THE EFFECT OF FASTING ON ANTI-INFLAMMATORY MEDIATORS, IL-1RA AND IL-1R2, AND THEIR ROLE IN RESISTANCE TO SICKNESS BEHAVIORS IN MICE

THE EFFECT OF FASTING ON ANTI-INFLAMMATORY MEDIATORS, IL-1RA AND IL-1R2, AND THEIR ROLE IN RESISTANCE TO SICKNESS BEHAVIORS IN MICE BY JENNIFER J. JOESTING THESIS Sumitted in prtil fulfillment of the requirements for the degree of Mster of Science in Animl Sciences in the Grdute College of the University of Illinois t Urn-Chmpign, 213 Urn, Illinois Adviser: Professor Gregory G. Freund

ABSTRACT Dietry regimens involving fsting hve long een linked to eneficil helth outcomes including; reduction in hert disese, dietes, improved mood nd cognition, nd resistnce to sickness ehviors such s norexi nd fever. The mechnisms underlying fsting-induced helth enefits nd ltertions in immunity nd sickness ehvior continues to remin deted despite over 2 yers of interest. This reserch project confirms tht cute dietry restriction (24 h wter-only fst) is le to ttenute IL-1β-induced norexi, in ddition to other sickness ehviors t erly stges of the cute phse response. We previously reported tht fsting ws le to reduce gene expression of inflmmtory IL-1α in the rin, here we looked t liver nd dipose in ddition to rin nd uncovered mjor up-regultion of IL-1 endogenous inhiitors, IL-1RA nd IL-1R2 in peripherl tissues. These findings imply tht ttenution in sickness ehviors oserved my e due to counter-regultion of IL-1 vi up-regultion of IL-1R2 nd/or IL-1RA shown in metoliclly ctive orgns, which is cple of lunting the centrlly-medited effects of induced peripherl chllenge. Our results further demonstrte tht the mechnisms involved in IL-1R2 nd IL-1RA up-regultion re independent of IL-1, TLR-4, IL-4 nd glucocordicoid signling. Here we lso demonstrte tht free ftty cids (FFA) re incresed in the plsm s consequence of fsting. Using plmitic cid injection to mimic fstings FFA increse, we elucidted novel mechnism y which IL-1R2 is up-regulted. This method showed incresed IL-1R2 gene trnscripts in the liver of mice. FFA signling, which produces n immune response, is shown here to e TLR-4-independent, implicting free ftty cid receptor 1 (FFAR1) s the key signling receptor inititing the nti-inflmmtory result of fsting documented in this study. ii

ACKNOWLEDGMENTS There re mny people to thnk for helping to mke this project possile. I m very thnkful to my dviser, Dr. Gregory G. Freund, for llowing me to do reserch in his l nd continue my eduction, nd lso for his guidnce nd support throughout this project. Additionl thnks to my committee memers, Dr. Jeffrey Woods nd Dr. F. Willim Simmons, for greeing to join the rce to my Msters without much notice. Thnks to ll memers of the Freund L, pst nd present, for their ssistnce, mentoring nd counseling on occsion. Specil thnks to my husnd Clint nd dughter Grcie, to my prents nd fmily, nd to my gret friends who encourged nd supported me throughout this process. I pprecite ll of your time, energy, resources nd love. And finlly, thnks to y girl Joesting #2 (due 4/16/13) for hopefully witing to come into this world until fter my defense nd thesis cceptnce. iii

TABLE OF CONTENTS CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW..1 CHAPTER 2: OBJECTIVES..3 CHAPTER 3: METHODOLOGY 4 CHAPTER 4: RESULTS..8 CHAPTER 5: TABLES, FIGURES AND FIGURE LEGENDS..16 CHAPTER 6: DISCUSSION.32 CHAPTER 7: CONCLUSIONS 38 REFERENCES.39 iv

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Within the lst 2 yers, the importnce of ody weight hs ecome physiologicl metric tht hs permeted the culturl psyche of nerly ll industrilized ntions. Weight loss hs ecome ner societl osession (Frser, 1997). Mny populr diet progrms initilly use significnt clorie restriction (CR) to fcilitte reltively rpid weight-loss (Volpe, 26) including the recent trend of detox nd/or clensing diets which my incorporte wter only fsting for up to 1 wk. In rod context, however, fsting is criticl element of mny religious nd culturl prctices (Brett & Nesit, 212; Slim, Al Suwidi, Ghdn, Alkilni, & Slm, 213) nd is recommended y prctitioners of lterntive nd complementry medicine s therpy for vriety of ilments (Fuhrmn, Srter, & Clro, 22; Goldhmer et l., 22; Michlsen, 21; Schmidt et l., 21). Cliniclly, dietry regimens tht incorporte intermittent fsting (IF) hve gined significnt populr ttention (The Fst Diet: Lose Weight, Sty Helthy, nd Live Longer with the Simple Secret of Intermittent Fsting, Dr. Michel Mosley) with humn studies showing potentil to reduce hert disese nd dietes (Horne et l., 28) nd to improve mood (Michlsen et l., 26; Michlsen, 21). Since fsting regimens pper to enefit cognition function in ging mice (Mrtin, Mttson, & Mudsley, 26) nd in the 3xTgAD mouse model of Alzheimer s disese, (Hlgpp et l., 27) interest in CR nd IF s wy to wrd off humn cognitive ging hs lso emerged (Mttson, 21). While overeting nd oesity re ssocited with vriety of mldies including type 2 dietes (T2D), hert disese, stroke (Kopelmn, 27), cncer (Louie, Roerts, & Nomur, 213) Alzheimer s disese (Lee, 211), nxiety/mood disorders (Kczmrczyk et l., 213; Simon et l., 26), nd declined cognition (Bressler et l., 213), precisely how overnutrition contriutes is not entirely cler. Emerging 1

evidence indictes tht oxidtive nd metolic stress in conjunction with dyslipedemi nd excess glucose stimultes the unfolded protein response (Scheuner & Kufmn, 28) nd ctivtes the NLRP3 inflmmsome (Vndnmgsr et l., 211). Thus, IL-1-medited inflmmtion is key consequence of overnutrition, whether indirectly provoked y cellulr injury (Gregor & Hotmisligil, 211) or directly triggered y excess nutrients cting s dnger signls (Lumeng, 213). Overnutrition-ssocited IL-1 is implicted in pncretic et cell loss in T2D, therogenesis in hert disese nd stroke nd neurodegenertion in Alzheimer s disese (Ci & Liu, 212) ut, in generl, IL-1 is criticl effecter of sickness symptoms tht include loss of ppetite, locomotion nd socil/environmentl enggement (Kelley et l., 23). Thus, meliortions tht inhiit IL-1 ioction my positively impct helth in those with either psychologicl (Mes, Song, & Yirmiy, 212; Rossi et l., 212) nd/or overtly orgnic diseses (Dyer, 23; Slon-Lncster et l., 213). We recently demonstrted tht mice fsted for 24 hrs hve reduced gene expression of rin IL- 1α (Lvin et l., 211), indicting tht CR my e physiologic wy to negtively regulte IL-1 ioction. Although strvtion ppers detrimentl to immunity, s evidenced y lipopolyscchride (LPS)-induced deth in ees nd mice (Fggioni, Moser, Feingold, & Grunfeld, 2; Moret & Schmid-Hempel, 2), fsting ppers to mitigte LPS-induced sickness symptoms in rts (Inoue, Somy, Poole, & Luheshi, 28). In ddition, fsting offers protection from ischemic (Mitchell et l., 21) nd hypoxic injuries (Go, Prenen, & Korf, 1988) which we nd others hve shown re medited in lrge prt y IL-1 (Chiu et l., 212; Johnson, O'Connor, Hrtmn, Tpping, & Freund, 27). Therefore, we exmined if the eneficil effects of fsting re due to IL-1 counter-regultion in oth the systemic nd neuroimmune systems. 2

CHAPTER 2 OBJECTIVES 1. Determine how fsting lters the immune system nd if there re ny cognitive or ehviorl effects ssocited with fsting. 2. Determine wht hppens to previously fsted immune system fter eing chllenged with IL-1β, nd elucidte whether these chnges re ssocited with ltertions in sickness ehviors with this immune stimuli. 3. Determine the mechnism y which fsting lters the immune system. 3

CHAPTER 3 METHODOLOGY Mterils- All regents nd chemicls were purchsed from Sigm-Aldrich (St Louis, MO) except s noted. All primers were purchsed from Applied Biosystems (Foster City, CA) Animls- Procedures were conducted on protocols pproved y the University of Illinois Institutionl Animl Cre nd Use Committee. IL-1 receptor 1 (IL-1R1) knock out (KO), toll-like receptor (TLR-4) KO, IL-4 KO nd C57BL/6J (wild type (WT)) mice were purchsed from Jckson Lortory (Br Hror, ME) nd red in-house. All KO mice were on C57BL/6 ckone. Mice were group housed ( 8 cge) in stndrd shoeox cges (length 46.9 cm; width 25.4 cm; height 12.5 cm) nd llowed wter nd food (Hrln XXX (Indinpolis, IN) d liitum except where otherwise noted. Housing temperture (72 F) nd humidity (45 55%) were controlled s ws 12/12 h reversed drk-light cycle (light = 22-1 h). Animls were scrificed using CO 2 except for those in which corticosterone ws mesured which were scrificed using ketmine/xylzine. Fsting nd mouse weight- As previously descried (Lvin et l., 211; York et l., 212), 2 dys prior to fsting, mice were singly housed. Fsting ws initited y plcing mice in new cge without food ut with d liitum wter. Mice were fsted for 24 h strting t 9 h. Mouse weight ws recorded immeditely efore nd immeditely fter fsting using n Ohus Adventurer Pro digitl scle (Prsippny, NJ). Injectles- Millipore recominnt mouse IL-1β (Billerci, MA) in 1 μl of sterile Cellgro PBS (Mnsss, VA) (vehicle) ws dministered intrperitonelly (IP) t dose of.9 μg/mouse immeditely 4

fter fsting. Mifepristone in BioUltr 4 polyethylene glycol (PEG) (vehicle) (PEG) ws dministered sucutneously (SC) t dose of 25 mg/kg immeditely prior to fsting. Plmitic cid in 5 μl of cstor oil (vehicle) ws dministered IP t dose of 3 μmoles. Quntittive PCR (qpcr)- As previously descried (Lvin et l., 211), nimls were perfused with 3 mls of ice cold PBS nd RNA isolted from homogenized tissues using Qigen RNesy Lipid Tissue Mini Kits (Vlenci, CA). RNA ws reverse trnscried using the Applied Biosystems High-Cpcity cdna Reverse Trnscription Kit. The TqMn Gene Expression primers used were: CD11 (Mm434455_m1), TNF-α (Mm443258_m1), IL-1α (Mm999996_m1), IL-1β (Mm9999961_mH), IL-1R1 (Mm434237_m1), IL-1RA (Mm1337566_m1) nd IL-1R2 (Mm439622_m1). qpcr ws performed on n Applied Biosystems 79 HT Fst Rel-Time PCR System using Applied Biosystems TqMn Universl PCR Mster Mix. To compre gene expression, prllel mplifiction of endogenous RPS3 (Mm656272_m1) ws performed. Rections with no reverse trnscription nd no templte were included s negtive controls. Reltive quntittive evlution of trget gene to RPS3 ws performed y compring ΔCts, where Ct is the threshold concentrtion. Locomotion- Spontneous locomotor ctivity ws mesured s previously descried (Lvin et l., 211, York et l., 212). At the times indicted, mice were video recorded in their home cge for 5 min under red light using using Sony Night Shot cple video cmer (Minto-ku, Tokyo). Distnce moved ws quntified using Noldus Informtion Technology EthoVision XT 7 utomted trcking softwre (Leesurg, VA). Socil Behvior- Socil explortion of novel juvenile ws mesured s previously descried (Sherry, Krmer, York, & Freund, 29). In rief, 4-wk old novel, conspecific mle juvenile (chllenge) 5

mouse enclosed in 3 x 3 x 3 inch wire mesh cge ws plced in the home cge of the dult (test) mouse for 5 minutes. Test mouse-initited explortory ehvior of the chllenge mouse enclosure ws video recorded under red light using using Sony Night Shot cple video cmer. Time spent exploring the chllenge mouse (nose within 2 cm region of interest (ROI) drwn round the cged juvenile) ws quntified using Noldus Informtion Technology EthoVision XT 7 utomted trcking softwre (Leesurg, VA). Blood glucose- Mouse til lood glucose ws determined in duplicte using n Aott Lortories AlphTRAK Blood Glucose Monitoring System (North Chicgo, IL) y methods we hve previously descried (Lvin et l., 211). Serum nd liver IL-1R2 nd IL-1RA- Serum IL-1R2 ws mesured using Millipore Milliplex MAP kit (Billeric, MA) following mnufctures instructions. Bed-sed fluorescent reporter signl ws detected on Luminex 1 System (Austin, TX). Serum IL-1RA ws mesured colorimetriclly in y ELISA using the R&D Mouse IL-1RA/IL-1F3 conjugte kit (Minnepolis, MN). Liver IL-1R2 nd IL-1RA were mesured from freeze-frctured liver y methods we hve previously descried (Chiu et l., 212). In rief, PBS perfused livers were liquid nitrogen frozen in freeze frcture uffer contining 1% glycerol, 5 mm NCl, 1 mm EDTA, 5 mm HEPES (ph 7.4) plus 1:2 dilution of Millipore protese inhiitor cocktil (Billeric, MA). The frctured liver ws homogenized using the TissueLyser II (Qigen, Vlenci, CA) t rottionl frequency of 3/sec for 2 min. Lystes were clrified t 16, x g for 15 min t 4º C nd the superntnt protein concentrtions determined using the Bio-Rd DC Protein Assy (Hercules, CA). Liver IL-1R2 nd IL-1RA ws expressed s pg of IL-1R2 per ng of superntnt protein. 6

Plsm corticosterone nd free ftty cids (FFAs)- Corticosterone ws mesured colorimetriclly y ELISA using the Acm corticosterone ELISA kit (Cmridge, MA) in plsm. FFAs were mesured colorimetriclly in plsm using the Rndox Nef Assy (Antrim, Irelnd) nd Beckmn-Coulter AU68 (Bre, CA) following mnufctures instructions. Sttistics- Individul ehviorl experiments were performed on seprte cohorts of mice. Biochemicl/qPCR experiments were performed on cohorts of mice not sujected to ehviorl testing. All dt re presented s men ± SEM. Dt were nlyzed using Sigm Plot 11.2 (Systt Softwre,Chicgo, IL). To test for sttisticl differences, one-wy nd two-wy ANOVAs were used with or without repeted mesurements where relevnt. Tukey s test ws used for post-hoc pir-wise multiple comprison procedures. Person product moment correltion ws used to compute correltion coefficient. Where indicted, rw dt ws trnsformed to ttin normlity or equl vrince vi log(s), ^2 or RANK simple trnsforms. All sttisticl nlysis included testing for time point x tretment interctions. Sttisticl significnce ws denoted t p<.5. 7

CHAPTER 4 RESULTS 1. Effect of fsting on gene expression Tle 1 shows tht CD11, TNF-α, IL-1α, IL-1β, IL-1R1, IL-1RA nd IL-1R2 gene trnscripts fter fsting re differentilly expressed in rin regions (hypothlmus, hippocmpus, cortex), nd peripherl tissues (liver, dipose). In ll rin regions, fsting cused CD11 gene trnscript to significntly downregulte ( p<.1 ll rin regions). Wheres oth dipose nd liver upregulted CD11 (p<.1 dipose, p=.32 liver; trnsformed to RANK for normlity). TNF-α ws consistently downregulted in ll tissues (p=.1 hypoth, p=.2 hippo, p<.1 cortex, p=.4 dipose, =.45 liver). IL-1α ws significntly downregulted in ll rin regions (p<.1 ll rin regions), ut dipose nd liver trnscript ws not significntly different due to fsting. IL-1β gene messge ws significntly downregulted in the hypothlmus (p<.6 hypoth) nd lso in dipose tissue (p=.8 dipose), ut ws not significntly different in ny other tissues. IL-1R1 ws significntly upregulted in ll tissues mesured (p=.8 hypoth,p=.31 hippo, p<.1 cortex, p<.1 dipose, p=.17 liver). IL-1RA messge ws significntly upregulted in hippocmpus (p=.46 hippo) s well s in the liver (p<.1 liver), ut ws downregulted in the dipose (p=.14), nd remined unchnged in hypothlmus nd cortex. IL-1R2 ws not significntly different in ny rin regions, ut ws significntly upregulted in oth dipose nd liver (p=.8 dipose, p=,16 liver), oth trnsformed to RANK for equl vrinces. 2. IL-1R2 nd IL-1RA protein increse in liver fter 24 h fst. Tle 2 shows tht, in greement with up-regulted gene trnscript of IL-1R2, mice sujected to 24 h fst hd incresed IL-1R2 protein in the liver, p=.4; dt trnsformed to log(s) for normlity. Also in greement with up-regulted gene trnscript of IL-1RA, mice sujected to 24 h fst hd incresed IL- 8

1RA protein in the liver, p<.1; dt trnsformed to log(s) for equl vrince. No significnt difference in serum IL-1R2 or IL-1RA protein due to fsting. 3. Fsting effects socil explortory ehvior in mice Fig. 1A shows tht 24 h fst cuses significnt incresed socil interction in mice (Fed 165.2± 13.9 vs. Fsted 21.5± 6.22). Min effect significnt t p=.15. Fig. 1B shows tht fsting hs no significnt effect on locomotor ehvior in mice (Fed 3155.± 267.4 vs. Fsted 2916.3± 19.6). No significnce t p=.484. There ws no significnce seen with dditionl dverse ehvior tests or cognitive deficits oserved when tested with these other prdigms: Open Field, Novel Oject Recognition, or Y-Mze (dt not shown). 4. Fsting induces resistnce to IL-1β-induced sickness ehvior. Fig. 2A shows tht fsted mice re resistnt to decresed socil explortory ehvior of juvenile 2 h fter IL-1β injection (Fed Sline 123.48 16.493s vs. Fsted Sline 172.414 9.737s vs. Fed IL-1β 62.721 13.82s vs. Fsted IL-1β 112.9 22.296s); min effects of tretment (P<.1) nd stte (P=.4), no tretment-stte interction (P=.98). Fig. 2B demonstrtes tht fsting mouse prior to injecting with inflmmtory IL-1β induces resistnce to sickness s shown y resistnce to decresed locomotor ctivity t 2 h, spontneous locomotor ctivity ws mesured 2 h post injection (Fed Sline 1213.374 36.764cm vs. Fsted Sline 112.946 115.244cm vs. Fed IL-1β 131.176 74.121cm vs. Fsted IL-1β 731.617 11.55cm); min effects of tretment (P<.1) nd stte (P=.46), tretment-stte interction (P<.1). Fig. 2C Shows tht mice sujected to 24 h fst were resistnt to IL-1β-induced sickness weight loss. Mice were weighed 2 h post injection nd compred to their initil weight ( h) prior to tretment (Fed Sline -.4.24g vs. Fsted Sline -.3.18g vs. Fed IL- 1β -1.28.18g vs. Fsted IL-1β -.24.67g); min effects of tretment (P=.16) nd stte (P=.2), tretment-stte interction (P<.7). Fig. 2D Shows tht fsting mice for 24 h prior to IL-1β 9

immune chllenge induces resistnce to IL-1β-induced decrese in lood glucose. Blood glucose ws mesured 2 h post injection nd compred to their initil lood glucose ( h) prior to tretment (Fed Sline -12. 18.898mg/dl vs. Fsted Sline -5.75 18.368mg/dl vs. Fed IL-1β -111.2 14.9mg/dl vs. Fsted IL-1β -11.4 7.619mg/dl); min effects of tretment (P=.4) nd stte (P=.3), tretment-stte interction (P<.7). 5. Effect of chllenging mice with IL-1β on rin region cytokine trnscripts Fig. 3A demonstrtes tht chllenging mice peripherlly with IL-1β induces IL-1R2 gene trnscript chnges in specific rin regions regrdless of prior 24 h fst. 2 h post injection, rin regions were hrvested (Hypothlmus-Hypoth, Hippocmpus-Hippoc, Cortex-Cortex), gene trnscripts were mesured s reltive fold mrna (Hypoth; Fed, Sline +.119 vs. Fsted, Sline 1.272 +.36 vs. Fed, IL-1β 1.817 +.189 vs. Fsted, IL-1β 1.877 +.233); min effects of tretment (P<.1) nd stte (P=.182), no significnt tretment-stte interction (P=.3). (Hippoc; Fed, Sline +.172 vs. Fsted, Sline 1.15 +.13 vs. Fed, IL-1β 1.811 +.245 vs. Fsted, IL-1β 2.29 +.312); min effects of tretment (P<.1) nd stte (P=.169), no significnt tretment-stte interction (P=.72). (Cortex; Fed, Sline +.3 vs. Fsted, Sline 1.274 +.68 vs. Fed, IL-1β 1.579 +.124 vs. Fsted, IL-1β 1.9 +.14); min effects of tretment (P<.1) nd stte (P=.2), no significnt tretment-stte interction (P=.614). Fig. 3B demonstrtes tht chllenging mice peripherlly with IL-1β induces IL-1RA gene trnscript chnges in specific rin regions regrdless of prior 24 h fst. 2 h post injection, rin regions were hrvested (Hypothlmus-Hypoth, Hippocmpus-Hippoc, Cortex-Cortex), gene trnscripts were mesured s reltive fold mrna (Hypoth; Fed, Sline +.442 vs. Fsted, Sline.461 +.37 vs. Fed, IL-1β 52.13+2.897 vs. Fsted, IL-1β 34.184 +3.63); min effects of tretment (P<.1) nd stte (P<.1), no significnt tretment-stte interction (P=.747), dt trnsformed to RANK for normlity. (Hippoc; Fed, Sline, +.412 vs. Fsted, Sline.87, +.267 vs. Fed, IL-1β 1

49.634 +8.322 vs. Fsted, IL-1β 31.42 +5.176); min effects of tretment (P<.1) nd stte (P=.245), no significnt tretment-stte interction (P=.525). (Cortex; Fed, Sline, +.63 vs. Fsted, Sline 1.118 +.175 vs. Fed, IL-1β 23.641 +3.136 vs. Fsted, IL-1β 16.56 +2.57); min effects of tretment (P<.1) nd stte (P=.339), no significnt tretment-stte interction (P=.82). Fig. 3C demonstrtes tht chllenging mice peripherlly with IL-1β induces IL-1α gene trnscript chnges in specific rin regions regrdless of prior 24 h fst. 2 h post injection, rin regions were hrvested (Hypothlmus- Hypoth, Hippocmpus-Hippoc, Cortex-Cortex), gene trnscripts were mesured s reltive fold mrna (Hypoth; Fed, Sline +.179 vs. Fsted, Sline.77 +.36 vs. Fed, IL-1β 13.471 +.613 vs. Fsted, IL-1β 1.354 +1.21); min effects of tretment (P<.1) nd stte (P=.13), no significnt tretment-stte interction (P=.7). (Hippoc; Fed, Sline, +.65 vs. Fsted, Sline.739 +.63 vs. Fed, IL-1β 7.638 +.557 vs. Fsted, IL-1β 7.665 +1.57); min effects of tretment (P<.1) nd stte (P=.21), no significnt tretment-stte interction (P=.2). (Cortex; Fed, Sline, +.74 vs. Fsted, Sline.546 +.74 vs. Fed, IL-1β 8.97 +.955 vs. Fsted, IL-1β 7.347 +1.362); min effects of tretment (P<.1) nd stte (P=.1), no significnt tretment-stte interction (P=.168). Fig. 3D demonstrtes tht chllenging mice peripherlly with IL-1β induces IL-1β gene trnscript chnges in specific rin regions regrdless of prior 24 h fst. 2 h post injection, rin regions were hrvested (Hypothlmus-Hypoth, Hippocmpus-Hippoc, Cortex-Cortex), gene trnscripts were mesured s reltive fold mrna (Hypoth; Fed, Sline +.412 vs. Fsted, Sline.425 +.67 vs. Fed, IL-1β 72.81 +14.91 vs. Fsted, IL-1β 69.313 +9.665); min effects of tretment (P<.1) nd stte (P=.61), no significnt tretment-stte interction (P=.91). (Hippoc; Fed, Sline, +.134 vs. Fsted, Sline.977, +.44 vs. Fed, IL-1β 11.289 +1.355 vs. Fsted, IL-1β 12.66 +2.138); min effects of tretment (P<.1) nd stte (P=.715), no significnt tretment-stte interction (P=.583). (Cortex; Fed, Sline, +.53 vs. Fsted, Sline.849 +.11 vs. Fed, IL-1β 11.119 +1.78 vs. Fsted, IL-1β 11

1.391 +1.48); min effects of tretment (P<.1) nd stte (P=.347), no significnt tretment-stte interction (P=.693). 6. Sustntil up-regultion in liver nd dipose IL-1R2 gene expression nd in liver IL-1RA gene expression nd their correltions to percent weight loss. Fig. 4A shows tht IL-1R2 is sustntilly upregulted in the liver fter 24 h fst. There is significnt difference in verge fold chnge in mrna in liver fed vs. fsted (1.82±.95 vs. 27.975± 13.853) significnt min effect, p<.1 run on CT vlues trnsformed to ^2 for normlity. Fig. 4B shows tht percent weight loss is negtively correlted to CT vlue, dt rn y Person Correltion test. The more weight the mouse lost during 24 h fst, the lower the CT vlue in PCR ws for IL-1R2 gene expression in liver, nd liver IL-1R2 correltion coefficient ws -.53, p=.2.. Fig. 4C shows tht IL-1R2 is sustntilly upregulted in dipose with significnt difference in verge fold chnge in mrna in dipose fed vs. fsted (1.118±.89 vs. 69.577± 21.829) significnt min effect, p<.1 run on CT vlues trnsformed to RANK for normlity. Fig. 4D shows tht percent weight loss is negtively correlted to CT vlue, dt rn y Person Correltion test. The more weight the mouse lost during 24 h fst, the lower the CT vlue in PCR ws for IL-1R2 gene expression in dipose. Adipose IL-1R2 correltion coefficient ws -.61, p<.1. Fig. 4E shows tht IL-1RA is lso sustnticlly upregulted in the liver fter 24 h fst nd there ws significnt difference in verge fold chnge in mrna in liver fed vs. fsted (1.219±.14 vs. 33.792± 6.292) significnt min effect, p<.1. Fig. 4F shows tht percent weight loss ws not significntly correlted to CT vlue for IL-1RA in liver, correltion coefficient -.36, p=.94. There ws no significnt correltion in ny groups to ge of mouse t fst (etween 9-14wks) nd CT vlue for IL-1R2 or IL-1RA, dt not shown. There ws lso no correltion etween strting weight t time of fst nd CT vlue for IL-1R2 or IL-1RA, dt not shown. 7. IL-1R2 nd IL-1RA gene expression on CD11+, CD11- nd heptocytes isolted from liver. 12

Fig. 5A shows tht IL-1R2 is primrily expressed only in those cells with CD11 when compred to CD11- cells. Although there ws no significnce etween fed or fsted in either isolted cell group, CD11+ cells hd significntly more expression of IL-1R2 thn CD11- cells (CD11+ Fed, +.26-.171 vs. CD11+ Fsted.92, +.285-.216 vs. CD11- Fed.1, +.-. vs. CD11- Fsted.1, +.-.); min effects of cell type (P<.1) nd stte (P=.24), no significnt cell typestte interction (P=.444). Fig. 5B shows tht IL-1RA is primrily expressed only in those cells with CD11 when compred to CD11- cells. Although there ws no significnce etween fed or fsted in either isolted cell group, CD11+ cells hd significntly more expression of IL-1RA thn CD11- cells (CD11+ Fed, +.221-.181 vs. CD11+ Fsted.62, +.51-.47 vs. CD11- Fed.27, +.2-.2 vs. CD11- Fsted.22, +.4-.3); min effects of cell type (P<.1) nd stte (P=.58), no significnt cell type-stte interction (P=.454). Fig. 5C shows tht IL-1R2 expression is not different due to the fsting stte in isolted heptocytes (Fed, +.337-.252 vs Fsted.847 +.223-.176). Fig. 5D shows tht IL-1RA expression is upregulted fter 24 h fst in heptocytes (Fed, +.199-.166 vs. 7.146 +2.59-1.857) p=.1. Fig5E shows tht llowing piece of liver to sit in uffer for 6 h fter hrvest, compred to extrcting RNA within 1 h, significntly decreses IL-1R2 messge (, +.219-.18 vs..13, +.1-.1), p<.1, nd lso significntly decreses IL-1RA messge (, +.175-.149 vs..437, +.22-.21), p=.9. 8. Effect of fsting on gene expression of IL-1R2 nd IL-1RA in liver nd dipose of knockout mouse models. Tle 3 shows tht IL-1R2 nd IL-1RA gene trnscripts fter fsting in the liver nd dipose re not impcted y knockout of IL-1R1, TLR-4 or IL-4 genes in mouse models. In IL-1R1 KO mice, fsting significntly up-regulted IL-1R2 mrna y 8 fold in the liver (p=.21), nd y 18 fold in the dipose (p=.7). Fsting of IL-1R1 KO mice lso significntly up-regulted IL-1RA mrna in the liver y 16 fold 13

(p<.1), nd hd no significnt difference in dipose IL-1RA. In TLR-4 KO mice, fsting significntly upregulted IL-1R2 mrna y 7 fold in the liver (p<.5), nd y 9 fold in the dipose (p=.2), dt trnsformed to RANK for equl vrinces. Fsting of TLR-4 KO mice lso significntly up-regulted IL-1RA mrna in the liver y 16 fold (p<.1), nd hd no significnt difference in dipose IL-1RA. In IL-4 KO mice, fsting significntly up-regulted IL-1R2 mrna y 8 fold in the liver (p<.5), nd y 22 fold in the dipose (p=.22). Fsting of IL-4 KO mice lso significntly up-regulted IL-1RA mrna in the liver y 13 fold (p<.1), nd hd no significnt difference in dipose IL-1RA. None of these groups were sttisticlly different from their WT controls (dt not shown). 9. Effect of fsting on plsm corticosterone levels in mice. Fig. 6A confirms tht 24 h fst cuses significnt incresed plsm corticosterone levels in mice (Fed 368.12± 28.59 vs. Fsted 754.828± 84.421). Min effect significnt t p=.4. Fig. 6B shows tht this increse in plsm corticosterone is significntly positively correlted (correltion coefficient.899) with percent ody weight lost y the mouse in 24 h fst, (p=.5). 1. Effect of inhiiting glucocorticoid receptor ctivity on IL-1R2 nd IL-1RA gene trnscripts in liver. Fig. 7A demonstrtes tht inhiiting glucocorticoid signling vi GCR ntgonist (Mifepristone) does not effect IL-1R2 gene trnscripts in the liver fter 24 h fsting period. Mouse liver ws hrvested fter 24 h fst nd IL-1R2 gene trnscripts were mesured s reltive fold mrna (Fed None, +.13 -.94; vs. Fsted None 4.247, +.623 -.543; vs. Fed Mifepristone 1.789, +.299 -.256; vs. Fsted Mifepristone 4.79, +.69 -.54; vs. Fed PEG 1.327, +.126 -.115; vs. Fsted PEG 3.471, +.661 -.555); min effects of tretment (P=.58) nd stte (P<.1), no significnt tretment-stte interction (P=.16). Fig. 7B lso demonstrtes tht inhiiting glucocorticoid signling vi GCR ntgonist (Mifepristone) does not effect IL-1RA gene trnscripts in the liver fter 24 h fsting period. Mouse liver ws hrvested fter 24 h fst nd IL-1RA gene trnscripts were mesured s reltive fold 14

mrna (Fed None, +.144 -.126; vs. Fsted None 8.939, +1.691-1.422; vs. Fed Mifepristone 1.236, +.157 -.14; vs. Fsted Mifepristone 9.521, +1.587-1.26; vs. Fed PEG 1.86, +.152 -.133; vs. Fsted PEG 5.182, +1.395-1.99); min effects of tretment (P=.77) nd stte (P<.1), no significnt tretment-stte interction (P=.161). 11. Effect of fsting nd plmitic cid injection on NEFA levels in mice. Fig. 8A shows fsting cuses plsm NEFAs to doule (Fed.927±.14 vs. Fsted 2.27±.161), nd this ws significnt t p<.1. Fig. 8B shows tht plmitic cid injection mimics fsting plsm NEFAs increse 2 h fter plmitic cid injection (Vehicle.881±.165 vs. Plmitic 1.63±.13); min effects of tretment (P=.3). 12. Liver IL-1R2 gene trnscription levels fter plmitic cid injection Fig. 9A shows tht IL-1R2 is upregulted in the liver of mice 2 h fter they re injected with plmitic cid. Plmitic injected mice hd significntly more expression of IL-1R2 in liver thn vehicle controls (Vehicle, +.213-.175 vs. Plmitic 5.6, +3.7-1.91); significnt min effect (P=.1). Fig. 9B shows tht IL- 1R2 is significntly incresed in the liver 2 h fter plmitic cid regrdless of TLR-4. (WT, Vehicle, +.113-.11 vs. WT. Plmitic 3.267, +.592-.51 vs. TLR-4, Vehicle.97, +.85-.78 vs. TLR-4, Plmitic 2.91, +.558-.468); min effects of tretment (P<.1) no significnt effect of genotype (P=.645), no significnt tretment-genotype interction (P=.75). 15

CHAPTER 5 TABLES, FIGURES AND FIGURE LEGENDS Tle 1 Effect of fsting on gene expression Tissue Hypothlmus Hippocmpus Cortex Adipose Liver Fed Fsted Fed Fsted Fed Fsted Fed Fsted Fed Fsted CD11 (.5,.5).577* (.3,.3) (.1,.1).599* (.3,.3) (.4,.4).54* (.4,.3) (.6,.6) 3.732* (.58,.5) (.1,.9) 6.16* (2.69,1.64) TNF-α (.18,.15).479* (.3,.2) (.9,.8).614* (.6,.5) (.7,.7).67* (.2,.2) (.28,.22).17* (.3,.2) (.1,.9).66* (.8,.7) IL-1α (.6,.6).567* (.3,.3) (.5,.5).565* (.2,.2) (.1,.9).496* (.2,.2) (.58,.36).295 (.4,.4) (.14,.12).857 (.3,.3) IL-1β (.17,.14).487* (.5,.5) (.8,.7).791 (.6,.5) (.12,.11).918 (.1,.9) (.36,.26).167* (.3,.3) (.12,.11).979 (.33,.25) IL-1R1 (.1,.9) 1.484* (.12,.11) (.2,.16) 1.6* (.18,.1) (.3,.3) 1.319* (.5,.5) (.11,.1) 2.334* (.35,.3) (.12,.11) 3.13* (.95,.69) IL-1RA (.32,.24).92 (.24,.19) (.11,.9) 1.6* (.33,.27) (.14,.12) 1.66 (.11,.1) (.24,.19).254* (.7,.6) (1.55,.64) 68.482* (38.6,24.7) IL-1R2 (.12,.11) 1.14 (.4,.4) (.9,.9).88 (.9,.8) (.4,.4) 1.61 (.6,.5) (.25,.2) 83.424* (79.,4.5) (.14,.12) 9.467* (19.3,6.35) Results re expressed s reltive fold chnge in mrna ( mrna), mens (SEM upper, SEM lower); n=4-9 * p<.5, significnt (One-wy ANOVA) 16

Tle 2 Effect of fsting on protein in liver (pg IL-1R2 or IL-1RA/ng totl protein) nd protein in serum (pg/ml) IL-1R2 IL-1RA Fed Fsted Fed Fsted Liver 8,475.2 27,14.4* 29,54.4 71,722.6* ±1,837.4 ±8,993.1 ±3,58. ±11,176.4 Serum 11,112.2 ±392.7 1,646.6 ±35.4 661.9 ±112.7 693.6 ±95.7 Results re expressed s mens (±SEM); Liver IL-1R2, n=7-8. Liver IL-1RA, n=13-16. Serum IL-1R2, n=8. Serum IL-1RA, n=8 * p<.5, significnt (One-wy ANOVA) Tle 2 IL-1R2 nd IL-1RA protein increse in liver fter 24 h fst. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed). IL-1R2 results re expressed s pg IL-1R2/ng totl liver protein, mens ( SEM); n=7-8. *p <.5. IL-1RA results re expressed s pg IL-1RA/ng totl liver protein, mens ( SEM; n=13-16. *p<.1. Serum IL-1R2 nd IL-1RA levels re not significnt, results re expressed s pg/ml. 17

Locomotor Activity (cm) Socil Interction (s) A 25 * 2 15 1 5 Fed Fsted B 4 35 3 25 2 15 1 5 Fed Fsted Figure 1. Fsting effects socil explortory ehvior, ut not locomotor ctivity in mice. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed). (A) Mice were introduced to novel juvenile mouse nd llowed to explore for 5 minutes. Results re expressed s seconds (s) spent eing socilly interctive, mens (±SEM); n=8. Significnt min effect (*p <.5). (B) Locomotor ctivity ws trcked for 5 minute intervl. Results re expressed s centimeters (cm) moved, mens (±SEM); n=6. 18

Totl Distnce Trveled (cm) Socil Interction (s) A 2 18 16 14 12 1 8 c 6 4 2 Fed Fsted Fed Fsted Sline IL-1β B 14 12 1 8 c 6 4 2 Fed Fsted Fed Fsted Sline IL-1β Figure 2 Fsting induces resistnce to IL-1β-induced sickness ehvior. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed) prior to IL-1β or vehicle (Sline) t h. (A) A juvenile ws introduced 2 h post injection, nd socil interction ws mesured s seconds (s) spent investigting juvenile for durtion of 5 minutes, mens (±SEM); n=8. Brs without common superscript re different (*p <.5). (B) 2 h post injection, spontneous locomotor ctivity ws mesured for 5 minutes. Results re expressed s totl distnce trveled in centimeters (cm), mens ( SEM); n=4. Brs without common superscript re different (*p <.5). 19

Chnge in Blood Glucose (mg/dl) Weight Chnge (g) C -.2 Sline IL-1β Fed Fsted Fed Fsted -.4 -.6 -.8-1 -1.2-1.4-1.6 D 2 Sline IL-1β Fed Fsted Fed Fsted -2-4 -6-8 -1-12 -14 Figure 2 (cont.) Fsting induces resistnce to IL-1β-induced sickness ehvior. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed) prior to IL-1β or vehicle (Sline) t h. (C) Mice were weighed 2 h post injection nd compred to their weight t h. Results re expressed s weight chnge from h in grms (g), mens ( SEM); n=4-5. Brs without common superscript re different (*p<.5). (D) Blood Glucose ws mesured 2 h post injection nd compred to h lood glucose mesurement for ech individul mouse. Results re expressed s chnge in lood glucose (mg/dl), mens ( SEM); n=4-5. Brs without common superscript re different (p<.5). 2

Reltive Fold ΔmRNA IL-1RA Reltive Fold ΔmRNA IL-1R2 A 3 2.5 2 c d 1.5 1.5 Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Hypoth Hippoc Cortex B 7 6 c 5 4 d 3 2 1 Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Hypoth Hippoc Cortex Figure 3. Effect of chllenging mice with IL-1β on rin region cytokine trnscripts. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed) prior to IL-1β or vehicle (Sline) t h. 2 h post injection, rin regions were hrvested (Hypothlmus-Hypoth, Hippocmpus-Hippoc, Cortex- Cortex). (A) IL-1R2 gene trnscripts were mesured nd compred to set control group within ech seprte region (Fed, Sline). Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper); n=4. Brs without common superscript re different (*p <.5). (B) IL-1RA gene trnscripts were mesured nd compred to set control group within ech seprte rin region (Fed, Sline). Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper); n=4. Brs without common superscript re different (*p <.5). 21

Reltive Fold ΔmRNA IL-1β Reltive Fold ΔmRNA IL-1α C 16 14 12 1 8 c c c c 6 4 2 Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Hypoth Hippoc Cortex D 1 9 8 7 6 5 4 3 2 1 c c Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Fsted, IL-1β Fed, IL-1β Fsted, Sline Fed, Sline Hypoth Hippoc Cortex Figure 3 (cont.). Effect of chllenging mice with IL-1β on rin region cytokine trnscripts. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed) prior to IL-1β or vehicle (Sline) t h. 2 h post injection, rin regions were hrvested (Hypothlmus-Hypoth, Hippocmpus-Hippoc, Cortex-Cortex).(C) IL-1α gene trnscripts were mesured nd compred to set control group within ech seprte rin region (Fed, Sline). Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper); n=4. Brs without common superscript re different (*p <.5). (D) IL-1β gene trnscripts were mesured nd compred to set control group within ech seprte rin region (Fed, Sline). Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper); n=4. Brs without common superscript re different (*p <.5). 22

Log(Rel. Fold mrna) IL-1R2 Liver Delt CT Vlue IL-1R2 Liver A 3 2 1-1 Fed Fsted B 16 14 12 1 8 6 4 2 5 1 15 2 25 % Weight Loss Figure 4. Sustntil up-regultion in liver nd dipose IL-1R2 gene expression, nd in liver IL-1RA gene expression nd their correltions to percent weight loss. These grphs represent collective description of dt from numerous cohorts of mice tht were either fst (Fsted) for 24 h, or continued to feed d li (Fed). Box plots re representtive of the summted reltive fold chnges in mrna trnsformed to log; the ox outlines the lower 1 st qurtile nd upper 3 rd qurtile, the verticl rs represent the lrgest/smllest non-outlier oservtion, the thick horizontl line represents the men of the reltive fold chnge vlues nd the thin horizontl line is the medin, nd lck circles represent outliers (A) Liver IL-1R2 gene trnscription results re summrized nd expressed s log (verge reltive fold chnge ( mrna)) for fed (n=29) nd fsted groups (n=31) *p<.1. (B) Percent weight lost per mouse ws correlted to their dct vlue for IL-1R2 in the dipose, significnt correltion ws oserved p<.1, n=3. 23

Log(Rel. Fold mrna) IL-1R2 Adipose Delt CT Vlue IL-1R2 Adipose C 3 2 1-1 Fed Fsted D 16 14 12 1 8 6 4 2 5 1 15 2 25 % Weight Loss Figure 4 (cont.). Sustntil up-regultion in liver nd dipose IL-1R2 gene expression, nd in liver IL- 1RA gene expression nd their correltions to percent weight loss. These grphs represent collective description of dt from numerous cohorts of mice tht were either fst (Fsted) for 24 h, or continued to feed d li (Fed). Box plots re representtive of the summted reltive fold chnges in mrna trnsformed to log; the ox outlines the lower 1 st qurtile nd upper 3 rd qurtile, the verticl rs represent the lrgest/smllest non-outlier oservtion, the thick horizontl line represents the men of the reltive fold chnge vlues nd the thin horizontl line is the medin, nd lck circles represent outliers. (C) Adipose IL-1R2 gene trnscription results re summrized nd expressed s log (verge reltive fold chnge ( mrna)) for fed (n=29) nd fsted groups (n=3) *p<.1.(d) Percent weight lost per mouse ws correlted to their dct vlue for IL-1R2 in the dipose, significnt correltion ws oserved p<.1, n=3. 24

Log(Rel. Fold mrna) IL-1RA Liver Delt CT Vlue IL-1RA Liver E 3 2 1 F 8-1 Fed Fsted 7 6 5 4 3 2 1 5 1 15 2 25 % Weight Loss Figure 4 (cont.). Sustntil up-regultion in liver nd dipose IL-1R2 gene expression, nd in liver IL- 1RA gene expression nd their correltions to percent weight loss. These grphs represent collective description of dt from numerous cohorts of mice tht were either fst (Fsted) for 24 h, or continued to feed d li (Fed). Box plots re representtive of the summted reltive fold chnges in mrna trnsformed to log; the ox outlines the lower 1 st qurtile nd upper 3 rd qurtile, the verticl rs represent the lrgest/smllest non-outlier oservtion, the thick horizontl line represents the men of the reltive fold chnge vlues nd the thin horizontl line is the medin, nd lck circles represent outliers (E) Liver IL-1RA gene trnscription results re summrized nd expressed s log (verge reltive fold chnge ( mrna)) for fed (n=29) nd fsted groups (n=31) *p<.1.(f) Percent weight lost per mouse ws correlted to their dct vlue for IL-1RA in the liver, no significnt correltion, n=31. 25

Rel. Fold mrna Rel. Fold mrna IL-1R2 Rel. Fold mrna IL-1RA Rel. Fold ΔmRNA IL-1R2 Rel. Fold ΔmRNA IL-1RA A 1.4 1.2 B 1.4 1.2 1 1.8.8.6.6.4.4.2.2 Fed Fsted Fed Fsted Fed Fsted Fed Fsted CD11+ CD11+ CD11- CD11- C 1.6 1.4 D 12 1 * 1.2 1 8.8 6.6 4.4.2 2 Fed Fsted Fed Fsted Heptocytes Heptocytes E 1.4 1.2 1.8.6 *.4.2 * 1 h 6 h 1 h 6 h IL-1R2 Liver IL-1RA Figure 5. IL-1R2 nd IL-1RA gene expression on isolted liver cells. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed), nd cells were isolted. (A) RNA ws extrcted from CD11+ nd CD11- cells isolted from liver, IL-1R2 results re expressed s reltive fold chnge ( mrna) compred to control CD11+ Fed group, mens (+ SEM Upper, - SEM Lower); n=8. Brs without common superscript re different (*p <.5). (B) IL-1RA results re expressed s reltive fold chnge ( mrna) compred to control CD11+ Fed group, mens (+ SEM Upper, - SEM Lower); n=8. Brs (C) RNA ws extrcted from heptocytes isolted from liver nd IL-1R2 gene expression ws mesured. Results re expressed s reltive fold chnge ( mrna), mens (+ SEM Upper, - SEM Lower); n=4. (D) IL- 1R2 gene expression ws mesured. Results re expressed s reltive fold chnge ( mrna), mens (+ SEM Upper, - SEM Lower); n=4. *p <.5. (E) Liver ws hrvested from mice nd RNA ws either extrcted immeditely, within 1 hour (1 h), or extrcted 6 hours fter hrvest (6 h). IL-1R2 nd IL-1RA gene expression ws mesured. Results re expressed s reltive fold chnge ( mrna), mens (+ SEM Upper, - SEM Lower); n=4. *p <.5. 26

Tle 3 Effect of fsting on gene expression of IL-1R2 nd IL-1RA in liver nd dipose of knockout mouse models. IL-1R2 IL-1RA Genotype Fed Fsted Fed Fsted IL-1R1 KO Liver (+.33,-.24) Adipose (+.24,-.19) 8.36* (+7.9,-3.82) 18.783* (+37.2,-9.31) (+.8,-.7) (+.14,-.12) 16.271* (+2.6,-1.82).589 (+.87,-.19) TLR-4 KO Liver (+.3,-.23) Adipose (+.28,-.24) 7.65* (+7.71,-3.83) 9.863* (+18.9,-6.48) (+.58,-.37) (+.43,-.31) 16.42* (+1.4,-6.37).759 (+.23,-.17) IL-4 KO Liver (+.19,-.16) Adipose (+.19,-.16) 8.129* (+1.9,-4.66) 22.768* (+35.5,-13.8) (+.3,-.23) (+.1,-.9) 13.678* (+5.4,-3.68).545 (+.24,-.17) Results re expressed s reltive fold chnge in mrna expression ( mrna), mens (+SEM upper, -SEM lower); IL-1R1 KO n=4. TLR-4 KO n=7. IL-4 KO n=4. * p<.5, significnt (One-wy ANOVA) 27

ng/ml Corticosterone ng/ml Corticosterone A 9 8 7 6 5 4 3 2 1 Fed * Fsted B 14 12 1 8 6 4 2 2 4 6 8 1 12 14 16 % Weight Loss Figure 6. Effect of fsting on plsm corticosterone levels in mice. Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed) efore lood ws hrvested. (A) Corticosterone ws mesured nd compred to control group (Fed). Results re expressed s ng/ml corticosterone, mens (±SEM); n=5-7. Significnt min effect (*p <.5). (B) Corticosterone levels in ng/ml were positively correlted to percent weight lost y mouse; n=5-7, p=.5. 28

Rel. Fold mrna IL-1RA LIVER Rel. Fold mrna IL-1R2 LIVER A 6 5 4 3 2 1 B 12 1 Fed Fsted Fed Fsted Fed Fsted None Mifepristone PEG c 8 6 c 4 2 Fed Fsted Fed Fsted Fed Fsted None Mifepristone PEG Figure 7. Effect of inhiiting glucocorticoid receptor ctivity on IL-1R2 nd IL-1RA gene trnscripts in liver. After injecting GCR ntgonist (Mifepristone), vehicle (PEG) or shm scruffing with no injection (None), mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed). (A) Liver IL- 1R2 gene trnscripts were mesured nd compred to set control group (Fed None). Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper, - SEM Lower); n=4-6. Brs without common superscript re different (*p <.5). (B) Liver IL-1RA gene trnscripts were mesured nd compred to set control group (Fed None). Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper, - SEM Lower); n=4-6. Brs without common superscript re different (*p <.5). 29

NEFA (mmol/l) NEFA (mmol/l) A 2.5 * 2 1.5 1.5 Fed Fsted B 2 * 1.5 1.5 Vehicle Plmitic Figure 8. Effect of fsting nd plmitic cid injection on NEFA levels in mice. (A) Mice were sujected to either 24 h fst (Fsted), or continued to feed d li (Fed) efore lood ws hrvested. Plsm NEFAs were mesured nd results re expressed s mmol/l, fed vs. fsted, mens (±SEM); n=3-4. Significnt min effect (*p<.1). (B) Mice were injected with plmitic cid or vehicle control nd 2 h fter injection lood ws hrvested. Plsm NEFAs were mesured nd results re expressed s mmol/l, Vehicle vs. Plmitic, mens (±SEM); n=6. Significnt min effect (*p <.5). 3

Rel. Fold mrna IL-1R2 Rel. Fold mrna IL-1R2 A 9 * 8 7 6 5 4 3 2 1 Vehicle Plmitic B 4.5 4 3.5 3 2.5 2 1.5 1.5 Vehicle Plmitic Vehicle Plmitic WT TLR-4 Figure 9. Liver IL-1R2 gene trnscription levels fter plmitic cid injection. Mice were injected with plmitic cid or vehicle control nd 2 h fter injection liver ws hrvested nd RNA ws extrcted. (A) Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper, -SEM Lower); n=6. Significnt min effect (*p <.5). (B) TLR-4 KO liver IL-1R2 gene trnscripts were compred to control group (WT, Vehicle). Results re expressed s reltive fold chnge in mrna, mens (+ SEM Upper, -SEM Lower); n=8. Brs without common superscript re different (*p <.1). 31

CHAPTER 6 DISCUSSION This project ws le to successfully identify immune regultors impcted y 24 h fst. As evidenced in Tle 1, fsting differentilly regulted trnscripts of cytokines in rin regions compred to liver nd dipose. This decoupling of immune phenotype of the rin indictes tht tissues involved in energy supply, during nutrient deprivtion, my hve the potentil to prevent, tret or excerte disese. CD11 is complement receptor nd hs minly een shown to hve centrl role in host defense in which it enhnces migrtion of leukocytes (ex. mcrophges nd microgli) from peripherl lood to sites of inflmmtion (Ktru et l., 29). Here we show tht CD11 gene trnscription is significntly down-regulted in ll rin regions, wheres CD11 is up-regulted in dipose nd liver in response to fsting. These perturtions my indicte rin preprtory shift to nti-inflmmtion. Different from CD11, TNF-α gene trnscription is consistently down-regulted throughout ll tissues investigted. TNF-α is produced y mcrophges nd is known est for its role in promoting inflmmtory responses, inducing fever nd norexi (McEwn, 22). It is logicl for fsting to downregulte genes responsile for inducing norexi in times of nutrient deprivtion. Mny chnges were seen in trnscription of the IL-1 fmily due to fsting. IL-1α ws downregulted in ll rin regions without significntly ffecting the peripherl tissues, nd IL-1β ws significntly decresed only in the hypothlmus nd dipose. These mrkers indicte generl dmpening of inflmmtion in these tissues. The IL-1 fmily is n importnt prt of the innte immune system nd its lnce is extremely essentil to medite inflmmtion nd tissue dmge. Becuse uncontrolled IL-1 is implicted in the pthogenesis of vriety of inflmmtory conditions nd diseses 32

(Dinrello, Simon, & vn der Meer, 212), mechnisms tightly controlling its potentilly potent ctivity t multiple levels is extremely importnt. For this reson, one wy IL-1 is counterlnced is y two distinct endogeneous inhiitors, IL-1RA nd IL-1R2. IL-1RA trnscription ws shown to e up-regulted in the hippocmpus nd liver, ut down-regulted in the dipose. IL-1R2 ws not up-regulted in ny of the rin tissues, ut ws insted sustntilly significntly up-regulted in oth dipose nd liver (Tle 1). This IL-1R2 up-regultion ws consistent throughout numerous experiments, ut produced extremely vrile fold chnges which we found to e relted to its correltion to percent ody weight loss during the 24 h fst (Fig. 4). IL-1RA is competitive inhiitor tht cn ntgonize the pro-inflmmtory effects vi inding with ner equl vidity s IL-1 to the signling IL-1 receptor (IL-1R1) without cusing ctivtion of the cells (Symons, Young, & Duff, 1995). Although less is understood out IL-1R2, it hs primrily een demonstrted to function s n importnt negtive regultor, cting like decoy receptor inding IL-1 oth on the plsm memrne nd s solule receptor in fluid phse, preventing the interction of IL-1 with IL-1R1 (Arend, 22; Bossu et l., 1995; Ruschmyr, Groves, & Kupper, 1997; Re et l., 1996). As Tle 2 shows, these mrna trnscription up-regultions were confirmed to trnslte into n increse in liver protein of oth IL-1RA nd IL-1R2. Interestingly, these increses in the IL-1 inhiitor proteins were not seen in serum of fsted mice. These results suggest these normlly secreted proteins re remining intrcellulr or remining in the interstitil fluid within the liver. More recently, studies hve elucidted new level of control for IL-1 in which IL-1R2 hs een shown to interct with precursor form IL-1α intrcellulrly (Chng, Su, & Lee, 29; Kwguchi et l., 26), nd this interction ws just confirmed in Cell where it ws found tht IL-1R2 is ound to pro-il-1α intrcellulrly during times of stress-induction, preventing IL-1 from further processing into its mture form. Binding of proil-1α nd IL-1R2 ws le to control nd prevent necrosis-induced sterile inflmmtion (Zheng, Humphry, Mguire, Bennett, & Clrke, 213). 33

Sickness Behviors re defined s dptive, physiologicl nd ehviorl chnges tht occur in response to infection nd inflmmtion (Hrt, 1988). Furthermore, drmtic reduction in food intke is commonly ssocited with illness during loclized nd systemic diseses (Hrt, 1988; Konsmn & Dntzer, 21), ut this disese-ssocited norexi is dependent on the feeding sttus, s it hs een shown tht the norexic effect is ttenuted when restricting food intke prior to IL-1β or turpentineinduced sickness (Kent, Rodriguez, Kelley, & Dntzer, 1994; Lrson, Romnoff, Dunn, & Glow, 22; Lennie, 1998; Mrosovsky, Molony, Conn, & Kluger, 1989). Acute strvtion hs lso een shown to ttenute the fever response to LPS in rts (Inoue et l., 28; Shido, Ngsk, & Wtne, 1989). Here we showed (Fig.2) tht fsting prior to peripherl IL-1B chllenge ws le to ttenute weight loss nd IL-1β- induced hypoglycemi t 2 h post injection. Fsted nimls were lso resistnt to reduction in locomotor ctivity nd socil ehvior. Proinflmmtory cytokines, such s those included in the IL-1 fmily, cn ct t the periphery to induce its own synthesis nd the synthesis of other cytokines llowing it to potentite or oppose its effects, nd these signls cn induce the expression of trnscripts of inflmmtory cytokines in the rin in response to peripherl stimuli (Sims nd smith, 21). Here, we uncover link etween dietry restrictions effect eing tht of up-regultion of IL-1 fmily ntgonistic meditors within the liver, which then lunts the centrlly-medited effects of induced peripherl chllenge. The importnce of the liver in trnsmitting cytokine signls from the periphery to the rin fter i.p.- injected inflmmtory stimuli hs een shown previously y those investigting the effects of sudiphrgmtic vgotomy. The vgus nerve, y which the liver is innervted, hs n importnt role in trnsmitting peripherl cytokine signls to the rin s evidenced y rodents injected i.p. with IL-1β, in which vgotomy ws le to sustntilly ttenute ny increse in IL-1β mrna in the rin, nd lock ehviorl effects of IL-1β injected i.p., wheres IL-1β mrna increse in liver ws not ffected nd the ehviorl effects were not different if injected i.v. or i.c.v. (Bluthe, Michud, Kelley, & Dntzer, 1996; Bluthe, Michud, Kelley, & Dntzer, 1996; Hnsen, Tishi, Chen, & Krueger, 1998). Although we did not 34

see n ttenution of IL-1α or IL-1β gene trnscription 2 h fter IL-1β peripherl chllenge in ny rin regions (Fig. 3), we did oserve significnt increse in IL-1R2 messge in fsted mice chllenged with IL-1β in the cortex, nd we lso sw significnt decrese in fsted mice IL-1RA in the hypothlmus 2 h fter IL-1B chllenge. Becuse of the nture of IL-1RA inhiiting IL-1, sources hve suggested tht incresed IL-1RA cn ctully e mrker of severity of inflmmtion rther thn expression of ntiinflmmtion (Pihljmki et l., 212). This implies for our results tht the decrese in IL-1RA in the hypothlmus is indictive of lesser inflmmtory process occurring in the fsted mice compred to fed. This dt suggests tht the liver, which we hve now found to e slly skewed nti-inflmmtory vi cute food deprivtion, plys mjor role in communicting inflmmtory signls to the rin fter n i.p. immune chllenge. When trying to elucidte mechnism s to how fsting up-regultes these nti-inflmmtory cytokines, we tried to look t which cells within the liver were expressing the messges. Fig. 5 shows tht we were unle to find significnt up-regultion of IL-1R2 or IL-1RA in either CD11+ or CD11- cell frctions, however, when we look t cellulr frctions of heptocytes, we were le to uncover IL-1RA up-regultion which is consistent with current findings (Gy, Gigley, Sipe, Arend, & Fntuzzi, 21). It ws then discovered tht ecuse processing necessry to isolte the cells of the liver took up to 6 hours for PCR(without fixtion), IL-1R2 ws rpidly degrded due to IL-1R2 trnscripts short hlf-life of only 11 min. (Zeisel et l., 211), nd this degrdtion is the prole cuse of our inility to see IL- 1R2 up-regultion in ny cellulr frction. This ws confirmed y setting out piece of liver tissue for 6 h (the time it tkes to isolte cells) nd compring it to piece of liver tht ws immeditely extrcted, in which our results show fter 6 h the IL-1R2 messge is prcticlly undetectle. 35