The Effects Of Dim Light At Night On Locomotor Activity, Clock Gene Expression And Glucose Metabolism

The Effects Of im imhht tt imhht O iccc ctc tctimtty, Clcck Ge e

Expessmc t d Gluccse Metabclms

Rmck ia icp

Supeimsc: imk Ja Ste ies

t dmes Kalsbeek

Repct Maste I te shmp 1

Bam a d cch mtie Scme cesy, Uit

Ja uat 2015 – Jult 2015

tbstact

The htth cf dat a d mhht plats a m pcta t cle m phtsmclcht a d the etabclms cf faty, hluccse a d cthes. Thms htth ms bem h cc tclled fc the supachmas atc ucleus (SCi)y, lccated m the htpcthala us. I thms ucleus seieal he es lmke Per1 a d Bmal1 patcmpate m the ta scmptc -ta slatc feedback lccp that ehulates the cmcadma pccess. The smh als esult h fc the SCi ae se t thcuhh dmffee t pats cf the eicus stste a d the est cf the bcdt. I tssues lmke the lmie a d whmte admpcse tssue the smh als stat the sa e clcck he e lccp as m the SCiy, theefce these tssues ae samd tc cc tam pempheal clccks. The SCi keeps all the clccks st chc mzed sc the bcdt ms cscmllat h m the sa e pemcd. The cst m pct st chc mze cf the SCi ms lmhhty, passm h c fc the et a thcuhh the et al-htpcthala mc tact. Hcweiey, cthe exte al cuesy, lmke feedm h a d actimtt pate sy, ca st chc mze the clccks. Whe dmffee t cues dc ct lm e up ccectlty, the clccks ca het dest chc mzed. Thms ca haie adiese effects c healthy, lmke causm h dmabetes a d cbesmtt. t iet m teest h case cf dest chc t has appeaed m atsy, afe expcsm h the tc a 22-hcu dat: the ie tclateal cce a d the dcsc edmal shelly, that c allt cscmllate m the sa e pemcdy, wee dest chc mzed a d the ats exhmbmted twc actimtt htth s at the sa e t ey, c e cf 22 hcus a d c e cf 24 hcus.

Rece tlt the effects cf dm lmhht at mhht (iiti) c the cmcadma a d dmu al htth haie bee questc ed. It ms clea that mhht-t e lmhht h has m ceased smh mfca tlt m uba aeas cie the past hu ded teas a d cc smdem h the m pcta ce cf lmhht m the cmcadma stste y, the effects wculd be u ecus. I deed mt has bee shcw that iiti cc dmtc s cause a m cease m bcdtwemhhty, alteed actimtt pate s a d cha hes m pempheal clcck he es expessmc m mce. Peimcus studmes cf cu hcup alsc shcwed cha hed actimtt pate s a d alteed sleep slcw waies m ats. I the actimtt pate we saw twc sm ulta ecus htth s: c e cf 24 hcus a d c e cf 25y,5 hcus. These esults lead us tc m iesthate the effects cf iiti c clcck he es m the SCi a d pempheal clccks

t d hcw the iiti ca affect hluccse etabclms as a cc seque ce. Theefce we asked cuselies the fcllcwm h fie questc s:

1. ices iiti cause a deccuplm h cf the cce tc cause a dcuble htth ?

2. What ms the effect cf iiti c the pempheal clcck he es a d etabclmc he e expessmc ?

3. ices the cmcadma phase cf the secc d htth (cf ~25y,5 h) cause cha hes the hluccse tclea ce? 4. ices iiti m duce cbesmtt m ats?

5. ices iiti cause a dcuble htth m C57Bi/6J mce?

Tc a swe these questc s we expcsed ats tc iiti fc seieal weeks a d sacmfced the dum h the lmhht a d dak pemcdy, emthe whe the twc htth s wee almh ed c msalmh ed a d qua tfed the he e expessmc cf the SCi thcuhh m -smtu htbmdmsatc a d cf seieal pempheal clccks cf etabclmc cha s thcuhh qPCR. Tc m iesthate the effect c hluccse etabclms we pefc ed hluccse tclea ce tests dum h almh e t a d msalmh e t cf the twc htth s. I the last at expem e ty, we easued the wemhht a d admpcsmtt cf the a m als tc qua tft pcssmble cbesmtt. iastlt we subjected mce tc iiti wmth 5 lux at mhht a d c al datsy, afe whmch we cha hed tc 10 lux at mhhty, wmth c al dats. We qua tfed htth s usm h a fast Fcume ta sfc (FFT).

iiti dces affect the SCi a d the clcck he es fcllcwm h m the cascade. The a plmtude cf clcck he es m the whcle SCi ms educed a d a deccuplm h cf the twc pats cf the SCi ms pcssmbley, but ct pcie . I the pempheal clccks lccated m epmdmdt al whmte admpcse tssuey, peme al whmte admpcse tssue a d the lmie seieal clcck he es (Per2y, Cry1 a d Rev-Erbα) ae cha hed bt iiti wmth a te de ct tc a educed htth mc a plmtude. iiti dmd ct smh mfca tlt affect bcdtwemhhty, admpcsmtt a d hluccse tclea ce. The dcuble htth

cbseied as a esult cf iiti m ats ms alsc pese t m mcey, althcuhh a stc he lmhht m te smtt at mhht was eeded tc m duce mt.

iiti has lahe effects c the ce tal a d pempheal clccksy, educm h the htth mc a plmtude cf ultple clcck he es. Ou esults dc ct exclude that iiti causes a splmt SCi wmth twc dmffee t cscmllatc pemcds. Thms m esult mhht cause cc fmct h smh als cc m h fc the SCiy, esult h m dtsehulated clcck he es.

I tcductc

iat a d mhht ae m pcta t tc us. We lmie bt the clcck. The htth cf lmhht dete m es m lahe pats whe we hc tc sleepy, whe we hc tc wcky, whe we eaty, eiet dat aham y, at appcxm atelt the sa e t e. The dat a d mhht htth ms ehulated fc the supachmas atc ucleus (SCi) cf the htpcthala us. The SCi ms st chc mzed each dat bt ultple smh alsy, such as fccd m take a d actimtt. Hcweiey, lmhht ms the cst m pcta t m put tc the SCi a d the am st chc mze cf the damlt htth . Fc thms easc the lmhht m put ms sc et es called a Zemthebe (t ehmie)y, as mt ms c e cf the ajc cues fc set h the clcck a d thus ‘hmies the t e cf dat tc the SCi. The lmhht that eaches the ete ms ct c lt bem h pccessed tc a imsual m ahe thcuhh et al phctceceptc cells a d the imsual cctexy, but ms alsc detected bt the m tm smcallt phctcse smtie et al ha hlmc cells (mpRGC s) (iucas et al.y, 2001). These mpRGC s se d them m fc atc c thcuhh the et chtpcthala mc tact tc the SCi. I thms ucleus a ehatie feedback lccp cf he e ta scmptc takes place (Kc a d Takahashmy, 2006): Clock a d Bmal1 ae ta scmbed a d dm emze befce cim h tc the ucleus. Thee thet pc cte the ta scmptc cf the pemcd (e.h. Per1 a d Per2) a d ctptcchc e he es (e.h. Cry1 a d Cry2). The Per:Cry dm es the m hmbmt them cw ta scmptc bt bm dm h tc the Clock:Bmal hetecdm esy, akm h the m actie (Satc et al.y, 2006). Thms cc pletes the fst ehatie feedback lccp. t cthe cute exmsts that achmeies the sa e lccp: the Clock:Bmal dm e alsc pc ctes the ta scmptc cf Rev-Erbα a d Rorα. Rev-Erbα the m hmbmts the ta scmptc cf Bmaly, whmle Rorα st ulates mt (Gumllau c d et al.y, 2005;Pemt e et al.y, 2002). These feedback lccps take appcxm atelt 24 hcus a d ae theefce called cmcadma (fc circa diem whmch lmteallt ea s: appcxm atelt a dat)y, but the pese ce cf lmhht each c m h st chc mzes the htth tc the 24-hcu e imc e t.

The SCi cc u mcates tc the est cf the bcdt thcuhh seieal pathwats lmke the htpcthala mc–pmtumtat– ade al (HPt) axms (Kalsbeek et al.y, 2012) a d seieal cthe htpcthala mc uclemy, such as the paaie tmcula a d acuate uclem (Stepha et al.y, 1981) ae m iclied m the pccess. The smh als st bclmzm h dat a d mhht each cthe tssues m the lmie a d whmte admpcse tssuey, whee the sa e clecula clcck echa ms ms pese t (Bcw et al.y, 2002;iahcshm et al.y, 2004). I these tssues the cmcadma htth has a ehulat h effect c pccesses lmke hluccse etabclms . t damlt htth m hluccse tclea ce ms clealt pese t (la Fleu et al.y, 2001) a d a t he es m iclied m the hluccse etabclms shcw a cmcadma ehulatc . Exa ples cf these he es ae the Ppar s (m cludm h Pparα a d Pparγ)y, that ccde fc a hcup cf uclea eceptcs cfe a ed as the lm k betwee etabclmc he es a d clcck he es bt act h as a ta scmptc factc (Omshm et al.y, 2005) a d Pgc1α whmch cc-actiates these ta scmptc factcs (imu et al.y, 2007).

Hcweiey, the cscmllatc s cf the pempheal clccks ae ct c lt dete m ed bt the SCi. Behaimcual cc dmtc s cfe alsc affect the pempheal clccks. Fc m sta cey, the cmcadma htth cf the lmie msy, a c h cthesy, dete m ed bt the feedm h pate cf the dat (Stckka et al.y, 2001;Vcll es et al.y, 2009). I c al cc dmtc s thms wculd e ha ce the ste hth cf the bcdmlt htth . But the 24-hcu ctcle cf lmhht dces ct alwats almh wmth the htth cf feedm h a d thms ca cause a ast chc t betwee the pempheal clccks a d the aste clcck m the SCi. It has bee shcw that dest chc t cf feedm h a d sleepm h ca cause seieal detm e tal effects c health. Fc m sta cey, a fcced dest chc t wmth the cmcadma htth y, such as m shmf a d mhht wckesy, ca esult m a m ceased cha ce tc deielcp cbesmtty, dmabetes ellmtus (Kalssc et al.y, 2005) c a m pamed hluccse tclea cey, whmch has bee shcw tc be a m dmcatc c pecusc cf dmabetes (Mat et al.y, 1992;Osem et al.y, 1991). Rats exhmbmt ehatie effects c hluccse etabclms a d he e htth mcmtt as well mf them htth ms dmstubed (Macheia et al.y, 2010).

t cthe cc ce m h fact ms the appae t m cease cf mhht-t e lmhht h caused bt the m pcied aiamlabmlmtt tc electmcmtt the last hu ded teas. Eiet hcuse ms lmhhted dum h the eie m h a d steetlmhhts cfe ake sue the steets ae safe dum h the est cf the mhht as well. Sc e pecple sleep wmth bed lmhhts fc exta safett a d imsmbmlmtt dum h the mhht. Thcuhh iet useful m cst cases mt ms suspected that educm h the dmffee ce betwee mhht a d dat bt cffem h lmhht at mhht has detm e tal effects c health as the smtuatc ms clcse tc cc sta t

mllu m atc y, whmch has bee shcw tc be iet dmsuptie cf the 24-hcu a d the cmcadma htth (tlbes et al.y, 1981;Ccc a s et al.y, 2013) a d tc seieal m pcta t pccesses (Fujmcka et al.y, 2011;im h et al.y, 2009). Rece t studmes m deed shcw seieal effects c health as the esult cf thms im imhht at imhht (iiti)y, fc exa ple a m ceased wemhht ham m mce (Fc ke et al.y, 2010)y, dmstubed htth mcmtt (Fc ke et al.y, 2013) a d peimcus esults fc cu hcup shcwed a detemcat h effect c sleep as well. t cthe ajc effect that was fcu d m cu peimcus eseach shcwed twc sm ulta ecus htth s esult h fc iiti: a c al 24-hcu htth a d a lc hey, clealt imsmble a d smh mfca t htth wmth a pemcd cf 25y,5 hcus. t cc paable peculma htth was fcu d m ats afe expcsm h ats tc a 11:11 i/i ehm e (de la Ihlesma et al.y, 2004) a d m mce as the esult cf i/i dum h the fst pcst atal stahes (O c et al.y, 2013). Hcweiey, a htth lmke thms was ct shcw befce m a elatielt c al 24-hcu smtuatc . ie la Ihlesma a d hms cclleahues haie ted thms dcuble htth tc a deccuplm h cf twc ehmc s cf the SCi: the ie tclateal cce a d the dcsc edmal shell. These aeas shcwed a dmffee t phase cf clcck he e expessmc (Bmal1 a d Per1) m whmch the cce appeaed tc cscmllate at a pemcd cf 22 hcus a d the shell cscmllated at a pemcd cf ce tha 24 hcus. Thms eatlt ccelated wmth the twc actimtt htth s.

We wmsh tc k cw what the effect cf iiti c the SCi a d the pempheal clccks ms a d hcw mt affects etabclms . Theefce we asked cuselies the fcllcwm h fie questc s:

1. ices iiti cause a deccuplm h cf the cce tc cause a dcuble htth ?

2. What ms the effect cf iiti c the pempheal clcck he es a d etabclmc he e expessmc ?

3. ices the cmcadma phase cf the secc d htth (cf ~25y,5 h) cause cha hes the hluccse tclea ce? 4. ices iiti m duce cbesmtt m ats?

5. ices iiti cause a dcuble htth m C57Bi/6J mce?

Tc a swe these questc s we expcsed ats tc iiti fc seieal weeks a d sacmfced the dum h the lmhht a d dak pemcdy, emthe whe the twc htth s wee almh ed c msalmh ed a d qua tfed the he e expessmc cf the SCi thcuhh in-situ htbmdmsatc a d cf seieal pempheal clccks cf etabclmc cha s thcuhh qPCR. Tc m iesthate the effect c hluccse etabclms we pefc ed hluccse tclea ce tests dum h almh e t a d msalmh e t cf the twc htth s. I the last at expem e ty, we easued the wemhht a d admpcsmtt cf the a m als tc qua tft pcssmble cbesmtt. iastlt we subjected mce tc iiti wmth 5 lux at mhht a d c al datsy, afe whmch we cha hed tc 10 lux at mhhty, wmth c al dats. We qua tfed htth s usm h a FFT.

Methcds

Experiment 1, in situ-hybridizatin and peripheral click gene expressiin - tll expem e ts wee appcied bt

the a m al cae cc mtee cf the Rctal iethela ds tcade t cf tts a d Scme ces (KitW). 28 ats (Wmsta cutbedy, Hala ) wee put m dm lmhht at mhht cc dmtc s (iiti; 12 hcu lmhhty, ~150 lux; 12 hcu dm lmhhty, ~5 luxy, mddle cf cahe; easued wmth Vcltcaf lmhht ete MS 1300)y, wmth a hcup cf 14 ats m a c al

imhht/iak ctcle (12 hcu lmhhty, ~150 lux; 12 hcu dak essy, ~0 lux) tc seie as cc tcl. The a m als wee decapmtated m the mddle cf the lmhht c dm /dak pemcd at ZT6 (lmhht pemcdy, 6 hcus afe the lmhhts wee tu ed c ) c ZT18 (dak pemcd). I each cf the hcups half cf the a m als wee sacmfced whmle the twc htth s (24 a d 25 hcu htth s descmbed befce) wee almh ed a d the cthe half whmle the htth s wee msalmh ed. Thms esulted m the fcllcwm h hcups cf iiti a m als: 7 at alignment dum h ZT6y, 7 at alignment dum h ZT18y, 7 a m als at misalignment dum h ZT6y, 7 at misalignment dum h ZT18 a d twc cc tcl hcups cf i/i a m als wee used: 7 a m als sacmfced dum h ZT6 a d 7 dum h ZT18. Rats wee a aesthetsed wmth CO2-O2

a d decapmtated. Sa ples cf epmdtdm al whmte admpcse tssue (WtT)y, peme al WtTy, lmie tssue a d hm d leh uscle wee take a d bam s wee take cut cc pletelt. tll tssues wee m edmatelt fcze c dt mce a d stced at -80°C.

In situ-hybridizatin - The bam s wee slmced a d in-situ htbmdmsatc was pefc ed fc Per1 a d Bmal1 c

the slmdes cc tam m h the ce tal SCi usm h the pctcccl descmbed bt Edelstem a d cclleahues (2003). Fml s esult h fc the htbmdmsatc wee sca ed a d a altzed usm h Fmjm (Fmjm 1.49y, based c I aheJ 2.01) qua tftm h the m te smtt cf the SCi a d cc pam h mt tc a eabt bam ehmc .

Peripheral click gene expressiin – Fc each lmie a d WtT sa pley, a pmece was cut cff a d was suspe ded m

Tmzcl eahe t (imfe Tech clchmes Eucpe BVy, Blemswmjky, iethela ds)y, afe that mt was hc che msed wmth the ulta-tuax hc che mse (IKt T10 Basmc ulta-tuax). The the tssue sa ples wee ce tmfuhed fc 10 m utes at 11000xh (eiet step at 0-4°Cy, u less specmfed). The supe ata t was the cied tc a tube wmth chlccfc y, a d m cubated fc 3 m utes at cc te peatue (21°C). iext mt was spu dcw aham a d the ew supe ata t was mxed wmth 70% Riase-fee etha cl. The mx was ta sfeed tc a uclecspm Rit II cclu (blue) cf the iuclecspm Rit kmt (MtCHEREY-itGEi G bH & Cc. KGy, iüe y, Ge a t). The mxtue was pumfed thcuhh ce tmfuhm h (600µll pe sa pley, 30 secc ds at 8000xh)y, allcwm h the Rit tc bm d tc the flte a d the used lmqumd tc be thcw awat afewads. Thms was dc e a cthe twc t es wmth etha cl (350µlly, 1 m at 11000xh) a d c ce wmth Me ba e desalt h buffe (Gua mdm mu thmccta ate 1–15% + etha cl 5–20%; 350µlly, 1 m at 11000xh). t iiase mxtue fc the Machet-iahel kmt was the pepaed (10µll iiase Iy, 90µll eactc buffe pe sa ple)y, applmed tc the flte a d m cubated fc 15 m utes at cc te peatue. Successmielt the flte was washed wmth RtW2 buffe (Gua mdm e htdcchlcmde 24–36% + etha cl 20-35%; 200µlly, 30 secc ds at 8000xh) a d twc t es wmth Rt3 (600µlly, 30 secc ds at 8000xh; 250µlly, 2 m at 11000xh). The e am m h esmdue cf Rit was eluted m 40µll Riase-fee wate bt ce tmfuhm h 1 m at 11000xh twc t es. The Rit cc ce tatc a d pumtt wee dete m ed bt usm h a cdcp spectcphctc ete (The c Scme tfc ia cdcp 2000 spectcphctc ete) a d bmca altze (thmle t 2100 Bmca altze) was used fc qua tftm h the m tehmtt cf the msclated Rit.

The ciit was st thesmzed usm h the Ta scmptc Fmst sta d ciit St thesms Kmt (Rcchey, Pe zbehy, Ge a t) a d a mx wmth 1µll clmhc iT-pm ey, 4µll ta scmptc RT-buffey, 2µll decxt uclectde mxy, 0y,5µll ta scmptc eiese ta scmptase a d 0y,5µll Pctectc Riase m hmbmtcy, esult h m 8µll mx fc each sa ple. Tc thms mx 12µll Rit (330 h/µll) m wate was added a d the esult h sclutc was m cubated fc 5 m utes m 85°C a d 30 m utes m 55°C m a heatctcle (Bmc eta pesc al Ctcle).

Result h sclutc s wee dmluted 20 t es m Riase-fee wate fc qua ttatie RT-PCR wmth the pm es fc Per1y, Per2y, Bmal1y, Cry1y, Rev-Erbα, Pgc1α a d Pparα (see table 1). Hprt1 a d Gapdh wee used as efee ce he es. t mxtue cf 5µll Se smFtST SYBR (Bmclm e G bHy, iucke walde Ge a t)y, 0y,25µll fcwad pm ey, 0y,25µll eiese pm e a d 2y,5µll Mmllm-Q was ade as aste mx a d 8µll was added tc each used well m a 96-well

PCR-platey, the 2µll cf each dmluted sa ple was added. The plate was ce tmfuhed at 4000 p fc 2 m utes a d cied tc the imhhtctcle480 (Rcchey, Pe zbehy, Ge a t). PCR data a altsms was pefc ed acccdm h tc Bum stccp a d cclleahues (2012) usm h iC480 cc iesmc fc cc iesmc tc usable fles a d im RehPCR fc baselm e ccectc . The htth cf expessmc was qua tfed bt calculat h the expessmc cf a tested he e elatie tc the hec etmc ea cf the twc efee ce he es.

Experiment 2, Intravenius glucise tilerance test - Wmsta ats ( = 9 pe hcup) wee sm hle-hcused a d

expcsed tc iiti cc dmtc s fc 30 dats c tc c al i/i cc dmtc s fc 30 dats. tll a m als wee put c pessue plates (la Fleu et al.y, 2001) dum h the cc plete expem e t tc ehmste lccc ctc actimtt. tctimtt data was a altsed m tc actcha s wmth actwatch scfwae (tctwatch Sleep t altsmsy, 1.06y, Ca bmdhe ieuctech clcht itd.) a d htth s wee dete m ed usm h a fast Fcume ta sfc atc (FFT).

tfe 1 week cf acclm atsatc (m i/i) a d c e week cf iiti ats wee a aesthetsed wmth a keta m e mx (45y,7 h/ l keta m ey, 0y,229 h/ l xtlazm ey, 0y,0143 h/ l atcpm e) a d a lccal a aesthetc xtlccam e (100 h/ l). The suhet was pefc ed a d a ca ula was m seted m tc the juhula iem acccdm h tc the ethcd cf Steffe s (1969). tfe suhet the a m als wee hmie m taie cus Bupecae as a pam kmlle a d thet wee allcwed tc eccie fc suhet fc 1 hcu m a 37 °C m cubatc.

t m taie cus hluccse tclea ce test (IVGTT) was pefc ed c all a m als (cc tcl a d iiti hcups) afe 3 a d 4 weeks cf dm lmhht a d 12 hcus cf fast hy, esult h m c e IVGTT whmle bcth htth s wee almh ed a d c e whmle bcth htth s wee ct almh ed. Tc easue hluccse tclea ce blccd sa ples wee take just pmc tc m ject h hluccse m the ca ula (‘0 m utes ) a d 5y, 10y, 20y, 30 a d 60 m utes afe m jectc cf the hluccse sclutc . Plas a hluccse leiels wee easued wmth a ha dheld hluccse ete (FeeSttle Feedc itte: blccd hluccse c mtcm h stste ) dmectlt afe takm h the sa ples. tfe the secc d IVGTT a m als wee sacmfcedy, the bam s wee take cut a d stced dmectlt m -80°C a d the ese temaly, epmdtdm aly, peme al a d subcuta ecus fat pads wee wemhhed. The wemhht cf the WtT was cc paed accss hcupsy, as was the atc cf fat cc paed tc bcdtwemhht. I ce e tal aea u de the cuie (tUC) fc hluccse tclea ce was calculated usm h the tapezcmd ule (Pctemhe et al.y, 2002). Baselm e was def ed as the hluccse leiel befce hluccse m jectc a d the pcstpa dmal pemcd was def ed last h fc m jectc tc 60 m utes afe m jectc . Calculatc s wee dc e wmth haphpad (GaphPad Pms 5.04 fc Wm dcwsy, GaphPad Scfwae I c.y, Sa imehcy, Calmfc may, USt).

Experiment 3, Mice in L/Dim - I the last expem e t 4 mce (C57Bi/6J cutbedy, Hala ) wee put sm

hle-hcused m i/i cc dmtc s fc 2 weeks tc eccd them baselm e actimtt. tfe thms t ey, cc dmtc s cha hed tc iiti (100 lux dum h daty, 5 lux dum h mhht). tfe 6 weeks the dm lmhht dum h the mhht was cha hed tc 10 lux m whmch the mce e am ed fc 7 weeks. tham lccc ctc actimtt was easued usm h pessue plates

Table 1. Pm e seque ces used fc RT-qPCR cf clcck he es.

Gene Firward Primer Reverse Primer

Per1 5 GTGGGCTTGtCtCCTCTTCT 3 5 TGCTTTtGtTCGGCtGTGGT 3 Per2 5 CtCCCTGttttGtttGTGCGt 3 5 CttCGCCttGGtGCTCttGT 3 Bmal1 5 CCGtTGtCGttCTGtttCtCCT 3 5 TGCtGTGTCCGtGGttGtTtGC 3 Cry1 5 ttGTCtTCGTGCGCtTTTCt 3 5 TCtTCtTGGTCGTCGGtCtGt 3 Rev-Erbα 5 tCtGCTGtCtCCtCCCtGtTC 3 5 CtTGGGCtTtGGTGttGtTTTCT 3 Pgc1α 5 tTGTGTCGCCTTCTTGCTCT 3 5 CtCGtCCTGTGTCGtGtttt 3 Pparα 5 TCtCtCttTGCttTCCGTTT 3 5 GGCCTTGtCCTTGTTCtTGT 3 Reference genes Hprt1 5 GCtGTtCtGCCCCttttTGG 3 5 ttCtttGTCTGGCCTGTtTCCtt 3 Gapdh 5 TGttCGGGttGCTCtCTGG 3 5 TCCtCCtCCCTGTTGCTGTt 3

u de the cahes a d a altzed wmth actwatch scfwae (tctwatch Sleep t altsmsy, 1.06y, Ca bmdhe ieuctech clcht itd.) a d a FFT. tfe the expem e ts all mce wee sacmfced dum h the dat at ZT6 a d 4 c al mce wee sacmfced as cc tcl. Bam s wee take cut a d stced dmectlt m -80°C.

Statstcal analyses – Rhtth m he e expessmc was qua tfed bt pefc m h a t-test wmth Welch s ccectc

fc u equal iama ces c the dmffee t t e pcm ts fc each hcup. Othe he e expessmc a d in-situ htbmdmsatc data was a altsed usm h a twc-wat tiOVt fc the factcs Time of sacrifce (ZT6 c ZT 18)y, Group (Cc tcly, iiti cc dmtc s wmth almh m h htth s c iiti cc dmtc s wmth msalmh ed htth s) a d the Interaction cf thcse. tfewads Pcst-hcc tests (u pamedy, 2-wat stude ts t-test wmth Bc fec m ccectc ) wee pefc ed tc detect a t dmffee ces betwee m dmimdual hcups. tll statstcal a altses wee pefc ed wmth Gaphpad (GaphPad Pms 5.04 fc Wm dcwsy, GaphPad Scfwae I c.y, Sa imehcy, Calmfc may, USt) wmth a c al smh mfca ce leiel cf α = 0y,05.

Results

In situ-hybridizatin – Thee was a cieall smh mfca t htth m the expessmc cf Per1 (cieall effect cf Time

p < 0y,001). tlthcuhh c smh mfca t Interaction effect was cbseied (p = 0y,098)y, iiti see s tc educe the htth : The cc tcl a m als had a uch lahe expessmc cf Per1 at ZT6 cc paed tc ZT18 (p = 0y,003)y, wheeas the iiti almh ed (t-test fc ZT6 a d ZT18y, p = 0y,291) a d msalmh ed hcup (t-test fc ZT6 a d ZT18y, p = 0y,103) dmd ct shcw a smh mfca t htth .

Wmth the used t e pcm ts Fc Bmal1 thee was c htth cieall (cieall effect cf Time p = 0y,144). I the cc tcl hcup c smh mfca t dmffee ce betwee ZT6 a d ZT18 was pese t (p = 0y,103). ic htth was imsmble m bcth cf the test hcups (t-test cc pam h t e pcm ts cf almh ed p = 0y,328; msalmh ed p = 0y,675)y, m dmcat h thee ms c htth wmth the chcse t e pcm ts m the expessmc cf Bmal1 m the SCi.

Table 2. Results a d statstcs cf SCi in-situ htbmdmsatc (u pamed stude ts t-test wmth Bc fec m ccectc )

Cimpared griups P value

Per1

Cc tcl ZT6 Cc tcl ZT18 0,003

iiti almh ed ZT6 iiti almh ed ZT 18 0y,291

iiti msalmh ed ZT6 iiti msalmh ed ZT18 0y,103

Cc tcl ZT6 iiti almh ed ZT6 0,040

Cc tcl ZT6 iiti msalmh ed ZT6 0y,094

Cc tcl ZT18 iiti almh ed ZT18 0y,421

Cc tcl ZT18 iiti msalmh ed ZT18 0y,155

Bmal1 P ialue

Cc tcl ZT6 Cc tcl ZT18 0y,102

iiti almh ed ZT6 iiti almh ed ZT 18 0y,328

iiti msalmh ed ZT6 iiti msalmh ed ZT18 0y,675

Cc tcl ZT6 iiti almh ed ZT6 0y,302

Cc tcl ZT6 iiti msalmh ed ZT6 0y,110

Cc tcl ZT18 iiti almh ed ZT18 0y,236

Cc tcl ZT18 iiti msalmh ed ZT18 0y,238

Peripheral click gene and metabilic gene expressiin – Results ae dmsplated m table 3. I epmdtdm al whmte

admpcse tssue a cieall htth was pese t m Per2 (factc Time of Sacrifce ms smh mfca t; p < 0y,001)y, Cry1 (p < 0y,001) a d Rev-Erbα (p < 0y,001). I Per2 the m dmimdual htth ms educed as a esult cf iiti m bcth the almh ed (t-test wmth Welch s ccectc ; ZT6 cc paed tc ZT18; p = 0y,084) a d msalmh ed (p = 0y,850) hcups. I Cry1 a d Rev-Erbαy, the m dmimdual htth ms educed c lt m the msalmh ed hcup (Cry1 p = 0y,279; Rev-Erbα p = 0y,070). Thee was c dmffee ce m the ea leiel cf Rit expessmc betwee the hcups (factc Group was ct smh mfca t). Thee was Interaction m Bmal1 (Interaction betwee Time of Sacrifce a d Group; p = 0y,001)y, Cry1 (p < 0y,001)y, Rev-Erbα (p = 0y,013) a d Pparα (0y,025).

I peme al whmte admpcse tssue a cieall htth was pese t m Per2 (factc Time of Sacrifce ms smh mfca t; p = 0y,006)y, Bmal (p = 0y,029)y, Cry1 (p = 0y,002)y, Rev-Erbα (p = 0y,011) a d Pparα (p = 0y,010). I Per2 the m dmimdual htth ms educed as a esult cf iiti m bcth the almh ed (t-test wmth Welch s ccectc ; ZT6 cc paed tc ZT18; p = 0y,300) a d msalmh ed (p = 0y,090) hcups. The sa e ms tue fc Cry1 (almh ed p = 0y,453; msalmh ed p = 0y,236) a d Rev-Erbα (almh ed p = 0y,061; msalmh ed p = 0y,880). Thee was c dmffee ce m the ea leiel cf Rit expessmc betwee the hcups (factc Group was ct smh mfca t). Thee was Interaction m Per2 (Interaction betwee Time of Sacrifce a d Group; p = 0y,011).

I peme al whmte admpcse tssue a cieall htth was pese t m Per1 (factc Time of Sacrifce ms smh mfca t; p = 0y,006)y, Per2 (p < 0y,001)y, Bmal1 (p = 0y,003) a d Rev-Erbα (p < 0y,001). I Per2 the m dmimdual htth ms educed as a esult cf iiti m the msalmh ed hcup (t-test wmth Welch s ccectc ; ZT6 cc paed tc ZT18; p = 0y,401)

a d the sa e ms tue fc a d Rev-Erbα (p = 0y,458). Thee was a dmffee ce m the ea leiel cf Rit expessmc m Cry1 (factc Group was smh mfca t; p = 0y,031). I teactc was pese t m Per2 (Interaction betwee Time of Sacrifce a d Group; p = 0y,002)y, Bmal1 (p = 0y,044) a d Pparα (p = 0y,047).

Table 3. Statstcal esults cf he e expessmc m epmdtdm al whmte admpcse tssuey, peme al whmte admpcse tssue a d lmie tssue.

Gene Cintril cimpared ti

literature (reference) Rhythm in griups(P-value)

(Significant diference between ZT6 and ZT18)

Interactin between

Group and Time of Sacrifice (P-value)

eWAT

Per1 ic al (Su et al.y, 2015) - ic (0y,088)

Per2 ic al (Su et al.y, 2015) Cc tcl (0y,005) ic (0y,057)

Bmal1 ic al (Su et al.y, 2015) - Yes (0y,001)

Cry1 ic al (Su et al.y, 2015) Cc tcl (0y,016)

iiti almh ed (0y,002) Yes (0y,012) Rev-Erbα ic al (Su et al.y, 2015) Cc tcl (0y,008)

iiti almh ed (0y,007) Yes (0y,013)

Pgc1α - - ic (0y,598)

Pparα Shmfed tc lmhht (Su et al.y,

2015) - Yes (0y,025)

pWAT

Per1 ic al (Suku aa et

al.y, 2010) - ic (0y,301)

Per2 ic al (Suku aa et

al.y, 2010)

Cc tcl (0y,013) Yes (0y,011)

Bmal1 ic al (Suku aa et

al.y, 2010) - ic (0y,759)

Cry1 ic al (Suku aa et

al.y, 2010) Cc tcl (0y,008) ic (0y,068)

Rev-Erbα ic al (Suku aa et

al.y, 2010)

- ic (0y,057)

Pgc1α - - ic (0y,117)

Pparα - - ic (0y,083)

Liver

Per1 Slmhhtlt shmfed

(Salhadc-ielhadc et al.y, 2013) - ic (0y,063)

Per2 ic al (t dc et al.y,

2009) Cc tcl (0y,009)iiti almh ed (<0y,001) Yes (0y,002)

Bmal1 ic al (t dc et al.y,

2009) - Yes (0y,044)

Cry1 ic al (t dc et al.y,

2009) - ic (0y,505)

Rev-Erbα ic al (Baclat et al.y, 2012)

Cc tcl (0y,015) iiti almh ed (0y,003)

ic (0y,100)

Pgc1α ic al

(Salhadc-ielhadc et al.y, 2013) - ic (0y,578)

Pparα Slmhhtlt shmfed

(Salhadc-ielhadc et al.y, 2013) - Yes (0y,047)

Glucise tilerance – Bcth easue e ts cf the cc tcl hcup shcwed a c al hluccse peakm h m edmatelt

afe the hluccse m jectc a d dmsplatm h a c al depessmc 30 m utes afe m jectc (fh. 6). iiti m he eal had c effect c the hluccse tclea ce cuie (p = 0y,956) a d c dmffee ce was pese t whe the htth s wee almh ed (p = 0y,981) c wee msalmh ed (p = 0y,921). The aea u de the hluccse tclea ce cuie

was alsc ct cha hed as a esult cf iiti (fh 7)y, bcth fc the almh ed (p = 0y,868) a d msalmh ed hcup (p = 0y,945).

iiti dmd ct cause a smh mfca t cha he m wemhht (p = 0y,948; fh. 8)y, whmch m dmcates that the ats dmd ct tc deielcp cbesmtt as a esult cf iiti. t te d see ed imsmble m the admpcsmtty, m dmcat h that iiti caused ce whmte admpcse tssue cc paed tc the tctal bcdt wemhht (fh. 9). Hcweiey, tdmpcsmtt dmd ct shcw a t smh mfca t esults m a t cf the tested admpcse tssue fat pads ( ese temal p = 0y,310; epmdtdm al p = 0y,180; peme al p = 0y,250; subcuta ecus p = 0y,235).

Actvity – Whmle the cc tcl ats e am ed tc haie a c al 24-hcu htth a d cthm h cha hed m the ccuse

cf the expem e t (fh. 10t)y, iiti clealt caused the appeaa ce cf the dcuble htth (fh. 10B)y, bt addm h the 25y,5-hcu htth as scc as iiti stated. Thms alsc shcws clealt m the pemcdcha s (fh. 11).

I Mmce a c al 24-hcu htth was imsmble m the cc tcl phase cf the expem e t. iiti wmth 5 lux at mhht c lt educed the actimtty, but iiti wmth 10 lux at mhht caused a dcuble htth (fh. 12)y, cc smst h cf the usual 24-hcu htth a d the 25y,5-hcu htth (fh. 13).

Table 4. Pemcd cf lccc ctc htth s cf mce m iiti.

Miuse Periid if first rhythm (strength

abive significance) Periid if secind rhythm (strength)

1 24y,2 (0y,013) 25y,1 (0y,010)

2 23y,7 (0y,057) 25y,4 (0y,033)

3 24y,0 (0y,020) 25y,2 (0y,007)

Cc clusmc

Because we haie twc easue e t pcm ts (ZT6 a d ZT18) m the in-situ htbmdmsatc a d the pempheal clcck he e expessmc expem e ts the cha hes imsmble m these expem e ts hmie a s all m dmcatc cf the whcle htth . We ca dmffee tate twc ttpes cf cha hes m the t e pcm ts we used: a eductc m ‘htth (lmke Per1 cf the in-situ a d the expessmc cf Rev-Erbα m epmdtdm al whmte admpcse tssue) a d a eductc cf he e expessmc m he eal (imke Cry1 m lmie tssue). Whe a eductc cf the htth ms cbseiedy, thee ae seieal pcssmble m tepetatc s. Fmst cf these ms sm plt a eductc m htth mc a plmtudey, wmth a s alle dmffee ce betwee the peak a d tcuhhy, whmle stll bem h at the sa e t e cf dat as befce iiti. The secc d pcssmble cause ms a shmf m the htth y, whmle etam m h the a plmtudey, thms mhht c mtself be caused bt a cha he m pemcd le hth c a cha he m c sety, althcuhh thet lcck the sa e m a shct t e pemcd. I thms expem e t mt ms m pcssmble tc pcm t tc c e causey, as bcth a cha he m a plmtude a d a shmf cf the htth ae equallt lmkelt tc esult fc a splmt SCi. t cha he m a plmtude mhht cc e tc be because cc fmct h smh als leaie the SCi a plmtude cculd be da pe ed because thee ms c st chc cus cutputy, the peaks m expessmc ae educed whmle the tcuhhs ae m ceased. t shmf mhht be caused mf c e cf the twc pats cf the SCi cscmllatesy, as htpcthesmzedy, wmth a lc he htth . The he e m questc m the specmfed tssue wculd the fcllcw the cscmllatc cf the cha hed pat cf the SCi. tlthcuhh iet u lmkelt a d ct shcw befcey, a thmd pcssmbmlmtt cculd be a dcuble htth m the expessmc cf a cetam he e. Thms ms m pcssmble tc qua tft wmth twc t e pcm ts a d wculd equme a lc h pemcd cf easue e t wmth ultple t e pcm ts each dat.

The cha he m the m -smtu htbmdmzatc cf the SCi pcies that the cscmllatc cf the SCi ms affected bt iiti. Fc Per1 a d the a plmtude ms educedy, fc Bmal1 a te d m dmcates the sa ey, theefce mt ms clea that iiti has a effect c the htth a d htth mc he e expessmc m the SCiy, a elt bt educm h the htth mc a plmtude cf the SCi as a whcle. Hcweiey, a clea dmffee ce m phase cf the twc pats cf the SCi was ct fcu d. We pesu e the cst m pcta t easc fc thms was that mt was had tc eallt lccate dmffee t pats cf the SCi m the sca s that wee used fc thms pupcse. The esclutc cf these sca s was ct hmhh e cuhh tc clealt dmst humsh the cce fc the shell wmthcut a t addmtc al stam m h fc the SCi m he eal. Theefce a dak patch m the sca y, see m hlt fc the dcsc edmal pat cculd ea the SCi was sm plt lccated bmt hmhhe m the m dmimdual a m al. Thms dces cf ccuse ct ea that thee ms c dmffee ce betwee the cce a d the shell. Hcweiey, ce scy, mt ms tc be expected that the aieahe htth mc a plmtude cf the whcle SCi educes as the twc dmffee t pats dc ct cscmllate m st chc t. Theefce mt ms stll pcbable that a deccuplm h cf the cce a d shell SCi causes the dcuble htth y, sm ce thms has bee fcu d befce tc be the cause cf a dcuble htth (de la Ihlesma et al.y, 2004). Cue tlt we ae wckm h c m pcie e t cf the sca sy, m cde tc clealt dmffee tate betwee the sub uclem. Whe the twc sub uclem cf the SCi dc ct cscmllate m st chc ty, mt ms lmkelt m cu setup that c e cf thcse ehmc s wmll shcw a pemcd cf 24 hcus a d the cthe a pemcd cf 25y,5 hcus. The ie tclateal sub ucleus cf the SCi ms affected dmectlt bt lmhht acccdm h tc lmteatue (t tle a d Smliey, 2005;Hast hs a d Hezchy, 2004) a d ms theefce the cst lmkelt ca dmdate fc the 24-hcu htth y, as all a m als wee stll expcsed tc a cbimcus dat- mhht ctcle wmth 24 hcus. The dcsc edmal sub ucleus cf the SCi ms the am cscmllat h pat a d ca cc t ue tc dc sc wmthcut lmhht m fc atc . The fee-u m h htth pese t m all a m als m ii ms lmkelt the esult cf the cscmllatc cf dcsc edmal ehmc cf the SCi. Fc thms easc thee ms a pcssmbmlmtt that the dcsc edmal SCi ms the cmhm cf the 25y,5-hcu htth m cu paadmh . It msy, hcweiey, lmkelt that a cthe feedback echa ms ms m fue cm h thms pat cf the SCiy, because the cmcadma actimtt htth cf ats has a s alle pemcd cf usuallt 24y,2 tc 24y,5 hcu (Stepha y, 1983). It ms pcssmble that the acuate a d/c paaie tmcula (PVi) uclem ae m fue cm h the SCi a d that fccd t m h ms m iclied m the secc d htth . Tc dete m e the effect cf the feedm h htth cc paed tc the actimtt htth mt wculd be best tc haie a ew hcup cf a m als expcsed tc iiti a d schedule them feedm h t esy, sc that fccd ms c lt aiamlable at mhht (the c al t m h). Thms wculd fcce a eat h htth cf 24 hcus a d mf the secc d htth cf 25y,5 hcus dmsappeas m thms setup mt wculd clealt m dmcate the 25y,5-hcu htth ms caused bt feedm h htth sy, lmkelt cmhm at h fc the acuate ucleus c the PVi. We ae pla m h thms as c e cf the fcllcw-up expem e ts tc dete m e the m fue ce cf the feedm h htth m iiti.

imke m mce (Fc ke et al.y, 2013) pempheal clcck he es ae dmstubed due tc iiti m ats. Hcweiey, the cha he ms less pc cu ced a d c lt few Group a d Interaction effects ae smh mfca t. Per2y, Cry1 a d Rev-Erbα he eallt expess a clea cieall htth m the used paadmh a d thms ms the case m all used tssue ttpes. The htth m these he es ms smh mfca t m the cc tcl cc dmtc s a d ms educed bt iiti emthe c lt m the msalmh ed hcup c bcth iiti hcups. tll these he es ae pm at clcck he es m iclied dmectlt m the ta scmptc -ta slatc al lccp cf the clecula clcck echa ms a d all haie a m hmbmt h effect c the ta scmptc cf cthe he es. It ms m teest h that Per1 a d Bmal1 ae ct as clea m them cha hes as the cthe clcck he esy, m facty, thee ms ct e cuhh dmffee ce m the cc tcl hcups tc shcw a smh mfca t htth . Thms ms lmkelt the esult fc the 2-t e-pcm t paadmh we usedy, as cst cf the clcck he es we usedy, dc ct shcw them peak expessmc at ZT6 c ZT18. Hcweiey, whe lcckm h at the haphs cf these he es thee see s a m dmcatc that the htth s mhht educe as a esult fc iiti. t cthe expem e t wmth appcxm atelt fcu t e pcm ts cculd e ha ce c pcie c dmspcie thms mdea wmth a lct ce cetam tt.

Thee ms c dmffee ce m the effect cf iiti c the htth cf clcck he es whe cc pam h he es that haie them peak expessmc dum h the lmhht pemcd wmth thcse that peak dum h the dak pemcd. iiti theefce dces ct affect he es expessed dum h dak ess ce. Thee ms lmtle dmffee ce m the effect cf iiti betwee the tssue ttpesy, the c lt imsmble dmffee ce ms whethe a htth ms pese t m the almh ed iiti hcup c ct; m peme al whmte admpcse tssue the almh ed iiti hcup he eallt has c smh mfca t htth whmle thms hcup dces haie a htth m the lmie a d epmdtdm al whmte admpcse tssues. It cculd be pcssmble that the cscmllatc s m peme al whmte admpcse tssue ae ct that stc h m he eal a d that eie as the esult cf a s all dmsuptc the he es lcss them htth mcmtt. Thms wculd ea the pempheal clcck m peme al whmte admpcse tssue ms ct exceedm hlt stc h. The ce etabclmc elated he es dc ct shcw a cha he as lahe m the clcck he es. Thms was tc be expected sm ce the pempheal clccks ae ct pat cf the ce tal clcck echa ms . Ge e expessmc m these ehmc s ms subjected tc dmffee t exte al factcsy, that ae cfe less cc smste t tha the lmhht m put that the SCi ecemies a d thcuhh bem h st ulated a d st chc mzed bt the SCiy, bem h late m the cascade cc es wmth a lcss m ste hth. I sc e cc tcl hcups the htth cf expessmc was dmffee t fc cthe lmteatue: Pparα m epmdtdm al whmte admpcse tssue a d Per1 a d Pparα deimate fc the expessmc htth s fc the atcles used fc the cc pamsc (Salhadc-ielhadc et al.y, 2013;Su et al.y, 2015). Hcweiey, m all these cases the peak cf the expessmc lmes appcxm atelt at ZT12y, m the mddle cf ZT6 a d ZT18. Thms ea s the steepest pat cf the cuie lmes at the t e pcm ts we tested. t s all cha he m expessmc y, pcssmblt due tc m dmimdual iamatc y, ca cause a see m hlt lahe effect m thms case.

Thee ms c m dmcatc that the hluccse tclea ce ms cha hed m a t cf the tested a m als due tc iiti. Theefce mt ca be samd that the cha he m clcck he es dces ct e telt f d mts wat tcwads the hluccse etabclms cf the a m als. The fact that the bcdt wemhht cf the ats dces ct cha he as a esult cf iitiy, was suspected; ealme u publmshed esults shcwed that ats dc ct exhmbmt cha he m bcdt wemhht cc paable tc mce m iiti. I the admpcsmtt data a slmhhtlt ce m teest h te d ca be see : thcuhh statstcallt cthm h cha hes m all hcupsy, all iiti ea pece tahes ae hmhhe tha the cc tcl ialuesy, wmthcut exceptc . Thms leads tc the pcssmbmlmtt that thee mhht be s all effect cf iiti c admpcsmtt that cculd becc e smh mfca t whe lahe hcups ae used c a lc he expcsue tc iiti ms used. Stll thee ms a u mstakable dmffee ce betwee the effect cf iiti c bcdt wemhht cf mce a d ats. It has bee shcw c ultple cccasmc s that feedm h dum h the est h phase causes cbesmtt m mce (Fc ke et al.y, 2010;Fc ke et al.y, 2013;Zha h a d Camy, 2011)y, but m ats thms has bee iet had tc shcw. I lm e wmth thms peimcus esults shcw thaty, whmle mce keep them tctal fccd m take at the sa e leiely, ats eat less whe c lt bem h fed dum h the est h phase. t cthe u publmshed expem e t shcwed that ats dc eat dum h them est h phase whe the dcuble htth fcces the tc. Cc bm m h these cbseiatc sy, mt appeas ats haie a stste tc ccpe wmth iet iamable fccd scucesy, sc cha hes m them fccd htth dc ct affect the m a iet ehatie wat.

tctimtt m mce ms alteed due tc iiti a d appeas educed m he ealy, thms ea s the mce ae affected bt the iiti eie thcuhh mt ms eelt 5 lux a d a dcuble htth ms ct imsmbley, thms ms alsc imsmble m the FFT. Thee ae seieal pcssmble expla atc s fc the fact that mce eed at least 10 lux tc deielcp a dcuble htth . Fmst cf ally,

the ats used m cu cthe expem e ts wee albm c Wmsta atsy, m stead cf the pmh e ted C57Bi/6J mce. tlbm c a m als haie ultple m pcta t dmffee ces cc paed tc c aly, pmh e ted a m alsy, lmke bem h ce susceptble tc lmhht cf a t km d. Theefce the ats pcbablt eed a lesse lmhht m te smtt tc shcw the effects cf lmhht. O e wculd the htpcthesmze that albm c mce wculd shcw esults cc paable tc thcse cf ats m the sa e setup. t cthe expla atc cculd be that se smtimtt tc lmhht m these cmcu sta ces ms m hee tlt dmffee t betwee specmesy, ea m h that mce m he eal ae less se smtie tc lmhht c mt dces ct affect them cmcadma clcck as uch. Theefce thet wculd eed a hmhhe lmhht m te smtt fc the sa e effect. If thms ms the casey, thms dmscepa ct cculd easmlt be sclied bt appltm h the used iiti paadmh tc albm c mce m stead cf pmh e ted c es. Usm h mce m he eal wculd be iet desmable as well m thms paadmh y, because mt allcws fc qua tftm h htth mc he e expessmc m a t tssue bcth in vivo a d in vitro wmth a PER2::iUC cc stuct. Thms cculd help m mde tftm h a dcuble htth m pempheal clccks wmth ce pecmsmc tha a 12-hcu m teialy, pcssmblt wmthcut sacmfcm h the a m al.

I cc clusmc mt ca be samd that iiti dces affect the SCi a d the clcck he es fcllcwm h m the cascade. The SCi ms affected m the se se that the a plmtude cf the htth cf Per1 a d Bmal1 deceases c aieahe fc the whcle SCi. I the pempheal clccks lccated m epmdtdm al whmte admpcse tssuey, peme al whmte admpcse tssue a d the lmie seieal clcck he es ae cha hed wmth a te de ct tc educed htth mc a plmtude. The dcuble htth pese t m lccc ctc actimtt ms ct clealt ecch mzable m bcth the SCi a d the pempheal clccksy, but ms ct excluded fc bem h pese t wmth the used expem e tal paadmh a d ethcds. The effects c bcdtwemhht a d admpcsmtt due tc iiti ae less pc cu ced a d ae ct statstcallt smh mfca t. Eie thcuhh te ds cculd be see m admpcsmtty, thee see s c effect c the tctal bcdtwemhht cf the a m als. t lahe sa ple smze c a m ceased expcsue tc iiti mhht tmeld ce smh mfca t esults m thms ehad. The dcuble htth cbseied as a esult cf iiti m ats ms alsc pese t m mcey, althcuhh a stc he lmhht m te smtt at mhht was eeded tc m duce mt.

Thms eseach shcws that a s all lmhht cf 5 tc 10 lux at mhht ca haie effects c dmffee t pats cf the cmcadma t m h stste a d pccesses that ae m fue ced bt mt. Thcuhh ct m hee tlt detm e taly, the cbseied effects ae a easc tc take mhht at lmhht m tc cc smdeatc whe eseachm h cmcadma a d dat-a d- mhht htth s c dmabetes ellmtus a d cbesmtt m mce a d ats.

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