Türk Biyokimy Dergisi [Turkish Journl of Biohemistry Turk J Biohem] 9; 34 () ; 3 38. Reserh Artile [Arştırm Mklesi] Yyın trihi 6 Mrt, 9 TurkJBiohem.om [Pulishe online 6 Mrh, 9] Effets of systemi leptin ministrtion on liver n plsm lipi peroxition in ol restrin stress [Soğuk hreketsizlik stresine sistemik leptin uygulmsının kriğer ve plzm lipi peroksisyonu üzerine etkileri] Çiğem Özer, Sevim Ern, Ayn Bül, 3 Z. Sevim Ern Gzi University, Fulty of Meiine, Deprtment of Physiology, Beşevler, Ankr 65, Turkey. Akeniz University, Shool of Helth Servies, Antly, Turkey. 3 Gzi University, Fulty of Meiine, Deprtment of Phrmology, Beşevler, Ankr 65, Turkey. Yzışm Aresi [Corresponene Aress] ABSTRACT Aim: Oxitive stress is stte to e n importnt mehnism of ol immoiliztion stress leing to tissue injury. The im of this stuy ws to investigte the effets of exogenous leptin ministrtion on plsm n hepti tissue lipi peroxition n ntioxint sttus in ol-restrint stress. Mteril n metho: In this stuy Wistr lino rts were ivie into four :. Control,. Stress, 3. Leptin ( μg/kg leptin, i.p. twie y, for 7 ys), 4. Leptin + stress. At the en of 7 th y Stress n Leptin + stress were expose to ol-restrint stress, inflite y pling the nimls in refrigertor t 4. o ±.5 o C for 4 h. At the en of the experiments nimls were kille uner thiopentl soium (5mg/kg) nesthesi. Plsm n liver mlonilehye, glutthione n totl nitri oxie levels were mesure. Results: Col-restrint stress inrese plsm n liver mlonilehye levels wheres erese glutthione n totl nitri oxie levels. Pretretment with leptin signifintly lowere mlonilehye levels n elevte glutthione n totl nitri oxie levels. Conlusions: Aute ol stress my le to oxitive stress y inresing the lipi peroxition while epleting the ntioxint pities. Pretetment with leptin exerte protetive effet on plsm n liver ginst ol restrint stress inue tissue injury, proly through inresing nitri oxie ontent. Key Wors: Col-restrint stress, leptin, lipi peroxition, glutthione, nitri oxie Prof. Dr. Ayn Bül Gzi Üniversity, Fulty of Meiine, Deprtment of Physiology, 65, Beşevler/Ankr TURKEY Tel: +9 3 693, Fx: +9 3 46 47, E-mil: ul@gzi.eu.tr [Reeive: 3 Novemer 8; Aepte: 3 Jnury 9] Kyıt Trihi: 3 Ksım 8; Kul Trihi: 3 Ok 9 ÖZET Amç: Oksitif stres, soğuk hreketsizlik stresinin neen oluğu oku hsrınki önemli meknizmlrn iriir. Çlışmnın mı soğuk hreketsizlik stresine eksojen leptin uygulmsının plzm ve kriğer okusun lipi peroksisyonu ve ntioksin urum üzerine etkilerinin inelenmesiir. Gereç ve Yöntem: Çlışm Wistr lino sıçnlr ört gru yrılı:. Kontrol,. Stres, 3. Leptin ( μg/kg leptin, i.p., güne iki ef, 7 gün süre ile) 4. Leptin+stres. 7. günün sonun Stres ve Leptin+stres gruplrı ortm sıklığı 4. o ±.5 o C oln uzolın 4 st süre ile soğuk-hreketsizlik stresine mruz ırkılılr. Deneylerin sonun hyvnlr tiyopentl soyum (5mg/kg) nestezisi ltın fe eililer. Plzm ve kriğere mlonilehi, gluttyon ve totl nitrik oksit üzeyleri ölçülü. Bulgulr: Soğuk hreketsizlik stresi plzm ve kriğere mlonilehit üzeyini rttırırken, gluttyon ve totl nitrik oksit üzeylerine zlmy neen olu. Leptin ile ypıln ön tevi mlonilehit üzeylerini önemli ölçüe üşürürken, gluttyon ve totl nitrik oksit üzeylerini e önemli ölçüe yükseltti. Sonuç: Akut soğuk stres lipi peroksisyonu rttırıp, ntioksin kpsiteyi skılyrk oksitif strese neen olilir. Leptin ile ypıln öntevi plzm ve kriğere soğuk hreketsizlik stresinin neen oluğu oku hsrın krşı koruyuu ir etki oluşturmuştur. Bu etki rtn nitrik oksit üzeyiyle ilişkili olilir. Anhtr Kelimeler: soğuk hreketsizlik stresi, leptin, lipi peroksisyonu, gluttyon, nitrik oksit http://www.turkjbiohem.om 3 ISSN 33 89X (eletroni) 5 4685 (printe)
Introution Stress is efine s onition in n orgnism tht results from the tion of severl stressors tht my e internl or externl origin (). It is well known tht ol exposure my e reflete in n elevte metoli rte n lso inrese proution of retive oxygen speies (ROS) (). When ROS proution exees the pities of protetion n repir mehnisms oxitive stress ours, resulting in mge to proteins n lipis (3). One the most importnt mging effet of free rils on tissues is lipi peroxition. It hs een emonstrte in some forms of stress, suh s exerise, strvtion, ol n wter-immersion restrint stress inresing the free ril genertion n lipi peroxition (4). Lipi peroxition n e evlute y the mesurement of mlonilehye (MDA) levels (5). Glutthione (GSH) is one of the importnt enogen ntioxints n prt from svenging free rils; lso plys role in the reution of vrious isulfie linkges n mintenne of proteins in proper oxiize-reue stte (6). Aute ol stress signifintly erese loo GSH levels n perturtion of GSH metolism in severl viserl orgns (7). Leptin, the 6 kd prout of o gene, is ytokinelike moleule. Inresing numer of evienes suggest tht leptin prt from the regultion of foo intke n energy homeostsis, prtiiptes in mny physiologil funtions inluing regultion of neuroenorine n immune systems, reproution, ngiogenesis n lipolysis (8). There hve een ontroversil reports out the oxint or ntioxint roles of leptin in ifferent tissues. It is well known tht leptin plys n importnt role in gstri muosl integrity n gstroprotetion (8). However, there is some eviene initing the orreltion of serum leptin levels with hepti firosis ut not with hepti stetosis or inflmmtion (9). It is lso known tht leptin stimultes enothelil nitri oxie (NO) proution () n intrvenous injetion of leptin inreses the plsm nitrite/nitrte onentrtion in ose-epenent mnner in normotensive rts (). Some stuies reporte tht NO is n importnt meitor of heptotoxiity n the high proution of NO uses tissue injury (, 3). However, in some moels of inflmtion, it hs een shown tht inhiition of NO inreses tissue ysfuntion or injury (4). Consequently, the pro-oxint n/or nti-oxint role of NO seems to e ontroversil. Although severl stuies hve investigte the effets of ol-restrint stress on the ntioxint system n inution of lipi peroxition in severl tissues, no informtion is ville regring the ntioxint effet of systemi leptin on ol restrint stress inue plsm n hepti tissue injury so fr. In the present stuy liver ws preferre for its high metoli rte. Thus, in the present stuy the effets of exogenous leptin ministrtion on ol-restrint stress inue plsm n hepti tissue mge were exmine y evlution of MDA, GSH n totl nitri oxie (NO) levels. Mteril n Methos Animls n Stuy Design The following experiments were pprove y the Ethil Committee of Gzi University for the re n use of lortory nimls. 3 mle Wistr lino rts weighing ± g were use in this stuy. They were fe stnr lortory iet n tp wter liitum n kept in room with ontrolle temperture ( ± o C), n :-h light-rk yle. They were ivie into four eh onsisting of 8 nimls n llowe free ess to stnr iet n wter liitum.. Control group; physiologil sline solution, PS, i.p. twie y, for 7 ys.. Stress group ; PS, i.p, twie y, for 7 ys. At the en of the 7 th y rts were expose ol-restrint stress. 3. Leptin group ; μg/ kg, reominnt rt leptin (CALBIOCHEM), i.p. twie y, for 7 ys. (8). 4. Leptin + stress group; μg/ kg leptin, i.p. twie y, for 7 ys. At the en of the 7 th y rts were expose ol-restrint stress. Animls were fste for 4 h efore the stress inution, ut hve h free ess to wter. A ol-restrint stress moel ws inflite y pling the nimls in refrigertor t 4. o ±.5 o C for 4 h (5). Control n leptin were not expose to stress. At the en of the experiments nimls were kille uner thiopentl soium (5mg/kg) nesthesi. Plsm ws seprte n store. The liver ws immeitely exise n kept in liqui nitrogen n store t 7 o C until susequent ssy. Biohemil etermintions Determintion of plsm lipi peroxie level Plsm lipi peroxie levels were estimte y the metho of Kurtel et l (6). Briefly, lipi peroxition ws quntifie y mesuring the formtion of thiorituri i retive sustnes (TBARS). Lipi peroxie level ws expresse in terms of MDA equivlent using n extintion oeffiient of.56 x 5 M - m - (7). Determintion of plsm RSH level The RSH levels were etermine y the metho of Kurtel et l. (6). The RSH levels were lulte ssuming molr extintion oeffiient of 3. mol - m - t 4 nm. Determintion of tissue MDA n GSH levels Hepti tissue smples were otine n frozen immeitely in liqui nitrogen, thn kept t 7 o C until nlyses were performe. Lipi peroxition ws quntifie y mesuring the formtion of thiorituri i retive sustnes (TBARS) (8). Lipi peroxie levels re expresse in terms of MDA equivlents using Turk J Biohem, 9; 34 () ; 3 38. 33
n extintion oeffiient of.56 x 5 mol - m -. The GSH levels were etermine y moifie Ellmn metho (9). The GSH levels were lulte using n extintion oeffiient of 3,6 mol - m -. Mesurements of MDA, GSH n RSH were rrie out t room temperture using spetrophotometer (UV 8, Shimzu, Jpn). Determintion of tissue n plsm NO levels NO levels were mesure with Elis reer y vnium hlorie (VCl 3 ) / Griess ssy. Prior to NO etermintion the tissues were homogenize in five volumes of phosphte uffer sline (ph=7) n entrifuge t g for 5 min..5 ml.3 M NOH ws e to.5 ml of the superntnt. After inution for 5 min. t room temperture,.5 ml of 5% (w/v) ZnSO 4 ws e for eproteiniztion. This mixture ws then entrifuge t 4 rpm for 5 min n superntnts were use for the ssys (). Nitrte stnr solution ws serilly ilute. After loing the plte with smples ( μl), ition of vnium III hlorie (VCl 3 ) ( μl) to eh well ws rpily followe y ition of Griess regents, sulphnilmie (SULF) (5 μl) n N- (-nphtyl) ethyleneimie ihyrohlorie (NEDD) (5 µl). After the inution (usully 3-45 min), smples were mesure t 54 nm using n ELISA reer. The sme proeure ws pplie to the plsm smples fter eproteiniztion ws rrie out. Sttistil Anlysis Dt re presente s mens ± S.D. Sttistil nlysis using y SPSS 3. pk for Winows. Vlues of p<.5 were regre s signifint. First of ll, for NO, GSH/RSH, n NO levels in liver n plsm, Kolmogorov-Smirnov test ws use to isply whether the t istriute normlly or not. Dt for those three prmeters ws normlly istriute in oth liver n plsm. Then ANOVA test ws employe for vrine nlysis, n there ws sttistilly signifint ifferene etween plsm n liver (p<.5). Followe y ANOVA test, Levene test ws employe for the equl vrine of eh vrile (MDA, GSH, NO) in four. Results of this test were isplye elow:. In liver Tissue: MDA n NO levels hve homogenous istriution, n Tukey Multiple Comprison test ws use for pirwise omprisons. Beuse of GSH levels showe non- homogenous istriution, Tmhne test ws employe. In plsm: MDA, RSH, n NO levels hve homogenous istriution, n Tukey Multiple Comprison test ws use for pirwise omprisons. Results Plsm MDA levels re shown in Figure. eing.56±. nmol/ml, 3.7±.5 nmol/ml,.37±. nmol/ml n.6±. nmol/ml in ontrol, stress, leptin n leptin + stress respetively. Plsm MDA levels were signifintly higher (p<.5) in the stress group thn the ontrol group. Plsm MDA levels were signifintly lower in the leptin group ompring ll the other three (p<.5). Leptin ministrtion (μg/kg i.p. for 7 ys) prior to ol-restrint stress le to signifint erese in plsm MDA levels s ompre to the stress group (p<.5). The ifferene were not sttistilly signifint n similr result ws otine etween the ontrol n the leptin+stress. Plsm RSH n NO levels re shown in Figure. n Figure 3. respetively. Plsm RSH levels were 45±.9 nmol/ml, 9.6±.9 nmol/ml, 47.5±.7 nmol/ml n 3.8±.7 nmol/ml in ontrol, stress, leptin n leptin + stress respetively. Plsm NO levels were 44.36 ±.7 μmol/l, 6.75±.8 μmol/l, 5.3±.4 μmol/l n 38.38±.9 μmol/l in ontrol, stress, leptin n leptin + stress respetively. Plsm RSH n NO levels were erese in fter ol-restrint stress (p<.5). In omprison to the ontrol group, systemi leptin ministrtion inrese the NO levels (p<.5), ut there ws no signifint hnge in RSH levels (Group 3). Pretretment with i.p. leptin resulte in signifint inrese in plsm RSH n NOx levels (Group 4) when ompre with the results mesure in tht group whih ws sujete to ol-restrint stress lone (group ) (p<.5, p<.5 respetively). The results out men liver MDA levels re shown in Figure 4. Tissue MDA levels were 46.68±.89 nmol/ g, 5.75± nmol/g, 39.35±.57 nmol/g n 47.37±.8 nmol/g tissue in ontrol, stress, leptin n leptin + stress respetively. Tissue MDA levels ws foun to e higher in the stress group when ompre with the ontrol group (p<.5). The most signifint erese in MDA levels were seen in leptin group (p<.5). Exogenous leptin signifintly erese MDA levels in the liver of ol-restrint stress tretment rts (Group 4, p<.5). The ifferene ws not sttistilly signifint etween the ontrol n the leptin + stress. Liver GSH n NO levels re shown in Figure 5. n Figure 6. respetively. Tissue GSH levels were 5.6±. μmol/g, 4.±.5 μmol/g, 6±.4 μmol/g n 4.6±. μmol/g tissue in ontrol, stress, leptin n leptin + stress respetively. Tissue NO levels were 3.73± μmol/g, 8.56±.49 μmol/g, 7.6±.48 μmol/g n.79±.8 μmol/g tissue in ontrol, stress, leptin n leptin + stress respetively. GSH n NO levels in the liver ws signifintly lower in stress group s ompre with the ontrol group (p<.5, p<.5 respetively). Leptin ministrtion prior to ol-restrint stress (group 4) le to signifint inrese in liver GSH n NO levels s ompre to the stress group (p<.5, p<.5 respetively). The most signifint inrese in NO level ws seen in the leptin group (Figure 6). Turk J Biohem, 9; 34 () ; 3 38. 34
Plsm MDA levels (nmol/ml) 4 3 Figure. MDA levels in the plsm of ontrol, stress, leptin n leptin+stress. Eh vlue represents the men ± S.D. of eight nimls per group. p<.5 : -, -, -, - - Plsm RSH levels (nmol/ml) 5 5 Figure. RSH levels in the plsm of ontrol, stress, leptin n leptin+stress. Eh vlue represents the men ± S.D. of eight nimls per group. p<.5 : -, -, -, -, - Plsm NOx levels (mirom/l) 6 5 4 3 Figure 3. NO levels in the plsm of ontrol, stress, leptin n leptin+stress. Eh vlue represents the men ± S.D. of eight nimls per group. p<.5 : -, -, -, -, -, -. Turk J Biohem, 9; 34 () ; 3 38. 35
Liver MDA levels (nmol/g) 6 5 4 3 Figure 4. MDA levels in the liver of ontrol, stress, leptin n leptin+stress. Eh vlue represents the men ± S.D. of eight nimls per group. p<.5 : -, -, -, -, -. Liver GSH levels (nmol/g) 8 6 4 Figure 5. GSH levels in the liver of ontrol, stress, leptin n leptin+stress. Eh vlue represents the men ± S.D. of eight nimls per group. p<.5 : -, -, -, -, -. Liver NOx levels (mirom/g) 4 3 Figure 6. NO levels in the liver of ontrol, stress, leptin n leptin+stress. Eh vlue represents the men ± S.D. of eight nimls per group. p<.5 : -, -, -, -, -. Turk J Biohem, 9; 34 () ; 3 38. 36
Disussion The physiologil omponents of stress response to ol overs metoli, irultory n hormonl proess; however, the ellulr n moleulr mehnisms meiting these responses remins to e eluite. Immoiliztion n ute ol stress re wiely use experimentl moel ompnie y onsierle erese in ntioxitive pity in nimls (, ). Lipi peroxition hs een implite in numer of eleterious effets n inrese in the levels of TBARS inite the enhne lipi peroxition leing to tissue injury n filure of ntioxint efense mehnisms to prevent the formtion of exess free rils (3). In the present stuy liver n plsm MDA levels were foun to e signifintly inrese in the rts expose to restrintol stress when ompre to the ontrols. These results re in greement with the previous finings whih re relte to stress-inue lipi peroxition in plsm n liver of the nimls (, 4). Our finings showe tht the ntioxint system ws lso ffete y ol-restrint stress. GSH is n importnt enogenous efense sustne ginst the retive oxygen speies (ROS) n the tissue GSH onentrtion reflets potentil etoxifition mrker. It hs een previously reporte tht ol stress reue the GSH levels in liver n in the other tissues in mie () n rts (5). Shustnov et l. hve reporte tht ol stress use n inhiition of ntioxint enzyme tivities suh s glutthione reutse in rin, liver n erythroytes (6). These results re in orne with the results of the present stuy, sine the olrestrint stress resulte in n tivtion of free ril ompnie y n umultion in liver n plsm of lipi peroxition prouts n erese of enogenous ntioxint mrker GSH. However, the inrese in plsm GSH level might e ue to the penetrtion of tissue GSH to plsm. Aoring to the results of the present stuy, it n e ssume tht the negtive reltion etween MDA n GSH levels in in the liver n plsm of the rts in the stress group my e the eviene of the suseptiility of the proteins to oxition. It hs een well known tht NO rils hve iret ntioxint effet through their retion with free rils n iron-oxygen omplexes. In ition to serving s stilizer n rrier of NO, S-nitrosoglutthione (GSNO) my hve protetive effets through trnsnitrosyltion retions. Moreover, it hs een suggeste tht NO n GSNO t like free ril svenger t moerte onentrtions whih re 5- times more potent thn tht of GSH (7). Perlt et l. reporte tht mtnos tivity n expression erese in liver n skeletl musle uring the first ys of the ol exposure (8). The positive orreltion etween ol stress n reue NOS tivity ws supporte y the finings of Zhu et l. (9), Lee et l. (3) in ifferent tissues. In the present stuy we oserve tht NO tivity oth in the plsm n liver ws foun to e signifintly erese uring ol-restrint stress. These results re lso in orne with the finings tht inite the evelopment of resistne to ol stress might e olishe y eresing NO (3). The erese of NO synthesis suggests n inility of the ells to synthesize GSH. Aoring to the results of the present stuy, leptin pretretment signifintly reue the olimmoiliztion inue inrese in MDA levels of plsm n liver. Although leptin ministrtion to norml rts i not hnge the plsm n liver MDA n GSH levels, use n inrese in plsm n tissue GSH levels otine from restrint-ol stress inue rts. There re ontroversil reports out the prooxint or ntioxint roles of leptin in ifferent tissues. This isrepny might e ue to the ifferent experimentl methos use n speies ifferenes. In some stuies leptin ministrtion, inrese lipi peroxition n inhiite ntioxint system in mie rin (3). But it hs een shown tht leptin, erese TBARS level of mie liver, hert n kiney (33), exerte potent gstroprotetive effet ginst ishemi-reperfusion (34) or ethnol inue stomh mge (35), suppresse poptosis in gstri muos (5) n liver (36). In this stuy leptin use n inrese in GSH, while erese MDA levels oth in plsm n liver tissue whih were foun to e erese n inrese respetively otine from rts expose to restrint-ol stress. Reently it hs een shown tht leptin uses n inrese in enothelil NO proution (37) n ontriutes to the heling proess of pepti uler y stimulting lol proution of NO (8) Our results re in orne with these finings. In the present stuy we oserve tht leptin uses n inrese in plsm n tissue levels of NO in norml rts n lso uses n inrese in the levels of NO whih is foun to e erese uring restrint-ol stress. These results inite tht restrint-ol stress uses n inrese in MDA levels n erese NO levels in plsm n liver tissues, erese GSH level in tissue while inrese in plsm. Leptin ministrtion uses n inrese oth in erese GSH n NO levels n ereses the inrement of MDA levels. In onlusion it n e ssume tht stress ffets the progression of severl isese vi the genertion of ROS n inhiition of ntioxint system s well s NO synthesis. Leptin proly y inresing NO synthesis prevents the inrese of MDA n erese in GSH levels vi ntioxint n tissue protetive effets. Aknowlegements We thnk Professor Dr. Semr Erş for her kin help on Sttistil Anlysis. Turk J Biohem, 9; 34 () ; 3 38. 37
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