ABSTRACT. Marek s disease virus (MDV) infection causes atherosclerosis, and prior

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1 ABSTRACT Title of Document: Directed By: COMPARATIVE STUDY OF LIPOPROTEIN METABOLISM IN MAREK S DISEASE SUSCEPTIBLE AND RESISTANT LINES Ping Yun, Mster of Science, 2010 Assistnt professor Dr. Jiuzhou Song, Deprtment of Animl nd Avin Sciences Mrek s disese virus (MDV) infection cuses therosclerosis, nd prior vccintion prevented the development of this disese. Two min strtegies to resist Mrek s disese (MD) hve been demonstrted: vccintion nd genetic resistnce. However, little is known bout the role of genetic resistnce in the progression of MDV induced therosclerosis. Atherosclerosis is primrily ssocited with lipoprotein metbolism. The purpose of this study ws to investigte whether lipoprotein metbolisms re different in distinct MD susceptible nd resistnt chicken lines. Here, we studied different bckgrounds of lipoprotein metbolism in the two lines nd the chnges of lipoprotein levels in response to MDV infection. The results showed tht during chicken growth, the increse in totl cholesterol ws mostly due to the incresing (LDL+VLDL) in MD susceptible line, wheres it ws minly due to the elevting HDL in MD resistnt line. These results suggested tht different lipoprotein metbolisms exist in MD susceptible nd resistnt lines. 1

2 Comprtive Study of Lipoprotein Metbolism in Mrek s Disese Susceptible nd Resistnt Lines By Ping Yun Thesis submitted to the Fculty of the Grdute School of the University of Mrylnd, College Prk, in prtil fulfillment of the requirements for Mster of Science 2010 Advisory Committee: Dr. Jiuzhou Song, Chir Dr. Zhengguo Xio Dr. Scott M Updike 2

3 Copyright by Ping Yun

4 Dediction To my prents, thnk you for ll your support ii

5 Acknowledgments Thnk you to my dvisor Dr. Song, who hs helped me gret del in developing my scientific thinking nd gve me solid support for my project. To the other members of my committee, Dr. Xio nd Dr. Updike, who hve given me vluble dvice for my Mster s work. Thnk you to the Song lb, for the help nd friendship you hve given me for the pst two yers. iii

6 Tble of Contents Dediction......ii Acknowledgements...iii Tble of contents...iv Chpter 1. Literture review...1 Introduction. 1 Mrek s disese nd therosclerosis...3 Lipoprotein nd therosclerosis.. 8 Adiponectin nd therosclerosis...16 Chpter 2: Comprison of lipoprotein metbolism in MD resistnt nd susceptible chicken lines...24 Introduction Experimentl design...26 Methods nd mterils Results...32 Phenotype nlysis...32 Plsm lipoprotein levels Adiponectin, dipor1 nd dipor2 mrna levels..41 Adiponectin, dipor1 nd dipor2 protein levels..45 Summry...55 Chpter 3: lipoprotein levels in response to MDV infection in MD resistnt nd susceptible lines.57 Introduction iv

7 Experimentl design Methods nd mterils Results...61 Phenotype nlysis...61 Plsm lipoprotein levels Summry...68 Chpter 4: Generl discussions..70 References..74 v

8 Chpter 1: Literture review Introduction Atherosclerosis is progressive disese chrcterized by lipid ccumultion in the lrge rteries, which cuses bout 50% of ll deths in westernized societies 1. It hs been demonstrted tht therosclerosis is complex process which involves the interction of severl genetic nd environmentl fctors, including dibetes, hypercholesterolemi nd cigrette smoking 2. Since therosclerotic lesions re thought to be initited by injury to the vsculr endothelium, considerble reserch hs been focused on studying the mechnisms responsible for this initil injury 2. Herpesviruses hve been proposed s potentil inititors of rteril injury. This sttement ws bsed on severl studies in the lte 1970 s, which reveled tht Mrek s disese virus (MDV), n vin herpesvirus, could induce therosclerosis in chickens 3-5. MDV is n oncogenic herpesvirus tht cuses severl syndromes in the chicken. It hs been demonstrted tht MDV infection cn induce occlusive therosclerotic lesions in lrge coronry rteries nd orts of infected chickens 5. MDV infection lso ffected enzymtic ctivities in rteril smooth muscle cells, which resulted in ortic lipid ccumultion in chicken, including cholesterol, cholesterol ester, tricylglycerol nd phospholipids 4. It hs been suggested tht the lipid ccretion in orts of MDV infected chickens ws possibly due to ltertions in cholesterol metbolism 4, 6. 1

9 Besides cholesterol metbolism, lipoprotein metbolism ws involved in therosclerosis 7, 8. Progressive ccumultion of cholesterol in the rteril wll cuses therosclerosis. Lipoproteins, s the key crriers of cholesterol through the circulting system, hve been reported to be ssocited with the development of therosclerosis. For instnce, the low density lipoprotein (LDL) ccumulted in the subendothelil mtrix, resulting in primry inititing event in therosclerosis 1. Moreover, epidemiologic evidence hs reveled LDL cholesterol s risk fctor for therosclerosis 9. Conversely, the high density lipoprotein (HDL) is strongly protective ginst therosclerosis. This protective effect results from the role of HDL in trnsporting excess cholesterol from fom cells to the liver, thus relieving the cholesterol ccumultion in the circulting system 10. Studies for the role of lipoprotein metbolism in therosclerosis induced by MDV infection will give better understnding for the initil process of therosclerosis. Furthermore, it is importnt to note tht, besides this niml model, studies in humns hve lso reported the epidemiologicl ssocition between herpesvirl infections nd ccelerted therosclerosis in hert trnsplnt ptients 11, 12. Multiple studies bout herpesvirl induced therosclerosis hs led to the hypothesis tht herpesviruses initite vsculr disese processes in humns 13, 14. Therefore, with this model of MDV infected chicken, elucidting the role of lipoprotein metbolism for herpesvirus induced therosclerosis could enble us to demonstrte the etiology nd pthogenesis of humn rteriosclerosis. 2

10 Mrek s disese nd therosclerosis Mrek s disese Mrek s disese (MD) ws first recognized by József Mrek in 1907 s generlized polyneuritis in chickens. It ws lso shown tht MD ws ssocited with viscerl lymphom in the lter 1920s 15,16. The clinicl disese is similr to the neoplstic (retrovirus-induced lymphtic leukosis) disese in chicken. A cler differentition between retrovirus- nd MDV-induced neoplsi ws mde in the lter 1960s, when the herpesvirus etiology of MD ws identified 17. It is importnt to note tht in the 1960s, the lymphoid form of MD becme serious problem with the expnsion nd intensifiction of poultry production. It ws only two yers fter the identifiction nd cell-culture isoltion of MDV 18,19 tht the use of ttenuted MDV ws introduced s highly effective vccine 20,21. The vccines chieved unprlleled success in preventing the disese nd provided lndmrk: the first effective nd widespred immune prophylxis ginst virus-induced cncer in ny species. However, over the lst 30 yers, the virus hs mutted to incresly virulent forms nd vccine breks begn to pper, which hs led to the requirement of new strtegies for controlling nd preventing the disese. Importntly, it is well known tht MD hs worldwide impct on the poultry industry. It hs been demonstrted tht MDV pthogenic strins re present in nerly every country from the multi-nnul niml disese sttus report by the Office Interntionl des Epizooties. Furthermore, the economic impct of MD hs been estimted to be US$ 1-2 billion nnully. Therefore, investigtion of MD hs remined essentil to this dy. Mrek s disese virus (MDV) 3

11 MDV is n oncogenic herpesvirus tht cuses severl syndromes in the chicken, its nturl host. MDV ws originlly clssified s Gmmherpesvirus becuse its biologicl behvior nd bility to infect lymphocytes re similr to Epstein-Brr virus ( γ-herpesvirus). However, electron-microscopy studies of the MDV genome found tht MDV is double-strnded liner DNA molecule 22 nd possesses repet structures, which re chrcteristic of Alphherpesvirine 23. Therefore, on the bsis of genomic properties, MDV is currently clssified s n lphherpesvirus. The current model of MDV pthogenesis describes the disese in terms of two phses: the erly cytolytic phse, nd the ltent nd tumor phse of MDV infection 24. In brief, the first step of MDV infection in the chicken is inhltion of the virus. Phgocytic cells in the respirtory route become infected 25, nd within 24 hours of uptke of MDV-crrier cells, severl tissues become infected, including the spleen, thymus nd the burs of Fbricius 26. Here, the virus meets B cells nd lter ctivtes CD4 + s its min primry trgets for the first phse of cytolytic repliction 27,28. After n erly cytolytic phse, MDV enters the ltent phse of infection from 6-7 dys post infection. During MDV ltency, the virl genomes present in the host cells, but there is no production of infectious progeny virus 29,30. MDV ltency is difficult to study since distinguishing ltently infected cells from trnsformed cells is nerly impossible. Subsequently, there is trnsformtion of ltently infected cells to lymphoblstoid tumor cells 15, 31, which is generlly considered s the ultimte consequence of interction of MDV with the host cells. Indeed, pthogenesis of MD is complex nd the life cycle of this fscinting herpesvirus remins to be elucidted, 4

12 such s the moleculr detils of T-cell trnsformtion nd the fctors tht control tumor formtion. Pthology of Mrek s disese MDV infection leds to pthologicl chnges in its host, which involve lymphoid, nervous nd other tissues of the chicken. The current pthology of MD is different from when it ws first described in 1907, due to the incresing virulence of MDV. Currently, there re severl pthologicl chnges known to occur fter MDV infection. Acute cytolytic infection It hs been found tht n cute cytolytic infection of lymphoid tissue ppers t 3 dys post infection, especilly in the burs, thymus nd spleen 32. B cells nd CD4 + nd CD8 + T cells re ctivted nd infected in these orgns, where the virus infection provokes n cute inflmmtory response 27. Moreover, the burs nd thymus exhibit severe regression of burs lymphoid follicles nd thymic cortex, respectfully, which result in weight loss in these orgns 33. However, inflmmtory response in the spleen results in increse in weight. Centrl nervous system nd peripherl neuropthy In 1959, Znder described new encephlitic syndrome termed s trnsient prlysis (TP). This clinicl syndrome ws relted to brin lesions, including cute vsculitis, vsogenic oedem nd perivsculr cuffing 34, 35. Subsequently, two further syndromes of centrl nervous system, persistent neurologicl disese (PND) nd lte prlysis, were identified by Gimeno in Additionlly, in 1967, Pyne 5

13 nd Biggs demonstrted peripherl nerve lesions, which were considered inflmmtory nd possibly indicted regression of the neoplstic lymphoprolifertion 37. Lymphomtosis Multifocl lymphoid prolifertion ppers one week fter MDV infection nd viscerl lymphoms ws involved in vrious tissues, such s the gonds, liver, kidney, spleen, hert, burs nd skeletl muscles. MD lymphom is cytologicl complex, which consists of trnsformed T cells nd severl other cells, including B cells, mcrophges nd nturl killer cells 38, 39. Atherosclerosis Atherosclerosis is complex process nd involves the interction of genetic nd environmentl fctors. It hs been demonstrted tht injury to the vsculr endothelium initites the therosclerotic lesions nd herpesviruses hve been described s potentil inititors for rteril injury 5. This theory ws bsed on studies from Fbricnt nd collegues in the 1970s, who found n ssocition between coronry therosclerosis nd MDV infection, n vin herpesvirus. The results from this group were: (1) MDV infection induced occlusive therosclerotic lesions in lrge coronry rteries nd orts of infected normocholesterolemic nd hypercholesterolemic chickens. In contrst, visible therosclerotic lesions were not found in uninfected normocholesterolemic nd hypercholesterolemic chickens. Histologicl evlution demonstrted tht rteril chnges were chrcterized by fibromusculr intiml thickening in the infected nimls 3, 5. 6

14 (2) At 4 nd 8 months fter MDV inocultion, MDV infected chickens hd significnt increse (P<0.05) in totl ortic lipid ccumultion, including cholesterol, cholesterol ester, tricylglycerol nd phospholipids, s compred with orts in uninfected chickens. It hs been demonstrted tht the lipid ccretion in orts of MDV infected chickens ws possibly due to ltertions in cholesterol metbolism 4, 6. Mrek s disese resistnt nd susceptible lines There re two min strtegies for controlling MD: vccintion nd genetic resistnce, which hve brought enormous benefits to the modern poultry industry 40. However, ggressive increse in MD vccintions drive MDV evolution to incresed levels of virulence, which rendered renewed interest to study genetic selection to improve MD resistnce. Differences in genetic resistnce to MD were first investigted by Asmundson nd Biely in 1930s 41. These results were subsequently confirmed by other surveys nd experiments 42, 43. Currently, two distinct genetic resistnces to MD hve been identified: chicken mjor histocomptibility complex (MHC) ssocited resistnce, nd non-mhc ssocited resistnce. MHC ssocited resistnce It hd been demonstrted tht the inheritnce of lleles in the B blood ws ssocited with incresed MD resistnce 44. Considering the B blood group locus s mrker for MHC, it hs been indicted tht genes within the MHC were responsible for MD 7

15 resistnce. Moreover, the N nd P lines were reported by Hutt nd Cole in , nd the differences in susceptibility between two lines were ssocited with their MHC 45. Non-MHC ssocited resistnce Besides the N nd P lines, the resistnt nd susceptible lines (line 6 nd 7) hve been reported by Stone t Est Lnsing in 1975, who lso provided the bsis for studying genetic resistnce. Importntly, line 6 nd 7 hve the sme MHC hplotype 46, which indicted tht differences in resistnce must be relted with non-mhc genes. There re drmtic differences in the size of primry lymphoid orgns between line 6 nd 7 (e.g., the lrger lobes of the thymus in line 7) 47. Furthermore, line 7 hs higher lymphoprolifertion trits thn line 6 (e.g., the lymphocyte response to mitogens in vitro) 48. The development of genomic mpping for chicken, together with DNA rrys nd interference RNA (RNAi), mkes it possible to identify the genes responsible for resistnce nd susceptibility to MD. Chicken genetic resistnce possibly provides more sustinble mens to prevent MD outbreks in the future. Lipoprotein nd therosclerosis Cholesterol Lipid orgniztion in biologicl membrnes is crucil for cell functions. Cholesterol is n importnt structurl element of cell membrnes nd subcellulr prticles in higher eukryotes 49, nd cts s n essentil determinnt for membrne orgniztion. For exmple, cholesterol helps to construct brrier between cellulr comprtments, modulte the fluidity of the cell membrne, regulte the biologicl function of membrne proteins nd is involved in trnsmembrne signling processes 50. In 8

16 ddition to its structurl role, cholesterol is n obligtory precursor for steroid hormones, vitmin D, nd bile cids; it is necessry for the ctivtion of severl neuronl signling molecules 51, 52. In nimls, only smll mount of circulting cholesterol comes from the diet, nd more thn 80 % is derived from endogenous synthesis 8. The most ctively synthesizing orgns re the liver nd intestine, where 3- hydroxy-3-methylglutryl coenzyme A reductse (HMGCR) ctlyses cetyl-coa to produce cholesterol. Additionlly, most circulting cholesterol is esterified with longchin ftty cids, nd free cholesterol constitutes minor component 49. Since cholesterol plys n indispensible role for norml cell function, orgnisms hve developed complex nd sophisticted mechnisms to regulte cholesterol levels within proper rnge 53. Cholesterol, synthesized minly in the liver nd intestine or obtined from food, is delivered throughout the body to exert its biologicl functions. For long-distnce trnsfer mong cells through the queous environment, cholesterol nd cholesterol ester, s hydrophobic molecules, must be shuttled by spheroidl mcromolecules clled lipoproteins. Therefore, it is not surprising tht lipoprotein cn profoundly influence cholesterol distribution or metbolism nd eventully modulte its functions. Lipoprotein structure nd clssifiction Lipoproteins re the mcromoleculr vehicles tht trnsport hydrophobic smll molecules throughout the queous environment 54. A lipoprotein is mde up of two prts: hydrophobic core nd hydrophilic cot 55, 56. The core of lipoprotein 9

17 contins polr components, such s tricylglycerols nd esterified cholesterol. The cot of lipoprotein consists of phospholipid monolyer with polr hed groups fcing the queous system. Severl mphiphilic molecules, such s free cholesterol (unesterified) nd polipoproteins, embed in the phospholipid lyer nd confer functionl properties to the lipoprotein 57. Lipoproteins re ssigned into four min clsses ccording to size, prticle density nd composition: chylomicrons, very low density lipoproteins (VLDLs), low density lipoproteins (LDLs) nd high density lipoproteins (HDLs). Chylomicrons nd VLDLs re minly used to crry tricylglycerols, wheres LDLs nd HDLs re used to shuttle cholesterol. (1) Chylomicrons Chylomicrons re the lowest density molecules with the lrgest dimeter (> 75 nm), nd primrily consist of tricylglycerols (80-88% weight by weight (w/w)) nd minor frction of protein (polipoprotein B isoform B48) 54. (2) VLDLs VLDLs, similr to chylomicrons, re minly composed of tricylglycerols (bout 45-50% (w/w)) nd lso contin unesterified nd esterified cholesterol. VLDLs hve prticle sizes rnging from nm in dimeter 54. The min physiologicl role of VLDLs is to trnsport tricylglycerols from the liver to peripherl tissues for use. The VLDLs remnnts re in prt clered by heptic receptors, but the min frction (70%) remining in the plsm is subsequently converted to LDL through the removl of 10

18 tricylglycerols by lipoprotein lipse (LPL) nd loss of polipoprotein E by cholesteryl ester trnsfer protein (CEPT) 58, 59. (3) LDLs LDLs re the ctbolism product of VLDLs nd hve prticle sizes rnging from nm in dimeter 54. Compred with chylomicrons nd VLDLs, LDLs primrily consist of cholesterol nd cholesterol esters, nd contin polipoprotein B-100. LDLs serve s the primry trnsport mechnism to deliver cholesterol from the liver to peripherl tissues, nd trnsport 70-80% of the circulting cholesterol in humns 60. LDLs re clered from the circulting system by the LDL-receptor pthwy 61. (4) HDLs HDL prticles re heterogeneous mixture with the smllest dimeter (< 12 nm). In contrst to other lipoproteins, HDLs re of reltively high protein content (pproximtely 50% (w/w)), contining primrily polipoprotein A1 nd A2 54. In ddition to high protein content, HDLs re composed of unesterified cholesterol nd tricylglycerols 62. HDLs serve s shuttle tht trnsports unesterified cholesterol from the peripherl tissues to the liver for excretion. Lipoprotein metbolism Lipoprotein metbolism is complex nd sophisticted network, including ssembly, secretion, processing nd ctbolism 8. In the smll intestine, dietry fts re bsorbed nd converted into tricylglycerols (TGs). TGs re pckged with cholesterol ester nd polipoprotein B isoform B48 to form chylomicrons. Chylomicrons re secreted 11

19 vi the lymphtic system, nd circulte in the bloodstrem until they interct with lipoprotein lipse (LPL). LPLs rpidly hydrolyze TGs in the chylomicrons into free ftty cid, leding to the formtion of chylomicron remnnts, which re clered by the liver vi n polipoprotein E receptor 8, 54. In VLDL nd LDL cholesterol metbolism, VLDLs re synthesized in liver cells (heptocytes) through pckging with TG, cholesterol nd polipoprotein B isoform B100. TG in VLDLs cn be lipolyzed by LPL, resulting in the relese of free ftty cids nd monocylglycerols from VLDLs to form VLDL remnnts. A smll mount of VLDL remnnts re clered by heptic receptors, wheres the mjority in the plsm re subsequently converted to LDL through CETP. LDL trnsports unesterified nd esterified cholesterol from the liver to the periphery. During the circultion, LDL prticles re endocytosed by peripherl cells nd removed by LDL receptors 54, 63, 64. In HDL cholesterol metbolism, polipoprotein A1 (ApoA1) is synthesized in the liver nd relesed to the periphery. These prticles interct with ATP-binding cssette A1 (ABCA1) trnsport proteins expressed in the peripherl cells. ABCA1 ctlyzes the trnsfer of unesterified cellulr cholesterol. Subsequently, ApoA1 ccumultes cholesterol nd phospholipids, leding to the genertion of nscent pre-hdl. The nscent pre-hdl is remodeled by lecithin-cholesterol cyltrnsferse (LCAT), phospholipid trnsfer protein nd CETP, thus completing the trnsformtion to mture HDL. These HDL prticles continue to pick up free cholesterol from the 12

20 peripherl tissues with the help of ATP-binding cssette trnsporter G1, nd then trnsport cholesterol to the liver. HDL prticles re removed from the bloodstrem vi the scvenger receptor B1, which is expressed by the liver 8, 54, 65. Lipoproteins, therosclerosis nd crdiovsculr disese Lipid metbolism is essentil for sustining norml cell function, nd disturbnce of this process cn hve serious consequences for orgnisms. Atherosclerosis nd crdiovsculr disese (CVD) re mjor diseses relted with cholesterol nd lipoprotein metbolism in humn 7, 8. Atherosclerosis is progressive disese which is chrcterized by the ccumultion of lipids in the lrge rteries. It hs been demonstrted tht therosclerosis cuses bout 50% of ll deths in westernized societies 1. Epidemiologicl nd genetic disorder studies hve reveled tht severl importnt environmentl nd genetic risk fctors re ssocited with therosclerosis nd CVD, such s higher LDL/VLDL levels, reduced HDL levels, elevted blood pressure, dibetes, obesity, fmily history, systemic inflmmtory, smoking nd lck of exercise 1. Of the fctors, the reltive bundnce of severl plsm lipoproteins seems to be of primry importnce 1, 8. Plsm lipoproteins nd CVD risk Clinicl trils hve shown tht elevted concentrtions of plsm LDL cholesterol re ssocited with incresed in coronry hert disese 66, 67. Plsm LDLs re normlly ctbolized through receptor-medited endocytosis. Chronic excess of plsm LDL interrupts rteril relxtion. Moreover, LDL prticles, which fil to be removed 13

21 through receptor-medited pthwy, cn be recognized by scvenge receptors on rteril-wll mcrophges 1, 68. Once LDLs re engulfed by mcrophges, they become oxidized nd induce toxic intermedites, leding to cytokine production nd txis of inflmmtory cells 68. In ddition, rteril-wll mcrophges loded with LDL become fom cells, which re components for therogenic plques, resulting in coronry hert disese 1. Unlike LDLs, HDL cholesterol levels re inversely correlted with crdiovsculr risk 69. Epidemiologicl studies show tht 1 mg/dl increse in HDL cholesterol contributes to 2-3% decrese in the risk of CVD 70. It is well known tht the primry physiologicl role of HDLs is reverse cholesterol trnsport (RCT) (i.e., the centripetl trnsport of cholesterol from the peripherl tissues to the liver). Cholesterol crried by HDLs prticles might be clered in the liver by the scvenger receptor B1 medited pthwy 71. Therefore, HDLs help to mintin totl circulting cholesterol within its correct rnge. Besides reverse cholesterol trnsport, HDLs hve vrious protective properties, including nti-inflmmtory, ntioxidtive, nticogulnt, nd ntiggregtory function tht provide potentilly protective mechnisms ginst CVD 72, 73. Plsm lipoproteins nd therosclerosis Progressive ccumultion of cholesterol in the rteril wll cuses therosclerosis. A clssic study reported in 1913 by Anitschkow nd Chltow demonstrted tht cholesterol-fed rbbits showed therosclerotic plques in their rteries, which 14

22 indicted cusl role of cholesterol in therosclerotic plque development 74. Subsequently, epidemiologicl studies confirmed tht incresed blood cholesterol concentrtions ccelerted the development of coronry hert disese 75. As the mjor crrier of cholesterol, LDLs hve been ssocited with the development of therosclerosis. It hs been demonstrted tht the erly lesions of therosclerosis re composed of subendothelil ccumultion of cholesterol-engorged mcrophges, termed fom cells, which hve key role in therogenesis 1. The ccumultion of LDL in the subendothelil mtrix initites n therosclerosis process. LDL retention in the subendothelium of vessel wll seems to be relted to the interctions between LDL constituent polipoprotein B (ApoB) nd mtrix proteoglycns 76. Besides LDL, lipoprotein (), prticle resembling LDL but contining ApoB nd polipoprotein(), cn retin in the intim nd promote therosclerosis 77. Furthermore, LDLs undergo modifiction, including oxidtion, ggregtion nd other mens, to induce series of biologicl responses tht result in therosclerosis 78. Aggregted LDL is mjor LDL modifiction in therosclerotic lesions, which delivers enormous mounts of cholesterol to mcrophges nd results in the formtion of fom cells 79. However, HDL is very protective ginst therosclerosis. The primry mechnism underlying this protective effect is the role of HDL in reverse cholesterol trnsport (crrying excess cholesterol from fom cells to the liver) 10. In ddition, HDL possesses ntioxidnt, nti-inflmmtory, nd ntithrombotic properties, which 15

23 contribute to protection ginst therosclerosis. For exmple, HDL interferes with n initil process of therogenesis by inhibiting LDL oxidtion. Recently, besides lowering LDL cholesterol levels, rising HDL cholesterol hs emerged s potentil strtegy to tret therosclerosis 71. Adiponectin nd therosclerosis Adiponectin, one of dipocytokines Adipose tissue is now considered to be not only pssive energy store, but lso n importnt endocrine orgn. Indeed, dipose tissue produces rnge of bioctive cytokines clled dipocytokines 80. Leptin, resistin, visftin nd diponectin re dipocytokines. Reserch conducted over the pst severl yers hs reveled tht dipocytokines cn ffect lipid metbolism 81 nd tht bsence or excess of individul dipocytokines cuses obesity, potentilly leding to dibetes nd crdiovsculr disese 82. Leptin is primrily involved in regulting food intke nd energy expenditure. Recent studies showed tht leptin exerts mny potentilly therogenic effects nd plys n importnt role in crdiovsculr diseses 83. It hs been found tht incresed levels of plsm resistin were positively correlted with crdiovsculr risk 84. Visftin, dipocytokine secreted by viscerl ft, mimics insulin ctivity by binding to the insulin receptor 85. Compred with the forementioned fctors, diponectin hs exhibited different biologicl properties. Unlike leptin nd resistin which re pro-inflmmtory cytokines, diponectin is considered to be n ntiinflmmtory cytokine 86 nd hs profound protective effects in the pthogenesis of 16

24 therosclerosis 87. Thus, diponectin is perhps the most promising of the dipocytokines for the development of therpeutic strtegies. Adiponectin Adiponectin ws identified independently by four reserch groups in 1995 nd Subsequently it ws lso termed s AdipoQ 88, Acrp30 (dipocyte complement-relted protein 30) 89, pm1 (dipose most bundnt gene trnscript 1) 90, nd GBP28 (geltin-binding protein 28) 91. Adiponectin is 30 kd dipocytokine hormone synthesized minly by dipose tissue in severl niml species. It is 244 mino cid protein nd consists of N-terminl collgen domin tht is responsible for building tertiry structure nd C-terminl globulr domin tht is importnt for mediting diponectin effects 92. Adiponectin exists s full-length protein s well s clevge frgment, which is known s globulr diponectin nd consists only of the C-terminl globulr domin 93. Full-length diponectin cn oligomerize vi the N-terminl collgen domin to form trimer (low-moleculr-weight diponectin), hexmer (middle-moleculr-weight diponectin), nd high-moleculr-weight diponectin 94. Adiponectin is minly synthesized by dipocytes. Recent studies hve found tht it is lso expressed in skeletl muscle cells 95, crdiomyocytes 96 nd endothelil cells 97. Adiponectin cdna in the chicken is % homologous to different mmmlin diponectin nd is expressed t the highest levels in dipose tissue, followed by the liver nd nterior pituitry 98. In humns, diponectin circultes t high concentrtions in serum rnging from 5-10 mg per ml nd ccounts for bout 0.01 % of totl plsm 17

25 proteins 99 (compred with leptin circulting t concentrtion of few ng per ml). It hs been reported tht plsm diponectin levels re influenced by different fctors, such s ge, gender nd lifestyle. Indeed, women hve higher plsm diponectin levels thn men 100. Some dietry fctors, such s soy protein nd fish oils, lso increse plsm diponectin levels 101. Adiponectin receptors Two diponectin receptors hve recently been identified (AdipoR1 nd AdipoR2). Both AdipoR1 nd AdipoR2 re seven trnsmembrne receptors, in which the N- terminus is internl nd the C-terminus is externl. These receptors re structurlly distinct from G-protein-coupled receptors 102. AdipoR1 is widely expressed, prticulrly in skeletl muscle, wheres AdipoR2 is most bundntly expressed in the liver 102. AdipoR1 nd AdipoR2 hve been found to be structurlly conserved in severl nimls, including the chicken 103. Moreover, two types of diponectin receptors hve been demonstrted to hve different ffinities for binding globulr nd full-length diponectin. AdipoR1 hs higher ffinity for the globulr diponectin wheres AdipoR2 minly engges with full-length diponectin 102. Globulr nd fulllength diponectin cn bind to both receptors nd medite the ctivtion of 5' denosine monophosphte-ctivted protein kinse (AMPK) 104,105 nd the peroxisome prolifertor ctivted receptor (PPARα) 106, thus exerting its biologicl functions. Different functions between AdipoR1 nd AdipoR2 in diponectin signl pthwys hve been reported. AdipoR1 my be ssocited with the ctivtion of 18

26 AMPK pthwys wheres AdipoR2 seems to be more tightly linked to ctivtion of the PPARα pthwy 107. Adiponectin s key meditor of diseses Adiponectin is hormone tht is secreted predominntly by dipocytes. Adiponectin cts s mjor nti-dibetic, nti-therogenic nd nti-inflmmtory dipocytokine. Adiponectin hs been shown to ply crucil roles in regulting glucose nd lipid metbolism 104, 108 s well s oxidtive stress 109 nd inflmmtion 110. Recent reserch hs reveled tht low plsm diponectin levels, known s hypodiponectinemi, re ssocited with vrious disese conditions. Adiponectin in therosclerosis nd crdiovsculr disese Adiponectin cts s mjor nti-therogenic nd nti-inflmmtory dipocytokine. Severl clinicl reports indicted tht low levels of diponectin re ssocited with the development of crdiovsculr disese 111, 112, 113. It hs been demonstrted tht inflmmtion is n essentil fctor in the initition nd development of therosclerosis 114. As previously mentioned, diponectin inhibits the secretion of inflmmtory cytokines, such s TNF in the therogenic process, thus suppressing the expression of TNF-induced dhesion molecules in endothelil cells 115. Adiponectin lso inhibits fom cell formtion 116 nd reduces prolifertion nd migrtion of smooth muscle cells 117. Considering tht therosclerosis is n inflmmtory disese 114, the C- rective protein (CRP), proinflmmtory mrker, is one of the most relible biomrkers to ssess crdiovsculr risk 118. It hs been found recently tht levels of 19

27 diponectin in plsm nd dipose tissue were negtively correlted with CRP concentrtions in ptients with coronry rtery disese 119. It hs been hypothesized tht diponectin plys both direct nd indirect roles in protection ginst crdiovsculr disese 120. There is incresing evidence tht the protection effect of diponectin is due to its involvement in regulting both lipid nd crbohydrte metbolism. It is importnt to note tht in the Women s Helth Study, cholesterol ws the strongest predictor for future crdiovsculr events, including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) nd totl cholesterol 121. Low levels of plsm diponectin hve been reported to be relted to smll dense LDL, high triglyceride, nd polipoprotein B (APOB) levels 122. It hs lso been found tht diponectin directly cts on vsculr endothelium nd protects ginst crdiovsculr disese in prt becuse of the reduction of lipid ccumultion in mcrophges 123. Furthermore, dministrtion of diponectin prevents therosclerosis in polipoprotein E (APOE)-deficient mice 124. Adiponectin in obesity, insulin resistnce nd dibetes It hs been demonstrted tht plsm diponectin levels re reduced in obesity, insulin resistnce, nd type 2 dibetes 125. Adiponectin expression in dipose tissue nd circulting levels in plsm re decresed in severl niml models of obesity 126. Studies in different humn popultions, including Jpnese nd Asin Indins, hve lso shown tht low plsm diponectin levels re predictors for future development of dibetes nd insulin resistnce 127,128,129. It hs lso been found tht simultneous 20

28 disruption of AdipoR1 nd AdipoR2 reduced diponectin-specific binding nd diponectin sensitivity. Conversely, the incresing expression of either AdipoR1 or AdipoR2 in the liver meliorted insulin resistnce nd dibetes 107. Administrtion of diponectin induced glucose-lowering effects nd meliorted insulin resistnce in mice 93,126,130, wheres diponectin-deficient mice exhibited insulin resistnt nd dibetic 108,131. It hs been reported tht the diponectin effect in insulin sensitivity my 104, 105,132 be regulted by incresing ftty cid oxidtion vi ctivtion of the AMPK nd PPARα pthwys 106,133. It is well known tht AMPK hs crucil role in regulting body weight, food intke, nd glucose nd lipid homeostsis, thereby controlling the systemic energy blnce 132. Moreover, AdipoR1 nd AdipoR2, the mjor receptors for diponectin, serve s key physiologicl regultors for the glucoselowering effect of diponectin 107. Adiponectin in inflmmtion In metbolic diseses, such s obesity nd dibetes, inflmmtion in the systemic microcircultion is widely known to led to orgn dmge nd other chronic complictions. Among mny proteins secreted by dipocytes, diponectin serves unique role s n nti-inflmmtory nd nti-therogenic fctor. It hs been demonstrted tht diponectin exerts vrious nti-inflmmtory effects, which include inhibition of pro-inflmmtory cytokine production, induction of nti-inflmmtory fctors, nd reduction of the expression of dhesion molecules 134. Adiponectindeficient mice hve higher expression levels of tumor necrosis fctor (TNF; proinflmmtory cytokine) mrna in dipose tissue nd higher plsm TNF 21

29 concentrtions thn diponectin-sufficient mice 131. In ddition, diponectin inhibits nucler fctor-κb (NF-κB) ctivtion in endothelil cells nd influences the function of mcrophges 135. Besides inhibiting the production of pro-inflmmtory cytokines, diponectin lso induces the production of severl importnt nti-inflmmtory cytokines, including IL-10 nd IL-1 receptor ntgonists, vi humn monocytes nd mcrophges 136. In the model of MDV-induced therosclerosis, Fbricnt et l. reported tht therosclerotic lesions were observed in lrge coronry rteries nd orts of both normocholesterolemic (reltively poor diet in cholesterol) nd hypercholesterolemic ( 2 % cholesterol supplement) chickens by infection with MDV wheres no rteril diseses developed in uninfected chickens fed either cholesterol-poor or cholesterolsupplemented diets 3, 5. These results provided direct evidence suggesting herpesviruses s potentil inititors of rteril injury. The sme group subsequently found tht MDV infection cused therosclerosis in the chicken while those nimls tht were previously vccinted nd lter chllenged with MDV did not exhibit totl ortic lipid ccumultion 4. These results suggested tht MD resistnce induced by vccintion could prevent the development of therosclerosis cused by MDV infection. Two min strtegies for resistnce of MD hve been demonstrted: vccintion nd genetic resistnce to MD 40. It hs been demonstrted tht prior vccintion with the turkey herpesvirus could prevent therosclerosis in MDVinfected chickens 4, 6. Therefore, it is very interesting to explore the role of genetic resistnce in the progression of herpesvirus-induced therosclerosis. Furthermore, the 22

30 role of lipoprotein metbolism in therosclerosis induced by MDV infection remins elusive lthough the role of cholesterol metbolism hs been demonstrted for decdes. The present study ws conducted to compre the effects on lipoprotein metbolism induced by MDV infection between MD resistnt nd susceptible chicken lines. We hypothesized tht lipoprotein metbolisms is different in distinct MD susceptible nd resistnt lines. To test this hypothesis, two experiments were designed 1) to determine whether differences exist in the bckground of lipoprotein metbolism between MD resistnt nd susceptible lines nd 2) to ssess whether the chnges of lipoprotein levels in response to MDV infection differ in the MD resistnt nd susceptible lines. Insights from these studies could provide us better understnding of the role of lipoprotein metbolism in herpesvirus induced therosclerosis, thus fcilitting the elucidtion of the etiology nd pthogenesis of humn rteriosclerosis. 23

31 Chpter 2: Comprison of lipoprotein metbolism in MD resistnt nd susceptible chicken lines Introduction Atherosclerosis is one of the min humn diseses involving cholesterol nd lipoprotein metbolism 7, 8. Although cholesterol ccumultion in rtery wlls is the defining chrcteristic of therosclerosis 91, lipoproteins lso ply n importnt role in the development of the disese. It hs been demonstrted tht LDL is risk fctor for therosclerosis 9, wheres HDL is protective fctor ginst therosclerosis 10. Rising HDL cholesterol nd lowering LDL cholesterol hve, thus, emerged s potentil strtegies to lower the risk of developing therosclerosis 10. MDV infection cn induce therosclerosis in chickens 3-5. It hs been reported tht MDV infection induced ortic lipid ccumultion, including cholesterol, cholesterol ester, tricylglycerol nd phospholipids 4. The lipid ccretion in orts of MDV infected chickens my hve been result of ltertions in cholesterol metbolism 4, 6. Lipoproteins, the key crrier of cholesterol through the circulting system, fundmentlly modulte cholesterol metbolism. Thus, lipoprotein metbolism my ply n importnt role in MDV- induced therosclerosis. We hypothesized tht MDV influences lipoprotein metbolism differently in MD susceptible nd resistnt lines. Therefore, we investigted whether the chnges of lipoprotein levels induced by MDV differ in the MD resistnt nd susceptible lines. First we determined whether bsl lipoprotein metbolism is different between MD resistnt nd susceptible lines. We exmined plsm lipoprotein levels nd nlyzed the phenotype in MD resistnt 24

32 nd susceptible lines. We lso determined the expression levels of diponectin nd its receptors, since diponectin, s protective mrker ginst therosclerosis, fundmentlly influences lipid metbolism

33 Experimentl design To investigte whether ny differences in plsm lipoprotein metbolism between line 6 3 nd line 7 2 exist, 2-month-old (growing period) nd 15-month-old (mture period) chickens from ech line were used for nlyzing phenotype, determining plsm lipoprotein levels, nd detecting mrna nd protein expression levels of diponectin nd its receptors. MD resistnt line (line 6 3 ) MD susceptible line (line 7 2 ) 2 months 15 months 2 months 15 months Phenotype nlysis, lipoprotein & diponectin levels Figure 1. Experimentl design for determining the differences of lipoprotein metbolism between line 6 3 nd

34 Methods nd mterils Phenotype dt At the end of ech study, nimls were weighed nd then scrificed for blood nd tissue collection. Tissue smples were weighed, nd immeditely frozen in liquid nitrogen nd stored t -80. The weight percentge of ech tissue ws clculted by the eqution: Tissue percentge = Tissue weight (g) Body weight (g) 100% Plsm HDL nd LDL/VLDL cholesterol levels Blood smples were drwn by crdic puncture nd plced in 10 ml tubes with EDTA. Plsm ws seprted from whole blood by centrifugtion t 2000 g for 20 min nd kept t 4 until nlysis. Plsm totl cholesterol, HDL cholesterol nd LDL cholesterol were mesured using HDL nd LDL/VLDL cholesterol quntifiction kit (BioVision, Exton, PA). Rel-time quntittive PCR Totl RNA ws extrcted from bdominl ft nd liver by using RNesy mini kit (Qigen Inc., Vlenci, CA) following the mnufcture s protocol. Totl RNA ws quntified ccording to UV bsorbnce (260/280 nm) using spectrophotometer ND (Thermo Fisher Scientific Inc., Wilmington, DE). Complementry DNA (cdna) ws synthesized by reverse trnscribing 800ng totl RNA using n oligo-dt (12-18) primer nd Superscript II reverse trnscriptse (Invitrogen). Primers used to 27

35 mplify diponectin, dipor1, dipor2 137 nd GAPDH (Glycerldehyde 3-phosphte dehydrogense) were described s follows: Adiponectin: Forwrd 5 -ACAGGTGCAGAAGGACCGAGAGGATT-3 Reverse 5 -AAGACAGAGCCGCTTGCTTGGTCAAC-3 AdipoR1: Forwrd 5 -GAATACACACCGAGACGGGCAACATCT-3 Reverse 5 -GCCCAAGACGCAGACAATGGAGAGGTA-3 AdipoR2: Forwrd 5 -GAGACTGGCAACATCTGGACGCATCTTC-3 Reverse 5 -TGCGATGCCCAGGACACAAATCACAAT-3 GAPDH: Forwrd 5 -TGACTTCAATGGTGACAGCC-3 Reverse 5 -ACTCCTTGGATGCCATGTGG-3 The products of the reverse trnscription rection were diluted (5 ) with ddh 2 O, nd 1 μl ws used s templte in rel-time polymerse chin rection (RT-PCR). The RT-PCR ws performed using SYBR Green RT-PCR kit (Qigen Inc.). The PCR rection ws crried out using the following progrm: 95 for 15 min, 40 cycles of 94 for 15 s, 57 for 30s nd 70 for 30s. For negtive control, wter ws used s templte in plce of single strnd cdna during RT-PCR. The quntittive PCR (qpcr) output provided the log-liner threshold vlues (C T ) for the threshold cycle. Smples from ech niml were mesured in duplicte to obtin verge C T vlues for ech mrna. Vlues were trnsformed to Ct vlue by normlizing gene expression to the housekeeping gene GAPDH using the following eqution: Ct = (Averge Ct-trget Averge Ct-GAPDH). The Ct vlue ws then clculted by subtrcting the smple Ct vlue from the smple with the highest expression level for controlling mplifiction efficiency. Results were then converted from log-liner to 28

36 liner terms by the function: 2 - Ct. The reltive mrna expression of diponectin, dipor1 or dipor2 in vrious groups ws compred. Western blot Protein extrction Protein extrcts from chicken bdominl ft nd liver were prepred s described previously 138, with slight modifictions. Briefly, g of bdominl ft nd liver were homogenized using pestle in 2 ml lysis buffer (10mM Tris-HCl, 150mM NCl, 1% Nonidet P-40, 0.5% sodium deoxycholte, 0.1% SDS) contining protese inhibitor cocktil (Sigm). The homogente ws shken in thermomixer t 1000 rpm for 2 hour t 4, nd the lystes were then centrifuged t 14,000 g for 20 min t 4. The superntnt ws collected, nd totl protein concentrtion ws mesured using Victor 1420 multilble counter (PerkinElmer). Chicken plsm ws used directly for western blot nlysis to determine protein levels of diponectin in the plsm. Western blot Protein smples were prepred for electrophoresis by heting with SDS-PAGE Lemmli buffer (50mM Tris-HCl, 10% glycerol, 2% SDS, 0.05% bromophenol blue, ph 6.8) contining 10% β-mercptoethnol (Promeg, Mdison, WI) for 10 min t 100. Proteins were seprted by SDS-PAGE in 10% (w/v) polycrylmide gels 139 t 120v for 90 min. After seprtion by SDS-PAGE, proteins were trnsferred onto PVDF membrne. The membrnes were blocked with SuperBlock T20 blocking buffer (Pierce, Rockford, IL) for 1 h t room temperture. The membrnes were then 29

37 incubted with primry ntibodies (rbbit nti-chicken diponectin, dipor1 nd dipor2 from Dr. Rmesh Rmchndrn) t 4 for 24 h. The ntibodies were diluted with SuperBlock T20 (diponectin in plsm nd bdominl ft (1:40,000); dipor1 nd dipor2 in liver (1:2,000) nd bdominl ft (1:500)). The membrnes were wshed three times with 1 Tris-Buffered Sline Tween-20 (TBST) for 5 min ech nd then incubted with secondry ntibody donkey nti-rbbit IgG (Snt Cruz Biotechnology) (diluted by 1:5,000) for 1 h t room temperture. The membrnes were then incubted with ECL chemiluminescense detection regent (GE Helthcre, Pisctwy, NJ). Chemiluminescent signl ws collected using ChemiDoc XRS imging system (Bio-Rd Lbortories, Hercules, CA). The protein bnds were quntified using Quntity One softwre (Bio-Rd). The moleculr weights of proteins were estimted using Pgeruler plus prestined protein ldder (Ferments interntionl Inc., Cnd). Stripping nd reprobing β-ctin Primry nd secondry ntibodies were removed from the membrnes by incubting with Western Blot Stripping Buffer (20 ml 10% SDS, 12.5 ml Tris-HCl, 0.8 ml β- mercptoethnol, 67.5 ml ddh2o, ph 6.8) for 40 min t 50. The membrnes were blocked with 5% nonft dried milk for 1 h nd then incubted with nti-β-ctin ntibody (Snt Cruz Biotechnology) (1:1,000 diluted with 1 TBST) t 4 for 24 h. The membrnes were incubted with donkey nti-mouse IgG (Snt Cruz Biotechnology) (1:10,000 diluted with 1 TBST) for 1 h t room temperture. The expression of β-ctin ws detected in the sme mnner s bove. 30

38 Sttisticl nlysis The dt were nlyzed with two-wy ANOVA followed by Tukey s test for comprison mong different groups using Sttisticl Anlysis System v.9.1 (SAS Institute, Cry, NC). Vlues shown re mens ± S.D (Stndrd devition). The sttisticl model for 2 month- nd 15 month-old chicken ws: Y = μ + Li + Aj + Li*Aj + ε i, j, k The line, ge nd their interction re treted s fixed fctors in the model. μ: grnd men; Li: the effect of the ith line (6 3 or 7 2 ) on Y; Aj: the effect of the jth ge (2 months or 15 months) on Y; ε i, j, k : rndom error. 31

39 Results Phenotype nlysis for 2 month- nd 15 month-old chickens from line 6 3 nd 7 2 To investigte whether there were differences in body weight nd tissue weight percentge between line 6 3 (L6 3 ) nd line 7 2 (L7 2 ), bdominl ft, brest muscle with bone, leg muscle with bone, liver, hert nd spleen smples were collected from chickens t 2 months nd 15 months of ge. The sttisticl model used to nlyze these phenotypes ws Y = μ + Li + Aj + Li*Aj + ε i, j, k ; including line, ge nd the interction between line nd ge, ll s fixed effects. The interctions between line nd ge were found to be non-significnt for body weight, brest muscle with bone, leg muscle with bone nd hert. The body weights were not sttisticlly different between L6 3 nd L7 2 in the two ge chickens. However, n obvious increse in body weight ws observed in 15 month-old chickens s compred to 2 month-old chickens in L6 3 ( ± vs ± g) nd L7 2 ( ± vs ± g) (Figure 2). We lso observed lower percentge of leg muscle with bone in L7 2 in compred to L6 3 in 15 month-old chickens (p < 0.01) (Figure 2d). L7 2 lso hd significntly higher hert percentge t 2 months nd 15 months of ge compred to L6 3 (p < 0.001) (Figure 2f). However, no difference between the lines ws pprent for the percentge of brest muscle with bone (Figure 2c). The interctions between line nd ge were significnt for bdominl ft (p<0.001), liver (p<0.001) nd spleen (p<0.05), so dt for the three phenotypes were nlyzed within ech ge. The bdominl ft percentge ws not different between L6 3 nd L7 2 t 2 months of ge (2.51 ± 0.55 vs ± 0.55 %), but L6 3 hd significntly higher 32

40 bdominl ft percentge thn L7 2 (7.58 ± 0.37 vs ± 0.37 %, p < 0.001) (Figure 2b) t 15 months of ge. L7 2 lso hd significntly higher liver weight percentge t 15 months of ge nd higher spleen weight percentge t 2 months of ge compred to L6 3 (p < 0.001) (Figure 2e, 2g). 33

41 Percentge (%) Percentge (%) Body weight (g) Line 6 3 Line Body weight 2000 b b months 15 months b c Abdoniml ft Brest muscle with bone b c b b b b 0 2 months 15 months 0 2 months 15 months 34

42 Percentge (%) Percentge (%) Percentge (%) Percentge (%) Line 6 3 d e Line 7 2 Leg muscle with bone Liver b b b 0 2 months 15 months 0 2 months 15 months f g Hert Spleen b b b months 15 months 0 2 months 15 months 35

43 Figure 2. Phenotype dt for 2 month- nd 15 month-old chickens. Body nd tissue weight were mesured for 2 month-old (n = 4) nd 15 month-old (n = 9) chickens in line 6 3 (blue br), nd for 2 month-old (n = 4) nd 15 month-old (n = 9) chickens in line 7 2 (purple br). Tissue percentges were clculted by ((Tissue weight) / (Body weight)) *100%. (), Body weight (g); (b), Abdominl ft percentge (%); (c), The percentge of brest muscle with bone (%); (d), The percentge of leg muscle with bone (%); (e), Liver percentge (%); (f), Hert percentge (%); (g), Spleen percentge (%). Vlues were shown s mens ± S.D. (mens ± stndrd devition). Letters identify differences mong groups; thus, two groups not shring lowercse letters re significntly different (two-wy ANOVA, Tukey s test, p<0.05). 36

44 Plsm lipoprotein levels for line 6 3 nd line 7 2 t 2 months nd 15 months of ge To determine whether there ws difference in lipoprotein metbolism between L6 3 nd L7 2, plsm lipoprotein concentrtions were mesured in 2 month- nd 15 monthold chickens (Figure 3). Results of totl cholesterol reveled tht L6 3 contined totl cholesterol 0.50 ± 0.11 μg/μl t 2 months of ge nd 0.84 ± 0.09 μg/μl t 15 months of ge, while L7 2 contined totl cholesterol 0.66 ± 0.14 μg/μl nd 1.36 ± 0.09 μg/μl, respectively (Figure 3). With respect to individul lipoprotein clsses, HDL cholesterol hd modest increse in both chicken lines during growth, but there were no significnt differences between the lines (p > 0.05) (figure 3b). As for the (LDL+VLDL) cholesterol levels, the interction between line nd ge ws significnt (p<0.001). We observed tht (LDL+VLDL) cholesterol levels in L6 3 nd L7 2 were not significntly different (p > 0.05) t 2 months of ge (Figure 3c). However, t 15 months of ge, L7 2 hd 247% more (LDL+VLDL) thn L6 3 (p < 0.001) (Figure 3c). Moreover, significnt interctions between line nd ge were lso observed in HDL rtio (p<0.01) nd (LDL+VLDL) rtio (p<0.01). Results of HDL rtio reveled no significnt difference between 2 month- nd 15 month-old chickens in L6 3 (0.62 ± vs ± 0.048, p > 0.05), wheres there ws significnt decrese in 15 month-old chickens (0.42 ± 0.05) compred to 2 month-old chickens (0.70 ± 0.07) in L7 2 (p < 0.05) (Figure 3d). Correspondingly, significnt increse in (LDL+VLDL) rtio ws observed in 15 month-old chickens compred to 2 month-old chickens in L7 2 (p < 0.05). 37

45 Concentrtion (ug/ul) Concentrtion (ug/ul) Line 6 3 Line 7 2 Totl cholesterol b months 15 months b HDL cholesterol months 15 months 38

46 Concentrtion (ug/ul) c Line 6 3 Line (LDL+VLDL) cholesterol b 2 months 15 months d Lipoprotein rtio 1.2 LDL+VLDL HDL L63-2months L63-15months L72-2months L72-15months 39

47 Figure 3. Plsm lipoprotein levels for 2 month- nd 15 month-old chickens. Lipoprotein concentrtions were mesured for 2 month-old (n = 6) nd 15 month-old (n = 9) chickens in line 6 3 (blue br), nd for 2 month-old (n = 4) nd 15 month-old (n = 9) chickens in line 7 2 (purple br). (), Totl cholesterol (μg/μl); (b), HDL cholesterol (μg/μl); (c), LDL+VLDL cholesterol (μg/μl); (d), Lipoprotein rtio. In (), (b), nd (c), vlues were shown s mens ± S.D. Letters identify differences mong groups; thus, two groups not shring lowercse letters re significntly different (twowy ANOVA, Tukey s test, p<0.05). 40

48 Adiponectin, dipor1 nd dipor2 gene expression Adiponectin hs been ssocited with fvorble effects on metbolism (i.e. reduced viscerl dipose mss, decresed plsm LDL cholesterol, nd incresed HDL cholesterol) 140. The biologicl effects of diponectin re minly medited by two receptors (dipor1 nd dipor2) 87. As our biochemicl experiments of plsm lipoprotein levels suggested tht totl cholesterol nd LDL cholesterol levels were different between the two lines, we further tested whether the levels of diponectin, dipor1 nd dipor2 were different in L6 3 nd L7 2. Using RT-PCR, we found no significnt difference in diponectin mrna levels in bdominl ft mong the four groups (L6 3-2months, L7 2-2months, L6 3-15months, L7 2-15months) (Figure 4). AdipoR1 mrna quntities in liver nd bdominl ft were slightly lower in L7 2 compred with tht in L6 3 (Figure 4b, 4c). Furthermore, dipor2 mrna quntities in liver nd bdominl ft were not significntly different (p > 0.05) between L6 3 nd L7 2 (Figure 4d, 4e). 41

49 Reltive mrna expression Reltive mrna expression Line 6 3 Line 7 2 Adiponectin in ft months 15 months b AdipoR1 in liver months 15 months 42

50 Reltive mrna expression Reltive mrna expression c Line 6 3 Line 7 2 AdipoR1 in ft months 15 months d AdipoR2 in liver months 15 months 43

51 Reltive mrna expression Line 6 3 e Line 7 2 AdipoR2 in ft months 15 months Figure 4. mrna levels of diponectin, dipor1 nd dipor2 by qpcr for line 6 3 (blue br) nd 7 2 (purple br) t 2 months nd 15 months of ge (n=4). (), Adiponectin in bdominl ft; (b), AdipoR1 in liver; (c), AdipoR1 in bdominl ft; (d), AdipoR2 in liver; (e), AdipoR2 in bdominl ft. GAPDH mrna expression ws used for normliztion. Vlues were shown s mens ± S.D. Letters identify differences mong groups; thus, two groups not shring lowercse letters re significntly different (two-wy ANOVA, Tukey s test, p<0.05). 44

52 Adiponectin, dipor1 nd dipor2 protein expression In light of the role identified for diponectin in regulting plsm lipoprotein levels, we next exmined protein expression levels for diponectin nd its receptors in L6 3 nd L7 2 using western blots (Figure 5). Duplictes from ech group were conducted in western blots. Lne 1 nd 2 were designed for L6 3-2months; Lne 3 nd 4 for L7 2-2months; Lne 5 nd 6 for L6 3-15months; Lne 7 nd 8 for L7 2-15months. Detection of β-ctin in the sme blot ws used for normliztion. Results were quntified by clculting the rtio of the density of the protein bnd nd β-ctin bnd. The results showed tht diponectin levels in bdominl ft were not significntly different mong the four groups (p > 0.05) (Figure 5), indicting tht L6 3 nd L7 2 my hve synthesized similr mount of diponectin s diponectin is primrily synthesized in bdominl ft 87. However, there ws significnt decrese in the level of diponectin in plsm in L7 2 (5.91 ± 0.58) compred to tht in L6 3 (11.41 ± 0.58) t 2 months of ge (p < 0.01, Figure 5b). Moreover, our results reveled tht L6 3-2 month-old chickens hd the highest level of diponectin, followed by L7 2-2 months, L6 3-15months, nd L7 2-15months. Interestingly, this trend seems to be inversely correlted to the chnge of totl cholesterol concentrtion within four groups, which showed the highest level in L7 2-15months, followed by L6 3-15months, L7 2-2months, nd L6 3-2months (Figure 3b). In order to determine the negtive correltion, plsm diponectin levels for four chickens from ech group were detected. We then nlyzed the correltion coefficient between plsm diponectin levels nd totl cholesterol concentrtions (Figure 3g). 45

53 The sttisticl nlysis showed tht correltion coefficient (r) ws when n outlier (for which totl cholesterol concentrtion is 1.62 ug/ul) ws removed from the dt set. These results re consistent with others report tht plsm diponectin levels is negtively correlted with totl cholesterol 141. We lso exmined protein expression levels of dipor1 nd dipor2 in liver nd bdominl ft. Quntittive results reveled tht there were no differences in dipor1 in the liver between the two lines t 2 months nd 15 months of ge (p > 0.05, Figure 5c). It should be noted tht no remrkble differences were observed between L6 3-2months nd L6 3-15months groups, wheres significnt decrese in dipor1 levels in the liver ws detected in L7 2-15months (4.06 ± 0.64) compred to L7 2-2months (7.49 ± 0.64) (p < 0.05, Figure 5c). Quntittive results of dipor1 in bdominl ft reveled no differences between the two lines in 2 month-old chickens (p > 0.05, Figure 5d). However, t 15 months of ge, L7 2 hd significntly higher level of dipor1 (6.21 ± 0.39) compred to L6 3 (3.54 ± 0.39) (p < 0.05, Figure 5d). Interestingly, L7 2-15months showed the lower level of dipor1 in liver nd the higher level of dipor1 in bdominl ft. No significnt differences in dipor2 expression levels were observed mong the four groups in both tissues (Figure 5e, 5f). 46

54 Reltive fold L63-2months L72-2months L63-15months L72-15months Adiponectin 30 kd β -ctin 42 kd L63-2months L72-2months L63-15months L72-15months Adiponectin protein levels in bdominl ft 47

55 Reltive fold b L63-2months L72-2months L63-15months L72-15months Adiponectin 30 kd b 6 4 bc c 2 0 L63-2months L72-2months L63-15months L72-15months Adiponectin protein levels in plsm 48