tapraid4/zh6-areg/zh6-areg/zh600707/zh65828d07a xppws S� 1 4/20/07 9: 48 MS: R-00603-2006 Ini: 07/rgh/dh A " #h!si$l %egul Integr &$ ' #h!si$l 2%&' R000 (R000) 200!$ 3. Originalarbeiten *irst publis#ed "arc# + 5) 200! doi' + 0$ + + 52,a-pregu$ 0060&$ 2006$ Clofibrate causes an upregulation of PPAR-� target genes but does not alter AQ: 1 expression of SREBP target genes in liver and adipose tissue of pigs Sebastian Luci, Beatrice Giemsa, Holger Kluge, and Klaus Eder Institut fu¨r Agrar- und Erna¨hrungswissenschaften, Martin-Luther-Universita¨t Halle-Wittenberg, Halle (Saale), Ger an! Submitted 25 August 2006 accepted in final form ! "arc# 200! AQ: 2 Luci S, Giemsa B, Kluge H, Eder K. Clofibrate causes an usuall0 increased 3#en baseline concentrations are lo3 1?62$ upregulation of PPAR-� target genes but does not alter expression of Effects of PPAR-� activation #ave been mostl0 studied in SREBP target genes in liver and adipose tissue of pigs$ A " #h!si$l rodents) 3#ic# ex#ibit a strong expression of PPAR-� in liver %egul Integr &$ ' #h!si$l 2%&' R000 (R000) 200!$ *irst publis#ed and s#o3 peroxisome proliferation in t#e liver in response to "arc# + 5) 200! doi' + 0$ + + 52,a-pregu$ 0060&$ 2006$ ./#is stud0 inves- PPAR-� activation 1&62$ Expression of PPAR-� and sensitivit0 tigated t#e effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor 1PPAR2-� and various to peroxisomal induction b0 PPAR-� agonists) #o3ever) var0 genes of t#e lipid metabolism in liver and adipose tissue of pigs$ An greatl0 among species 1+ %) 2+ 2$ 7n contrast to rats and mice) experiment 3it# + 4 pigs 3as performed in 3#ic# pigs 3ere fed eit#er 3#ic# are #ig#l0 sensitive to induction b0 peroxisome prolif- a control diet or t#e same diet supplemented 3it# 5 g clofibrate,5g for erators) guinea pigs) mon5e0s) pigs) and #umans are relativel0 24 da0s$ Pigs treated 3it# clofibrate #ad #eavier livers) moderatel0 insensitive 12+ ) &2) &%) ?42$ 7n t#ese nonproliferating species) increased mR6A concentrations of various PPAR-� target genes in expression of PPAR-� in t#e liver is muc# lo3er and t#e liver and adipose tissue) a #ig#er concentration of &-#0drox0but0rate) response of man0 genes to PPAR-� activation is 3ea5er t#an and mar5edl0 lo3er concentrations of trigl0cerides and c#olesterol in in proliferating species 142$ plasma and lipoproteins t#an control pigs 1# � 0$ 052$ mR6A con- 7n contrast to rodents) t#ere is little information to date about centrations of sterol regulator0 element-binding proteins 1SREBP2-+ t#e effects of PPAR-� agonists on lipid metabolism in pigs) Insig -( Insig-) and -2) insulin-induced genes 1 2 and ) and t#e SREBP 3#ic# are not onl0 of agricultural importance but are also a target genes acet0l-CoA carbox0lase) &-met#0l-&-#0drox0glutar0l- CoA reductase) and lo3-densit0 lipoprotein receptor in liver and valuable model for stud0ing t#e lipid metabolism because of adipose tissue and mR6A concentrations of apolipoproteins A-7) A-77) t#eir close relations#ip to #umans 162$ 7t #as been s#o3n t#at AQ: 3 and C-777 in t#e liver 3ere not different bet3een bot# groups of pigs$ treatment of pigs 3it# clofibrate stimulates mitoc#ondrial and 7n conclusion) t#is stud0 s#o3s t#at clofibrate treatment activates peroxisomal � -oxidation in liver) muscle) and 5idne0 1&?) 562$ PPAR-� in liver and adipose tissue and #as a strong #0potrigl0ceri- "oreover) it 3as found t#at pigs express functional PPAR-� in demic and #0poc#olesterolemic effect in pigs$ /#e finding t#at mR6A t#e liver) and several target genes induced in t#e liver b0 concentrations of some proteins responsible for t#e #0polipidemic PPAR-� activation #ave been identified 142$ ;o3ever) effects action of fibrates in #umans 3ere not altered suggests t#at t#ere 3ere of PPAR-� activation on lipid concentrations in plasma and certain differences in t#e mode of action compared 3it# #umans$ 7t is liver of pigs #ave not 0et been investigated$ 7n contrast to rats) also s#o3n t#at PPAR-� activation b0 clofibrate does not affect mice) or #umans in 3#ic# PPAR-� is predominant in liver #epatic expression of SREBP target genes involved in s0nt#esis of 1+ + 2) pigs ex#ibit also a #ig# expression of PPAR-� in adipose trigl0cerides and c#olesterol #omeostasis in liver and adipose tissue of pigs$ tissue) and it #as been suggested t#at pigs #ave a considerable capacit0 for � -oxidation in adipose tissue 1+ &2$ /#e effect of peroxisome proliferator-activated receptor-� c#olesterol trigl0cer- PPAR-� agonists on gene expression of PPAR-� target genes AQ: 4 ides in adipose tissue of pigs) #o3ever) #as not 0et been investi- gated$ Recent studies 1+ !) 2&) 25) &&2 in rodents suggested t#at *7BRA/ES ARE A 8R9:P 9* ;<P9=7P7>E"7C agents t#at #ave been in activation of PPAR-� in@uences #epatic trigl0ceride s0nt#esis clinical use for several decades in #umans 1?62$ 7t is 3ell and c#olesterol #omeostasis b0 interacting 3it# gene expres- establis#ed t#at t#ese agents act as s0nt#etic agonists of per- sion or proteol0tic activation of sterol regulator0 element- oxisome proliferator-activating receptor-� 1PPAR-�2) a nu- binding proteins 1SREBPs2) 5e0 regulators of lipid s0nt#esis clear receptor also activated b0 natural ligands suc# as free and #omeostasis$ SREBP-+ preferentiall0 activates genes re- fatt0 acids or some eicosanoids$ PPAR-� is an important Auired for fatt0 acid s0nt#esis) 3#ereas SREBP-2 preferen- regulator of cellular fatt0 acid upta5e and intracellular fatt0 tiall0 activates t#e =>= receptor gene and various genes acid transport) mitoc#ondrial and peroxisomal fatt0 acid oxi- reAuired for c#olesterol s0nt#esis 1222$ SREBPs are s0nt#e- dation) 5etogenesis) and gluconeogenesis 1?42$ 7n #umans) t#e siBed as inactive precursors bound to t#e endoplasmatic retic- most pronounced effect of fibrates is a decrease in plasma ulum membranes$ *or activation to occur) membranes #ave to trigl0ceride-ric# lipoproteins$ Concentrations of lo3-densit0 be cleaved b0 t3o resident proteases 3it#in t#e 8olgi) 3#ic# AQ: 5 lipoprotein 1=>=2 c#olesterol generall0 decrease in individuals seAuentiall0 cleave t#e SREBPs and release t#e amino-terminal 3it# elevated baseline plasma concentrations) and plasma b;=;-Cip-containing domain from t#e membrane) allo3ing it #ig#-densit0 lipoprotein 1;>=2 c#olesterol concentrations are to translocate to t#e nucleus and activate transcription of target Address for reprint reAuests and ot#er correspondence' D$ Eder) 7nstitut fuEr Agrar- und ErnaE#rungs3issensc#aften) "artin-=ut#er-:niversitaEt ;alle- /#e costs of publication of t#is article 3ere defra0ed in part b0 t#e pa0ment Fittenberg) Emil-Abder#alden-Str$ 26) >-06+ 04 ;alle 1Saale2) 8erman0 of page c#arges$ /#e article must t#erefore be #ereb0 mar5ed Hadvertise entI AQ: 12 1e-mail' Dlaus$ ederG land3$ uni-#alle$ de2$ in accordance 3it# + 4 :$ S$ C$ Section + !&? solel0 to indicate t#is fact$ AQ: 12 #ttp' ,,333$ a-pregu$ org 0&6&-6+ + %,0! J4$ 00 Cop0rig#t K 200! t#e American P#0siological Societ0 R+ 16 tapraid4/zh6-areg/zh6-areg/zh600707/zh65828d07a xppws S� 1 4/20/07 9: 48 MS: R-00603-2006 Ini: 07/rgh/dh R2 E**EC/S 9* C=9*7BRA/E /REA/"E6/ 76 P78S genes$ 7nsulin-induced genes 1Insig2-( and Insig-) are modu- diet per da0$ /#e amount of diet administered 3as � + 5O belo3 t#at lators of SREBP activit0 15&) 552$ /#e0 bloc5 t#e proteol0tic consumed ad libitum b0 pigs of a similar 3eig#t 1as assessed in a cleavage and transcriptional activation of SREBP$ 7n pigs) in previous stud02$ /#erefore) t#e diet offered 3as completel0 ta5en in AQ: ( contrast to rodents in 3#ic# lipogenesis ta5es place primaril0 b0 all pigs in t#e experiment$ >uring t#e feeding period) t#e amount of diet offered eac# da0 3as increased continuousl0 from ?00 to + )200 g$ in t#e liver 1+ 5) ??2) adipose tissue is t#e ma-or site of /#e pigs #ad free access to 3ater via nipple drin5ing s0stems$ /#e lipogenesis$ 7t #as been s#o3n t#at SREBP-+ and its target experimental diets 3ere administered for 24 da0s$ All experimental gene fatt0 acid s0nt#ase) one of t#e 5e0 enB0mes of de novo procedures described follo3ed establis#ed guidelines for t#e care and fatt0 acid s0nt#esis) are expressed at a #ig#er level in pig use of laborator0 animals and 3ere approved b0 t#e local veterinar0 adipose tissue t#an in pig liver 1+ 2) + &2$ F#et#er a lin5 exists office$ AQ: ) also in pigs bet3een PPAR-� activation and gene expression or Sa 'le c$llecti$n* After completion of t#e feeding period) t#e proteol0tic activation of SREBPs in liver or adipose tissue) animals 3ere 5illed under lig#t anest#esia$ *our #ours before eut#a- 3#ic# in turn could in@uence lipid s0nt#esis) is presentl0 nasia) eac# pig 3as fed its respective diet$ After deat#) blood 3as un5no3n$ collected into #epariniBed pol0et#0lene tubes$ Plasma 3as obtained b0 /#e ob-ective of t#e present stud0 3as to investigate t#e centrifugation of t#e blood 1+ )+ 00 g at ?NC for + 0 min2$ Plasma effects of clofibrate treatment on expression of genes involved lipoproteins 3ere separated b0 step-3ise ultracentrifugation 1"i5ro- :ltraBentrifuge Sorvall Products) Bad ;omburg) 8erman02 at in lipid metabolism in liver and adipose tissue of pigs$ Fe 3ere %00)000 g at ?NC for + $ 5 #$ Plasma densities 3ere ad-usted b0 sodium particularl0 interested in to 3#at extent clofibrate upregulates c#loride and potassium bromide) and t#e lipoprotein fractions � � AQ: * PPAR-� target genes in liver and adipose tissue of pigs and + $ 006 5g,l Lver0 lo3-densit0 lipoproteins 1Q=>=2 plus c#0lomicronsM) 3#et#er t#ere is an interaction