gene expression n prllels the lrge inrese in IL6 gene expression. This rise the question s to whether these ipokines my be ontributing to the reltive insulin resistne in gluose metolism t h. Tretment with n nti-pig TNF ntiboy resulte in reuing the resistne of gluose metolism to insulin, but only t h fter tretment; the ntiboies to IL6 i not lter the insulin resistne (t not presente). Inlusion of the ntiboies throughout the h permitte bining of preforme or tissue surfe TNF, s well s subsequently the synthesize n serete TNF. These t suggest tht the inution of TNF following tretment ontribute to the insulin resistne etete h fter the tretment ene. How stimultes the expression n relese of TNF is not ler. The use of yloextrins to extrt holesterol from veole is not subtle metho. Numerous reeptors n signling moleules re ffete, isturbe or reyle s result of the proess. Therefore, the potentil signl for this inrese in ipokine gene expression nnot be sertine t this time. Wht is unntly ler is tht metolilly stresses the ell, resulting in n inrese in TNF expression tht n hve n impt on insulin sensitivity n 1695

8 Rmsy, Blomberg n Cpern thus gluose metolism. A mjor purpose of this experiment ws to fin replement for lbumin, s it hs shown to inue ytokine expression by rt ipose tissue (Shlesinger et l., 26). However, the present stuy hs emonstrte tht is not stisftory substitute for lbumin in experiments to ssess ipose-erive ytokines. A serh for more pproprite substitute for lbumin is still neessry. In vivo, infusion of solutions ontining 5% porine lbumin use nphylxis, wheres 5% solution of i not (Wry-Chen et l., 21). The lk of n nphylti response to in tht stuy woul suggest tht iposeerive ytokines my not mke signifint ontribution to the overll peripherl response to when ssesse reltive to the observe ute elevtion in TNF in the present stuy. Aknowlegments The uthors thnk M. Stoll for her ssistne with the RNA extrtions n rel-time PCR. The uthors thnk A. Shnnon for her ssistne with niml olletion. The uthors lso thnk the swine her stff, USDA-Beltsville, for their work in niml re. 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Moleulr Enorinology 2, Prpl S, Krlsson M, Thorn H n Strålfors P 21. Cholesterol epletion isrupts veole n insulin reeptor signling for metoli ontrol vi insulin reeptor substrte-1, but not for mitogen-tivte protein kinse ontrol. Journl of Biologil Chemistry 276, Rmsy TG 23. Porine leptin inhibits lipogenesis in porine ipoytes. Journl of Animl Siene 1, Rmsy TG n Rihrs MP 2. Hormonl regultion of leptin n leptin reeptor expression in porine subutneous ipose tissue. Journl of Animl Siene 2, Rmsy TG n Azin MJ 27. Comprison of gene expression in len ontemporry n rossbre obese swine. Aipoytes 2, Rmsy TG n Cpern TJ 29. Ontogeny of ipokine expression in neontl pig ipose tissue. Comprtive Biohemistry n Physiology Prt B 152, Rmsy TG, Stoll MJ n Cpern TJ 21. Aipokine gene trnsription level in ipose tissue of runt piglets. Comprtive Biohemistry n Physiology Prt B 155, Rsouli N n Kern PA 2. Aipoytokines n the metoli omplitions of obesity. Journl of Clinil Enorinology n Metolism 93 (suppl. 1), S6 S73. Run H, Zrnowski MJ, Cushmn SW n Loish HF 23. Stnr isoltion of primry ipose ells from mouse epiiyml ft ps inues inflmmtory meitors n own-regultes ipoyte genes. Journl of Biologil Chemistry 27, Shlesinger JB, vn Hrmelen V, Alberti-Huber CE n Huner H 26. Albumin inhibits ipogenesis n stimultes ytokine relese from humn ipoytes. Amerin Journl of Physiology 291, C27 C33. Strkie R, Ostrowski SR, Juffre S, Febbrio M n Peersen BK 23. Exerise n IL-6 infusion inhibit enotoxin-inue TNF-lph proution in humns. FASEB Journl 17, 6. Thorn H, Stenkul KG, Krlsson M, Ortegren U, Nystrom FH, Gustvsson J n Strlfors P 23. Cell surfe orifies of veole n loliztion of veolin to the neks of veole in ipoytes. Moleulr Biology of the Cell 1, Winer J, Jung CK, Shkel I n Willims PM Development n vlition of rel-time quntittive reverse trnsriptse-polymerse hin retion for monitoring gene expression in ri myoytes in vitro. Anlytil Biohemistry 27, 1 9. Wry-Chen D, Cpern TJ n Steele NC 21. 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