Effects of Rosiglitazone on Inflammation in Otsuka Long-Evans Tokushima Fatty Rats

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1 Originl Article Koren Dibetes J 21;34: doi: 1.493/kdj pissn eissn Effects of Rosiglitzone on Inflmmtion in Otsuk Long-Evns Tokushim Ftty Rts Jin Woo Lee 1, Il Seong Nm-Goong 1, Je Geun Kim 2, Chng Ho Yun 3, Se Jin Kim 1, Jung Il Choi 2, Young IL Kim 1, Eun Sook Kim 1 1 Deprtment of Internl Medicine, Ulsn University Hospitl, Ulsn University Collge of Medicine, 2 Biomedicl Reserch Center, Ulsn University Hospitl, 3 Deprtment of Biologicl Sciences, University of Ulsn, Ulsn, Kore Bckground: Inflmmtion plys role in the response to metbolic stress in type 2 dibetes. However, the effects of rosiglitzone on inflmmtion of skeletl muscle hve not been fully exmined in type 2 dibetes. Methods: We investigted the effects of the insulin-sensitizing nti-dibetic gent, rosiglitzone, on the progression of skeletl muscle inflmmtion in Otsuk Long-Evns Tokushim Ftty (OLETF) type 2 dibetic rts. We exmined the expression of serologic mrkers (serum glucose, insulin nd free ftty cid) nd inflmmtory cytokines (tumor-necrosis fctor-α, interleukin [IL]-1β nd IL-6) in OLETF rts from erly to dvnced dibetic stge (from 28 to 4 weeks of ge). Results: Serum glucose nd insulin concentrtions were significntly decresed in rosiglitzone-treted OLETF rts compred to untreted OLETF rts. Rosiglitzone tretment significntly decresed the concentrtions of serum inflmmtory cytokines from 28 to 4 weeks of ge. The mrna expression of vrious cytokines in skeletl muscle ws reduced in rosiglitzone-treted OLETF rts compred with untreted OLETF rts. Furthermore, rosiglitzone tretment resulted in the downregultion of ERK1/2 phosphoryltion nd NF-κB expression in the skeletl muscle of OLETF rts. Conclusion: These results suggest tht rosiglitzone my improve insulin sensitivity with its nti-inflmmtory effects on skeletl muscle. Keywords: Dibetes mellitus, type 2; Inflmmtion; Muscle, skeletl; Rts, inbred OLETF; Rosiglitzone INTRODUCTION Activtion of inflmmtion by metbolic stress in dibetes is ssocited with insulin deficiency cused by destruction of pncretic bet cells nd insulin resistnce in dipose tissue nd liver [1,2]. Adipocytes, which secrete severl dipokines (leptin, resistin, tumor-necrosis fctor [TNF]-α, interleukin [IL]-6, nd PAI-1), induce insulin resistnce nd inflmmtion throughout the entire body through decrese in diponectin [3]. The secretion of cytokines cused by the ctivtion of NF-κB lso induces insulin resistnce in ftty liver [4]. Some studies hve reported tht TNF-α is positively ssocited with insulin resistnce, which is ccompnied by dibetes nd obesity [5,6]. In some studies tht investigted the reltionship between insulin resistnce nd TNF-α, increses in TNF-α were found in the skeletl muscle of ptients who hd high insulin resistnce [7]. In ddition, TNF-α inhibits ctivtion of the insulin receptor tyrosine kinse by blocking insulin receptor substrte (IRS) phosphoryltion nd Akt substrte 16 phosphoryltion, which re importnt proteins in the insulin signling pthwy in skeletl muscle [8-1]. Bsed on these results, it seems tht n increse in TNF-α is not just byprod- Corresponding uthor: Eun Sook Kim Deprtment of Internl Medicine, Ulsn University Hospitl, Ulsn University Collge of Medicine, 29-3 Junh-dong, Dong-gu, Ulsn , Kore E-mil: es1@unitel.co.kr Received: Nov. 3, 29; Accepted: Mr. 3, 21 This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Copyright 21 Koren Dibetes Assocition

2 Lee JW, et l. uct of inflmmtion, but my directly influence inflmmtion of skeletl muscle. Thizolidinedione (TZD) is n insulin sensitizer tht functions not only to enhnce the effect of insulin by controlling gene trnscription through binding with nucler receptor peroxisome prolifertor ctivted receptor (PPAR)-γ, but lso to inhibit inflmmtion of dipose tissue, immunologic cells nd blood vessels through severl inflmmtory pthwys (STAT, AP-1 nd NF-κB pthwy) [11-13]. In this study, we investigted the role of skeletl muscle inflmmtion in the pthogenesis of insulin resistnce in type 2 dibetes in the Otsuk Long Evns Tokushim Ftty (OLETF) rt, well-known type 2 dibetes niml model. OLETF rts progress to hyperglycemi in postntl week 18 nd fibrosis of pncretic bet cells in postntl week 4 [14]. We lso investigted whether the nti-inflmmtory effect of rosiglitzone, which hs been shown in dipose tissue nd inflmmtory cells, is lso present in skeletl muscle. METHODS Lbortory nimls Five-week-old OLETF rts (n = 21) nd Long-Evns Tokushim Otsuk (LETO) rts (n = 11) were obtined from the Otsuk Phrmceuticl Co. Ltd. (Tokushim, Jpn). Ech rt ws housed in seprte cge nd ws fed niml feed. The cges were mintined t constnt temperture of 22 ± 2 C nd the light-drk cycle ws djusted utomticlly every 12 hours. To extrct tissues before the experiment, 5 rts from the OLETF group nd 5 rts from the LETO group were scrificed. Between the 28th nd 4th weeks, LETO rts (n = 6) were fed normlly, nd OLETF rts were divided into non-treted group (n = 8) nd rosiglitzone-treted (GlxoSmithKline Phrmceuticls, Phildelphi, PA, USA) group (3 mg/kg body weight, n = 8) (Fig. 1). At the 4th week, weight nd urine glucose levels were mesured, nd the rts were scrificed to collect gstrocnemius muscle smples. The body weights of the nimls were mesured every 4 dys t 4 o clock in the fternoon between the 8th nd 4th weeks. Serologic tests At the 28th nd 4th weeks, fsting blood ws extrcted from the herts of OLETF nd LETO rts. Plsm ws seprted from whole blood nd refrigerted t -7 C. Serum glucose level ws mesured by glucose oxidse rection method using n enzyme regent (Asn Phrmceuticl Co., Seoul, Kore). The concentrtion of insulin ws mesured by enzyme-linked immunosorbent ssy (ELISA) kit (Shibygi Co., Ishihr, Jpn). The concentrtion of free ftty cids ws mesured by the kinetic lkline picrte method using n enzyme rection kit (Asn Phrmceuticl Co.). Homeostsis model ssessment (HOMA)- insulin resistnce (IR) nd HOMA-β (HOMA bet cell function) were estimted using the following formul: [Fsting insulin (μiu/ml) fsting plsm glucose (mmol/l)]/22.5; HOMA-β = 2 C fsting insulin (μu/ml)/fsting glucose (mg/ dl) 63 [15]. Mesurement of inflmmtory cytokines At the 28th nd 4th weeks, the concentrtions of TNF-α, IL- Scrifice for 1st smple Scrifice for 2nd smple LETO (n = 11) 28 week (n = 5) No treted group 4 week (n = 6) OLETF (n = 2) 28 week (n = 5) Rosiglitzone tretment (3 mg/kg) Once dy / 12 weeks No treted group Rosiglitzone treted group 4 week (n = 8) 4 week (n = 8) Fig. 1. Experimentl schedule in Otsuk Long-Evns Tokushim Ftty (OLETF) nd Long-Evns Tokushim Otsuk (LETO) rts. 192 Koren Dibetes J 21;34:

3 Anti-inflmmtion of rosiglitzone in OLETF rts 1β nd IL-6 were mesured from the smpled blood plsm using n ELISA kit (ebioscience, Sn Diego, CA, USA). Expression of inflmmtory cytokine genes To extrct gstrocnemius muscle smples, the rts were scrificed by intrperitionel injection of 2.5% tribromoethnol t the 28th nd 4th weeks. The extrcted gstrocnemius smples were immeditely frozen with liquid nitrogen nd ground with homogenizer. Totl RNA ws seprted from the ground tissues using TRIzol regent. The cdna ws synthesized using oligo-(dt) primer nd AccuScript TM high fidelity 1ST strnd cdna synthesis kit (Strtgene) t 65 C for 5 minutes, t 42 C for 1 hour nd t 7 C for 15 minutes. For the PCR rection, 1 PCR buffer (1 mm Tris-HCl, ph 8.3, 5 mm KCl,.1% Triton X-1), 25 μm dntp mix, 1 U Tq polymerse (Tkr) nd specific primer for ech cytokine were mixed with 1 µg cdna. The rection ws incubted t 94 C for 3 seconds for denturtion, t C for 3 seconds for nneling, nd t 72 C for 3 seconds for extension. This cycle ws repeted times. ACTIN ws used for the comprison with PCR rection. The primer sequences for the PCR rection re shown below. Actin; Forwrd, 5 -TCA TGA AGT GTG ACG TTG ACA TCC GT-3 ; Reverse, 5 -CCT AGA AGC ATT TGC GGT GCA CGA TG -3, TNF- α ; Forwrd, 5 -AAA GCA TGA TCC GAG ATG TG-3 ; Reverse, 5 -AGC AGG AAT GAG AAG AGG CT- 3, IL-6: Forwrd, 5 -CCG GAG AGG AGA CTT CAC AG-3 ; Reverse, 5 -AGA ATT GCC ATT GCA CAA C-3, IL-1β; Forwrd, 5 -CAT CTT TGA AGA AGA GCC CG-3 ; Reverse, 5 - GGG ATT TCG TTG TTG CTT GT-3. Inflmmtory signl molecules Ground gstrocnemius muscle liquid, which ws extrcted from the 28th nd 4th week, ws wshed twice with HBSS buffer, nd then lysed on ice with RIPA buffer (5 mm HEP- ES, ph 7.4, 15 mm NCl, 1% deoxycholte, 1 mm EDTA, 1 mm PMSF, 1 mg/ml protinin). To observe NF-κB expression, the nucleus ws extrcted using NE-PER nucler nd cytoplsmic extrction regent (Pierce, Rockford, IL, USA). The protein ws mesured by the Brdford method. The sme quntity (2 mg) of protein ws mixed with 2X smple buffer (1 mm Tris- HCl, ph 6.8, 2 mm dithithreitol, 4% SDS,.2% bromophenol blue, 2% glycerol). Then, the mixture ws seprted with 1% SDS-PAGE. After electrophoresis, the protein ws trnsferred to nitrocellulose membrne t 4 C, 3 V for 16 hours. The membrne ws blocked with 1% skim milk t room temperture for 1 hour, nd then the membrne ws rected for 2 hours t room temperture with dilution of monoclonl ntibody for β-ctin, ERK1/2 MAPK or NF-κB (Cell Signling Technology; #911, #336); ntibodies were diluted with Tris-buffered sline contining.5% tween (TBS-T). The membrne ws then wshed three times with TBS-T. Then the membrne ws rected with HRP-conjugted nti-rbbit IgG secondry ntibody t room temperture for n hour, nd bnds were visulized with n enhnced chemiluminescence kit nd X-ry film (Amershm Phrmci Biotech, Sn Frncisco, CA, USA). Sttisticl nlysis All results re shown s the men nd stndrd devition bsed on three experiments. Sttisticl nlysis of the difference between groups ws nlyzed by one-wy ANOVA (GrphPd Prism progrm; GrphPd Softwre Inc., Sn Diego, CA, USA) nd Student t-test. A P vlue less thn.5 ws considered sttisticlly significnt. RESULTS Chnges in body weight The weights of the OLETF nd LETO rts were mesured every 4 weeks between the 18th nd 4th week. The weights of both OLETF nd LETO rts decresed with time until the 36th week. An insignificnt decrese in weight in normlly fed OLETF rts ws observed t the 4th week. The increse in the weight of the OLETF rts ws greter thn tht of the LETO rts, regrdless of rosiglitzone dministrtion. At the 36th week, the verge weight of the LETO rts ws ± 7.7 g, while tht of rosiglitzone-treted OLETF rts ws ± 18.6 g, nd tht of untreted OLETF rts ws ± 18. g. At the 4th week, the verge weight of the LETO rts ws ± 8.7 g, while tht of untreted OLETF rts ws ± 48.9 g nd tht of the rosiglitzone-treted OLETF rts incresed up to ± 19. g (Fig. 2). The weight gin of the rosiglitzone-treted OLETF rts ws greter thn tht of the untreted OLETF rts t the 32nd week. However, the difference ws not sttisticlly significnt until the 4th week. Chnges in serologic mrkers To investigte the chemicl chnges relted to the dministrtion of rosiglitzone, blood ws obtined from both OLETF nd LETO rts t the 28th nd 4th week. Concentrtions of Koren Dibetes J 21;34:

4 Lee JW, et l. 3 body weight (g) Weeks Fig. 2. Chnges of body weight in Long-Evns Tokushim Otsuk (LETO) nd Otsuk Long-Evns Tokushim Ftty (OLETF) rts. Body weights were mesured t 28, 32, 36, nd 4 weeks, nd chnges re represented s the verge weights on indicted dys. Vlues re presented s men ± stndrd devition. ROSI, rosiglitzone. P <.1 s compred to LETO rts. free ftty cid (FFA), insulin nd glucose were mesured from the plsm (Fig. 3). At the 28th week, the fsting glucose concentrtions of the LETO nd OLETF rts were ± 4.1 mg/dl nd ± 44.2 mg/dl, respectively. The glucose level ws higher in the OLETF rts thn in the LETO rts. At the 4th week, the glucose level of the LETO rts ws ± 26.8 mg/dl, while tht of the rosiglitzone-treted OLETF rts ws ± 42.5 mg/dl, nd tht of the untreted OLETF rts ws ± 44.4 mg/dl. The glucose level of the rosiglitzonetreted OLETF rts decresed significntly compred with tht of untreted OLETF rts. At the 28th week, the insulin concentrtions of the LETO rts nd OLETF rts were.94 ±.1 ng/ml nd 2.66 ±.4 ng/ ml, respectively. The insulin concentrtion of the OLETF rts incresed more thn tht of the LETO rts (P <.5). At the 4th week, the insulin concentrtion of the LETO rts ws 1.89 ± 1.6 ng/ml, tht of the rosiglitzone-treted OLETF rts ws 2.1 ± 1.4 ng/ml nd tht of the untreted OLETF rts ws 1.89 ± 1.6 ng/ml. The insulin concentrtion decresed significntly in the rosiglitzone-treted group (P <.5, Fig. 3). There were no sttisticlly significnt chnges in HOMA-β nd HOMA-IR mong the LETO rts, rosiglitzone-treted nd untreted OLETF rts (dt not shown). The men vlues of HOMA-IR mong the LETO rts, rosiglitzone-treted nd untreted OLETF rts were.8 ±.35, 2.4 ± 1.5 nd 1.83 ± Glucose (mg/dl) Insulin (ng/ml) FFA (mg/dl) weeks 4 weeks b 28 weeks 4 weeks 28 weeks 4 weeks Fig. 3. Chnges in concentrtion of serologicl mrkers in Long-Evns Tokushim Otsuk (LETO) nd Otsuk Long- Evns Tokushim Ftty (OLETF) rts. Glucose, insulin nd free ftty cid in ser of LETO or OLETF rts were mesured by colorimetric nd enzymtic ssy t 28 nd 4 weeks. Vlues were represented s men ± stndrd devition. FFA, free ftty cid. P <.5 nd b P <.1 s compred to ech group. ROSI is n bbrevition for rosiglitzone. 194 Koren Dibetes J 21;34:

5 Anti-inflmmtion of rosiglitzone in OLETF rts.86, respectively. Insulin resistnce incresed more in untreted OLETF rts thn in LETO rts. There ws trend of decresed insulin resistnce in rosiglitzone-treted OLETF rts compred with untreted OLETF rts (dt not shown). At the 28th week, the FFA concentrtions of the LETO nd OLETF rt groups were ± 77.9 mg/dl nd ± 76.7 mg/dl, respectively (Fig. 3). At the 4th week, the FFA concentrtion of the LETO rts ws 55.3 ± 82.7 mg/dl, tht of the untreted OLETF rts ws ± 18.3 mg/dl nd tht of the rosiglitzone-treted OLETF rts ws 28.1 ± 46.6 mg/dl. At the 4th week, the FFA level ws lower in the OLETF group compred to the LETO group. In contrst, decrese in the FFA level ws not observed in the rosiglitzone-treted group compred with the untreted group. Chnges in inflmmtory cytokines Serum TNF-α concentrtions t the 4th week were ± 8.3 pg/ml in the LETO rts, ± 64.3 pg/ml in the untreted OLETF rts, nd ± pg/ml in the rosiglitzonetreted OLETF rts (Fig. 4). The TNF-α concentrtion of the OLETF rt group ws significntly incresed compred with tht of the LETO rt group t the 4th week. In the OLETF rt group, TNF-α secretion ws significntly decresed by rosiglitzone dministrtion. For IL-1β t the 4th week, the men concentrtion in the LETO rt group ws significntly lower thn tht in the untreted OLETF rt group (125.6 ± 7.9 pg/ml vs ± 3.6 pg/ml, respectively). The IL-1β concentrtion of the rosiglitzone-treted OLETF rt group t the 4th week, ws ± 5.7 pg/ml, which ws significntly lower thn tht of the untreted group (Fig. 4). For IL-6 t the 4th week, the men concentrtion in the LETO rt group ws significntly lower thn tht of the untreted OLETF rt group (261.8 ± 19.5 pg/ml vs ± 49.1 pg/ml, respectively). The rosiglitzone-treted rts hd significntly lower IL-6 levels (79.1 ± 61.2 pg/ml) compred with untreted rts (Fig. 4). IL-1b (pg/ml) TNF- (pg/ml) IL-6 (pg/ml) , b b 28 weeks 4 weeks 28 weeks 4 weeks b 28 weeks 4 weeks Chnges of inflmmtory cytokine gene expression in skeletl muscle The gene expression of inflmmtory cytokines in the OLETF rt group ws higher thn tht of the LETO rt group (Fig. 5). Cytokine gene expression ws lower in the rosiglitzone-treted OLETF rt group thn in the untreted OLETF rt group, lthough the difference ws not sttisticlly significnt. Fig. 4. Chnges in inflmmtory cytokine levels in Long-Evns Tokushim Otsuk (LETO) nd Otsuk Long-Evns Tokushim Ftty (OLETF) rts. Tumor-necrosis fctor (TNF)-α, interleukin (IL)-1β, IL-6 levels in ser of LETO or OLETF rts were mesured by enzyme-linked immunosorbent ssy t 28 nd 4 weeks. Vlues re represented s the men ± stndrd devition. ROSI, rosiglitzone. P <.5 nd b P <.1 s compred to ech group. Koren Dibetes J 21;34:

6 Lee JW, et l TNF- IL-1b mrna expression (%) IL-6 IL-6 TNF- IL-1b Fig. 5. Chnges in inflmmtory cytokine gene expression in Long-Evns Tokushim Otsuk (LETO) nd Otsuk Long-Evns Tokushim Ftty (OLETF) rts. The expression of Tumor-necrosis fctor (TNF)-α, interleukin (IL)-1β, IL-6 mrna in skeletl muscle tissues of LETO nd OLETF mice were determined by RT-PCR t 4 weeks. There re no significnt differences between the groups. This is representtive figure from three independent experiments. ROSI is n bbrevition for rosiglitzone perk1/2 ERK1/2 b-ctin NF-kB p65 b-ctin 1 2 p-erk1/2 ctivity NF-kB ctivity LETO OLEFT OLETF +ROSI LETO OLEFT OLETF +ROSI A B Fig. 6. Chnges in levels of inflmmtory signling molecules in Long-Evns Tokushim Otsuk (LETO) nd Otsuk Long-Evns Tokushim Ftty (OLETF) rts. (A) ERK1/2 phosphoryltion in cytosol nd (B) NF-kB p65 expression in nucleus of skeletl muscle tissues were determined by Western blot nlysis t 4 weeks. This is representtive figure from three independent experiments. There re no significnt differences between the groups. ROSI is n bbrevition for rosiglitzone. Chnges in inflmmtory signl molecules expression To investigte the effects of rosiglitzone dministrtion on the inflmmtory pthwy, expression of signling molecules in ERK1/2 MAPK nd NF-κB pthwys ws exmined in skeletl muscle tissue of LETO nd OLETF rts by Western blot (Fig. 6). The phosphoryltion of ERK1/2 MAPK of the LETO rts ws shown to increse compred with tht in the OLETF rts. The phosphoryltion of ERK1/2 ws lowered by rosiglitzone tretment in OLETF rts (Fig. 6A). In ddition, for NFκB, there ws n increse in the expression of the NF-κB p65 subunit in the nucleus in OLETF rts, nd this ws decresed by dministrtion of rosiglitzone (Fig. 6B). 196 Koren Dibetes J 21;34:

7 Anti-inflmmtion of rosiglitzone in OLETF rts DISCUSSION Skeletl muscle minly uses glucose s n energy fuel. Insulin resistnce of muscle, cused by ccumultion of ftty cid nd reduced expression of glucose trnsporter, plys significnt role in the pthogenesis of type 2 dibetes. Norml muscle tissues re not involved in the inflmmtory process, except for in the condition of muscle trophy, which is seen in cchexi or chronic inflmmtion of muscles. Humn leukocyte ntigen (HLA) clss Ι, HLA clss ΙΙ nd secretion of inflmmtory cytokine increse when proinflmmtory stimulus is given to norml muscle tissues [15]. TNF-α mrna expression fter delivery ws higher in gesttionl dibetes ptients thn in ptients with norml glucose tolernce, nd this ws lso relted to insulin resistnce [16]. The inflmmtory process ws observed in the skeletl muscle of dibetes ptients, in whom chronic systemic inflmmtion is known to be present. However, the degree of inflmmtion of skeletl muscle in reltion to insulin resistnce in dibetes hs never been shown. An increse in dipose tissue cused by obesity ctivtes the JNK nd NF-κB pthwy, nd lso induces inflmmtion in liver through secretion of severl inflmmtory cytokines. This phenomenon is known to contribute to hyperglycemi through inpproprite inhibition of gluconeogenesis in liver [4]. Our hypothesis ws tht incresed inflmmtion in skeletl muscle tissues of dibetes ptients contributes to increses in insulin resistnce in type 2 dibetes, just s there is inflmmtion in liver tissues in dibetes ptients. Then, we exmined inflmmtion mrkers in skeletl muscle tissue nd plsm in OLETF rts, which is n niml model of type 2 dibetes. In this experiment, t the 4th week, gene expression of inflmmtory cytokines (TNF-α, IL-1β nd IL-6) nd expression of ERK1/2 MAPK nd NF-κB in the gstrocnemius muscle ws incresed in OLETF rts compred to LETO rts. Incresed expression of the trnscriptionl fctors of inflmmtory pthwys such s ERK1/2 MAPK nd IκB/NF-κB in the nucleus is known to be involved in the pthogenesis of insulin resistnce in skeletl muscle. There hve been other studies tht observed ctivtion of NF-κB cused by reduction of inhibitor of κbβ (IκB) t skeletl muscle of type 2 dibetes ptients is relted with insulin resistnce [17]. Activtion of NFκB in dipose tissue, which is cused by TNF-α stimulus, induces insulin resistnce through inhibition of insulin signling [18,19]. The mode of ction of TZD hs not verified completely, but it is known to bind with PPAR-γ selectively in dipose tissue, muscle tissue nd liver tissue, which re the trget orgns of insulin ction. It ccelertes gene expression of glucose trnsporter (GLUT)-1, GLUT-4 nd ctivtes differentition of dipose tissue nd inhibits expression of TNF-α, heptic glucokinse. Finlly, it improves insulin resistnce nd recovers glucose homeostsis in the humn body. In this experiment, we found tht rosiglitzone plys role in reducing glucose nd insulin levels mong vrious biochemicl mrkers. We observed n improvement in insulin resistnce, lthough there ws not n increse in insulin secretion bility. Rosiglitzone-treted OLETF rts gined their weight grdully until the 4th week, while untreted OLETF rts lost weight fter the 4th week, which is considered to be negtive side effect of rosiglitzone. It is known tht TZDs hve n nti-inflmmtory effect through inhibition of the trnscription process in the inflmmtion pthwy. PPAR-γ expression ws bundntly found in dipose tissue, mcrophges nd endothelil cells of blood vessels nd it ws lso reported tht TZDs decrese NF-κB expression t colonic epithelil cells in inflmmtory bowel disese [2]. However, its nti-inflmmtory effect on skeletl muscle, which is the trget orgn of insulin ction, hs not been clrified yet. It is known tht rosiglitzone hs n effect on severl inflmmtory pthwys, such s STAT, AP-1, nd NF-κB [21]. Rosiglitzone inhibits inflmmtory cytokine secretion t monocytes nd mcrophges by interruption of NF-κB in these inflmmtory pthwys [11-13]. In our study, t the 4th week, inflmmtory cytokines (TNF-α, IL-1β nd IL-6) were reduced significntly in rosiglitzone-treted OLETF rts in comprison to untreted OLETF rts. The expression of inflmmtory cytokine mrna (TNF-α, IL-1β nd IL-6) ws found to be decresed in rosiglitzone-treted OLETF rts. The inflmmtory pthwys of ERK/MAPK nd NF-κB were inhibited t skeletl muscle of rosiglitzone-treted OLETF rts. This study result shows tht rosiglitzone, which increses the expression of glucose trnsporters nd to decrese the ccumultion of lipids t skeletl muscle [22,23], lso interrupts the expression of inflmmtory cytokine (TNF-α, IL-1β nd IL-6) mrna nd thus contributes to the inhibition of inflmmtory pthwy of ERK/MAPK nd NF-κB t skeletl muscle [24,25]. However, there is still doubt tht only inhibition of NF-κB pthwy t skeletl muscle is sufficient to improve insulin resistnce in the whole body, becuse previous study showed n opposite result bsed on n experiment with muscle-specific IKK2 (IκB kinse) knockout mice [26]. The nti-inflmmtory effect of rosiglit- Koren Dibetes J 21;34:

8 Lee JW, et l. zone on insulin resistnce must be studied more extensively through the mesurement of GLUT-4 expression, glucose receptor nd IRS-1 in the skeletl muscle of OLETF rts. In ddition, it is necessry to investigte whether Rel trnsloction, Rel DNA binding nd IκBα degrdtion, which re considered to be involved in the pthogenetic mechnisms tht inhibits NF-κB pthwy, re relted to the nti-inflmmtory effect of rosiglitzone. In this experiment, we did not observe improvement in FFA level, which is well-known effect of rosiglitzone in other tissues. Although the weight of OLETF rts incresed, the FFA level did not increse in the OLETF rts compred to the LETO rts between the 28th nd 4th week. Thus, we were unble to detect n improvement in lipids. However, the nti-inflmmtory effect of rosiglitzone ws observed. It hs lredy been shown tht low dose rosiglitzone exerts n nti-inflmmtory effect independently of improvements in lipid nd insulin resistnce in type 2 dibetes [27]. In this study, we observed the reltionship between chnges in skeletl muscle inflmmtion nd chronic inflmmtion in hyperglycemic sttus. Rosiglitzone directly inhibited the inflmmtory process in the skeletl muscle in our rt model, which is considered to be component of the pthogenesis of insulin resistnce in skeletl muscle. These effects might help to improve insulin resistnce, not only in skeletl muscle, but lso in the entire body. ACKNOWLEDGEMENT This study ws supported by grnt of the Koren Dibetes Assocition (28) nd ws funded by Ulsn University Hospitl (Biomedicl Reserch Center Promotion Fund, UUH- 27-7). REFERENCES 1. Donth MY, Storling J, Berchtold LA, Billestrup N, Mndrup- Poulsen T. Cytokines nd bet-cell biology: from concept to clinicl trnsltion. Endocr Rev 28;29: Shoelson SE, Lee J, Goldfine AB. Inflmmtion nd insulin resistnce. J Clin Invest 26;116: Ahim RS, Flier JS. Adipose tissue s n endocrine orgn. Trends Endocrinol Metb 2;11: Ci D, Yun M, Frntz DF, Melendez PA, Hnsen L, Lee J, Shoelson SE. Locl nd systemic insulin resistnce resulting from heptic ctivtion of IKK-bet nd NF-kppB. Nt Med 25;11: Winkler G, Slmon F, Hrmos G, Slmon D, Speer G, Szekeres O, Hjos P, Kovcs M, Simon K, Cseh K. Elevted serum tumor necrosis fctor-lph concentrtions nd bioctivity in type 2 dibetics nd ptients with ndroid type obesity. Dibetes Res Clin Prct 1998;42: Mishim Y, Kuym A, Td A, Tkhshi K, Ishiok T, Kibt M. Reltionship between serum tumor necrosis fctor-lph nd insulin resistnce in obese men with type 2 dibetes mellitus. Dibetes Res Clin Prct 21;52: Sghizdeh M, Ong JM, Grvey WT, Henry RR, Kern PA. The expression of TNF lph by humn muscle: reltionship to insulin resistnce. J Clin Invest 1996;97: Hotmisligil GS, Perldi P, Budvri A, Ellis R, White MF, Spiegelmn BM. IRS-1-medited inhibition of insulin receptor tyrosine kinse ctivity in TNF-lph- nd obesity-induced insulin resistnce. Science 1996;271: Plomgrd P, Bouzkri K, Krogh-Mdsen R, Mittendorfer B, Zierth JR, Pedersen BK. Tumor necrosis fctor-lph induces skeletl muscle insulin resistnce in helthy humn subjects vi inhibition of Akt substrte 16 phosphoryltion. Dibetes 25;54: Bouzkri K, Zierth JR. MAP4K4 gene silencing in humn skeletl muscle prevents tumor necrosis fctor-lph-induced insulin resistnce. J Biol Chem 27;282: Jing C, Ting AT, Seed B. PPAR-gmm gonists inhibit production of monocyte inflmmtory cytokines. Nture 1998; 391: Ricote M, Li AC, Willson TM, Kelly CJ, Glss CK. The peroxisome prolifertor-ctivted receptor-gmm is negtive regultor of mcrophge ctivtion. Nture 1998;391: Mohnty P, Aljd A, Ghnim H, Hofmeyer D, Tripthy D, Syed T, Al-Hddd W, Dhinds S, Dndon P. Evidence for potent ntiinflmmtory effect of rosiglitzone. J Clin Endocrinol Metb 24;89: Kwno K, Hirshim T, Mori S, Sitoh Y, Kurosumi M, Ntori T. Spontneous long-term hyperglycemic rt with dibetic complictions: Otsuk Long-Evns Tokushim Ftty (OLETF) strin. Dibetes 1992;41: Ngrju K, Rben N, Merritt G, Loeffler L, Kirk K, Plotz P. A vriety of cytokines nd immunologiclly relevnt surfce molecules re expressed by norml humn skeletl muscle cells under proinflmmtory stimuli. Clin Exp Immunol 1998;113: Koren Dibetes J 21;34:

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