RBP4 functions as a hepatokine in the regulation of glucose metabolism by the circadian clock in mice

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1 Dietologi () 59:5 DOI.7/s ARTICLE functions s heptokine in the regultion of glucose metolism y the circin clock in mice Xing M, & Zn Zhou, & Yqiong Chen & Yuting Wu & Yi Liu Receive: August 5 /Accepte: Octoer 5 /Pulishe online: Novemer 5 # Springer-Verlg Berlin Heielerg 5 Astrct Aims/hypothesis As one of the key ipokines, retinol ining protein () is suggeste to positively correlte with insulin resistnce; however, not ll clinicl stuies support this ssocition. Although some explntions re propose for this iscrepncy, the temporl spect of secretion hs not een consiere. Aryl hyrocron receptor nucler trnsloctor-like (lso known s BMAL) n its trget D site-ining protein () re oth pivotl trnscription fctors of the circin core clock. Given the overwhelming presence of circin control in metolism n the principl role of the liver in secretion, we hypothesise tht my oscillte uner the control of BMAL n ct s heptokine, prticipting in the mintennce of glucose homeostsis y the circin clock. Methos We use liver-specific Bml (lso known s Arntl)- knockout mice n recominnt enoviruses expressing short-hirpin RNA (shrna) specific for Dp or Rp in the liver. Xing M n Zn Zhou contriute eqully to this work. Electronic supplementry mteril The online version of this rticle (oi:.7/s5-5-7-) contins peer-reviewe ut uneite supplementry mteril, which is ville to uthorise users. Yi Liu liuyi@sis.c.cn Key Lortory of Nutrition n Metolism, Institute for Nutritionl Sciences, Shnghi Institutes for Biologicl Sciences, Chinese Acemy of Sciences, Yueyng Ro, Shnghi, People s Repulic of Chin Shnghi Institutes for Biologicl Sciences, University of the Chinese Acemy of Sciences, Shnghi, People s Repulicof Chin Results isplye iurnl oscilltions in the liver n plsm, which were mpene in liver-specific-bml-knockout mice. BMAL regulte heptic expression vi its irect trget,. Heptic knockown of or increse whole-oy insulin sensitivity in mice in time-of-yepenent mnner. Conversely, heptic overexpression of reverse the insulin-sensitising effects of liver-specific epletion of BMAL. Conclusions/interprettion Our results not only provie novel mechnism for circin regultion of, ut lso unveil criticl role of, cting s heptokine in the regultion of glucose metolism y the circin clock. Keywors BMAL. Circin clock.. Glucose metolism. Insulin sensitivity. Arevitions BMAL Aryl hyrocron receptor nucler trnsloctor-like ChIP Chromtin immunoprecipittion CLOCK Clock circin regultor CREB camp responsive element ining protein D site-ining protein DBS -ining site Gpse Glucose--phosphtse Green fluorescent protein L-Bml / -specific eletion of Bml PCK Phosphoenolpyruvte croxykinse, cytosolic Retinol ining protein -Δ Retinol ining protein without signl peptie Rp-Luc Rp-luciferse

2 Dietologi () 59:5 55 RNAi shrna SIBS RNA interference short-hirpin RNA Shnghi Institutes for Biologicl Sciences Unspecific RNAi Wil-type Zeitgeer time woul isply iurnl oscilltions tht woul e regulte y BMAL, n tht my e require for the regultion of glucose metolism y the circin clock. With this in min, we stuie liver-specific-bml-knockout mice, circin-clock-isruption mouse moel tht hs n insulin-sensitive phenotype. In ition, we took vntge of enoviruses tht selectively inhiit the expression of Dp or Rp in the mouse liver. Introuction Retinol ining protein (), specific crrier for retinol in the circultion [], is prouce n relese minly y heptocytes (%) [] n ipocytes (%) []. A previous stuy hs reporte tht elevtion of ipocyte-secrete contriutes to the evelopment of insulin resistnce in mice []. Clinicl stuies hve shown tht circulting levels re increse in iniviuls with impire glucose tolernce n type ietes, n correlte inversely with insulin sensitivity [ 7]. However, these ssocitions re not supporte y ll stuies in humn, s severl stuies foun no correltion etween circulting levels n insulin resistnce, impire glucose tolernce or type ietes [ ]. This iscrepncy suggests limite unerstning of the reltionship etween n insulin resistnce. Although liver is the principl source of circulting [], it remins unknown whether heptic plys role in moulting glucose homeostsis. The circin clock is n intrinsic time-keeping system tht enles orgnisms to pt their physiologicl n ehviourl rhythms in response to the externl environment []. While the light-responsive centrl clock resies in the suprchismtic nucleus, iniviul clocks re present in the peripherl tissues [ 7]. The current moleculr moel for the circin clock is se on trnscriptionl feeck loop in which ryl hyrocron receptor nucler trnsloctor-like (BMAL)/clock circin regultor (CLOCK) heteroimers rive the trnscription of cryptochrome n (Cry n Cry) n perio circin clock, n (Per, Per n Per) y ining to the E-oxes on their promoters. In turn, the cryptochrome (CER)/perio (PER) complex represses the trnscriptionl ctivity of BMAL/ CLOCK, which genertes intrinsiclly oscilltory signls [ ]. This core clock mchinery regultes the rhythmic expression of ownstrem genes, lrgely y coorinting with other trnscription fctors, incluing their irect trgets (e.g. D site-ining protein (Dp), Pprα [lso known s Ppr], n Pprγ [lso known s Prg]) [9]. However, little is known out how these clock components influence in the liver n plsm. While recent stuies strongly suggest n importnt role of circin clocks in glucose homeostsis [, ], the unerlying mechnism remins to e elucite. Thus, we hypothesise tht the levels of Rp mrna or protein Methos Animl experiments Bml lox (lso known s Arntl tmweit /J) were purchse from the Jckson Lortory (Br Hror, ME, USA). Bml lox mice were mte with the C57BL/ J strin expressing Cre-recominse to generte mice with liverspecific eletion of Bml (L-Bml / ), s escrie y Lmi et l []. / miceweregenerteonc57bl/j-genetic ckgroun y SIDANSAI Biotechnology (Shnghi, Chin) using trnscription ctivtor-like effector nuclese (TALEN)- meite gene trgeting, s escrie y Chen et l []. The - to -week-ol mle C57BL/ mice were purchse from Shnghi Lortory Animl Centre (Shnghi, Chin). All mice were house n re in the niml fcility t Shnghi Institutes for Biologicl Sciences (SIBS) t ± C, mintine on h/ h light/rk cycle n fe liitum for t lest weeks efore the stuy. Recominnt enovirus ws elivere y systemic til-vein injection to mice. At 5 ys fter virl infection, mice were sujecte to glucose n insulin tolernce tests. See electronic supplementry mteril (ESM) Methos for further etils. All niml cre n use proceures were in ccornce with guielines of the Institutionl Animl Cre n Use Committee of the Institute for Nutritionl Sciences (INS), SIBS n the Chinese Acemy of Sciences (CAS). Plsmis Full-length mouse cdnas, Rp-luciferse (Rp-Luc) n shrna plsmi constructions were clone into the vectors inicte using stnr moleculr iology techniques. See ESM Methos for etils. For cloning primers n oligonucleoties, see ESM Tles n. Aenoviruses n eno-ssocite viruses Aenoviruses encoing, retinol ining protein without signl peptie (-Δ), control green fluorescent protein (),, BMAL, with Rp RNA interference (RNAi), Dp RNAi, Bml RNAi n control unspecific RNAi enoviruses were generte through homologous recomintion etween linerise trnsfer vector pad-trck n the enovirl ckone vector pad-esy, s escrie y He et l []. Viruses were purifie y the CsCl metho n ilyse ginst PBS uffer contining % glycerol s escrie y Becker et l [5]. The Rp RNAi eno-ssocite virus ws generte y Oio Technology (Shnghi, Chin).

3 5 Dietologi () 59:5 Culture of primry heptocytes Primry heptocytes were prepre from mice y collgense perfusion techniques. See ESM Methos for further etils. Quntittive RT-PCR Quntittive RT-PCR ws use to etermine the reltive expression levels of mrnas. See ESM Methos n ESM Tle for further etils. Antioies n western lot Anti-BMAL (5) n nti- () were otine from Acm (Cmrige, UK). Cellulr (97-) ws purchse from Epitomics (Burlingme, CA, USA) n plsm ntioy ws gift from Y. Liu (Institutes for Nutritionl Sciences, Shnghi, Chin). Tissue n cell proteins were lyse in hypotonic uffer (% Noniet P-, 5 mmol/l Tris-HCl,. mmol/l EDTA, 5 mmol/l NCl, ph 7.). See ESM Methos n ESM Tle for further etils. Chromtin immunoprecipittion Chromtin immunoprecipittion ssys were one s escrie y Sun et l []. See ESM Methos for further etils. The primer sequences re shown in ESM Tle 5. Genertion of reporter cell lines Reporter cells n clonl lines were generte s previously escrie [7]. See ESM Methos for further etils. Bioluminescence recoring n t nlysis ALumiCycle luminometer (LumiCycle, Actimetrics, Wilmette, IL, USA) ws use for ioluminescence recoring. The LumiCycle Anlysis progrm version.5 (Actimetrics, Wilmette, IL, USA) ws use to etermine circin vriles, s escrie elsewhere []. Luciferse ssy The luciferse reporter ssy for HEK9T cells hs een escrie previously [9]. Sttisticl nlysis Results re shown s men±sem. Sttisticl nlysis ws performe using one-wy or twowy ANOVA or two-tile unpire Stuent s t test where pproprite. Differences were consiere sttisticlly significnt t p<.5. All experiments were performe on t lest two inepenent occsions. Results Heptic BMAL eficiency mpens Rp expression To explore the ily chnges in Rp expression, we nlyse the mrna levels of Rp n Bml in the livers of mice kille t h intervls roun the clock (Fig., ). The ccumultion of Rp mrna n protein isplye roust iurnl rhythms in the livers of wil-type () mice, peking roun Rp mrna (fol) c Plsm Plsm g Reltive ioluminescence (counts/s) Rp-Luc Bml mrna (fol) BMAL Time (ys) L-Bml -/- the onset of night (zeitgeer time [], Fig., c). Interestingly, plsm levels lso oscillte cross the h light/rk cycle, ut with n h phse vnce compre with its heptic expression (Fig. c). Furthermore, liver-specific Reltive mplitue (fol) Rp mrna (fol) BMAL () L-Bml -/- () L-Bml -/- (BMAL) e.5..5 pge Bml RNAi f Dp mrna (fol) Perio (h) pge Bml RNAi Fig. BMAL regultes the rhythmic oscilltion of Rp in the liver. L-Bml / n littermte mice were kille t h intervls cross the h light/rk cycle. Quntittive PCR nlysis of reltive mrna levels of () Rp n () Bml in the liver. Vlues were normlise to Gph n re presente reltive to the lowest control, set t. Blck imons, ; white circles, L-Bml /. Grey shing n rk rs inicte rk perios (n= ). p<.5 n p<. with two-wy ANOVA. (c) Immunolotting, with the ntioies inicte, of liver homogentes or plsm t the s inicte. (injecte with A-gfp), L-Bml / (injecte with A-gfp) nl-bml / (injecte with A-Bml) mice were kille t h intervls cross the h light/rk cycle. () Immunolotting, with the ntioies inicte, of liver homogentes or plsm t the s inicte. (n= ). Quntittive PCR nlysis of reltive (e) Rp n (f) Dp mrna levels in the livers. Blck imons, (); white circles, L-Bml / (); lck circles, L-Bml / (BMAL). (g) Bioluminescence rhythm of Rp-Luc collecte from NIHT cells trnsfecte with pge (GeneErse shrna expression vector, see ESM Methos for further etils) or Bml RNAi. Perio n reltive mplitue re presente. n=; lck lines, pge; grey lines, Bml RNAi. p<. with Stuent s t test. Dt re presente s men±sem

4 Dietologi () 59:5 57 knockout of Bml significntly ttenute the mplitue of Rp mrna n protein rhythms in the liver n plsm without ltering their phses (Fig. c). In contrst, the oscilltion of Rp mrna in white ipose tissue ws little ffecte in L-Bml / mice (ESM Fig, ). Moreover, the mpe oscilltion of in L-Bml / mice ws restore to full-lown rhythmicity in the liver n plsm y ectopic expression of heptic BMAL (Fig. f). Consistently, Bml RNAi-meite knockown mrkely ttenute the mplitue of the oscilltion of Rp-Luc in NIHT cells, with little effect on its perio s monitore y rel-time ioluminescence imging (Fig. g). However, to our surprise, the results of the chromtin immunoprecipittion (ChIP) ssy showe no presence of BMAL on the Rp promoter (ESM Fig. c, ). trnsctivtes Rp in the liver Further nlysis of the Rp promoter sequence showe homology of the nucleotie sequence locte from to 9 in the Rp gene with the consensus -ining site (DBS; ESM Fig. ) require for the recruitment of trnscription fctor ; heptic expression of the ws olishe in L-Bml / mice (ESM Fig. ). We then teste the ility of to rive Rp trnscription through these puttive DBSs. Overexpression of significntly stimulte Rp-Luc ctivity in ose-epenent mnner (Fig. ), wheres eletion of the puttive DBSs olishe -inuce ctivity (Fig. ). ChIP nlysis confirme the presence of on these DBSs in the Rp promoter (Fig. c). Consistently, we oserve tht the mplitue of the oscilltion of Rp-Luc ws gretly ttenute y Dp RNAi (ESM Fig. c) in NIHT cells y using rel-time ioluminescence imging. Moreover, enovirl expression of Dp (A-Dp) significntly enhnce the expression of in primry heptocytes. Conversely, Dp RNAi enovirus (A-Dpi) ecrese Rp mrna levels, which le to reuction in levels of oth expression n secretion of protein in primry heptocytes (ESM Fig. -f). In vivo investigtion revele tht A-Dpi significntly ecrese the mplitue of protein n mrna oscilltion in mouse liver (Fig. f). Furthermore, enovirl overexpression of Bml or Dp ws sufficient to restore oth the levels n secretion of in Bml / primry heptocytes, suggesting tht is involve in the mechnism y which BMAL regultes (Fig. g). Heptic moultes glucose metolism To etermine the effects of heptocyte-erive in regulting glucose homeostsis, we prepre n Rp RNAi enovirus (A-Rpi), which specificlly reuce Rp expression levels in the liver ut not in white ipose tissue (ESM Fig.,). Compre with mice expressing unspecific RNAi (A-), levels of resting glucose (the loo glucose level in mice fe liitum) were significntly lower in -knockown mice Rp-Luc ctivity (fol) (ng) Rp mrna (fol) g 5 5 Rp-Luc ctivity (fol) e Dp mrna (fol) L-Bml -/- BMAL ΔDBS ΔDBS ΔDBS+ ΔDBS Cellulr FLAG-BMAL FLAG- Meium Loing control roun most time points (,,, ) except n (Fig. ). Consistently, Rpi significntly increse insulin sensitivity in mice t, wheres these effects were not oserve t (Fig., c). These two sets of t inicte tht moultes glucose metolism minly uring resting/fsting perios (ytime for mice) when heptic gluconeogenesis is stimulte. Inee, nlysis of f creltive recruitment on Rp promoter (fol) DBS DBS Dpi Reltive protein levels (fol) BM BM L-Bml -/- L-Bml -/- Cellulr Meium Fig. Rp trnscription is regulte y in the liver. () Reltive luciferse ctivity of Rp-Luc in the presence of ng n ng oses of in 9 T cells. Vlues were normlise to β-gl n re presente reltive to control, set t ; p<. with Stuent s t test. () Reltive luciferse ctivity of Rp-promoter mutnts in the presence of vector expressing in 9 T cells., wil-type Rp-promoter; ΔDBS n ΔDBS, Rp promoter with eletion of the puttive -ining site (ATTATGTTTT) n (GTTACGAAAG); respectively. White rs, plsmi cytomeglovirus promoter (pc)dna; lck rs, ; p<. with Stuent s t test. (c) ChIP nlysis showing the reltive recruitment of to DBS n DBS on the promoter of Rp in mouse primry heptocytes synchronise y exmethsone ( μmol/l) for h. White rs, nti-; lck rs, nti-; p<. with Stuent s t test. Mice injecte with A- or A-Dpi enovirus were kille t h intervls cross the h light/rk cycle. Quntittive PCR nlysis of reltive mrna levels of () Rp n (e) Dp in liver. Blck imons, ; white circles, Dpi. Grey shing n rk rs inicte rk perios (n= ); p<.5 n p<. with two-wy ANOVA. (f) Immunolotting, with the ntioies inicte, of liver homogentes or plsm t the s inicte. (g) Immunolotting nlysis of proteins in or Bml-null primry heptocytes infecte with enovirus expressing, BMAL n. Heptocytes were synchronise s efore. Reltive protein levels of re presente. Vlues re normlise to or loing control n re presente reltive to control, set t ; p<.5 n p<. with Stuent s t test. Dt re presente s men±sem., glycerlehye -phosphte ehyrogense Plsm

5 5 Dietologi () 59:5 c , e f g A-li Fst h A-li Fst h Gpse mrna (fol) Pck mrna (fol) Rp mrna (fol) PCK p-creb CREB Rpi Rpi Rpi Rpi Fig. Temporl effects of heptic knockown on glucose metolism. mice were injecte with enovirus expressing Rpi orcontrol.() Resting glucose levels were mesure roun inicte s (n= ). () GTTn(c) ITTwereperforme roun n (n= ). Mice were fste for h efore GTT n h efore ITT. AUCs were mesure. Blck imons n rs, ; white circles n rs, Rpi; p<.5 n p<. with one-wy ANOVA. ( f) Quntittive PCR nlysis of reltive Gpse, Pck n Rp mrna levels in the liver. White rs, -liitum-fe ; htche rs, -liitum-fe Rpi mice; grey rs, fste ; lck rs, fste i mice; p<.5 n p<. with Stuent s t test. Dt re presente s men±sem. (g) Immunolotting of liver homogentes with the ntioies inicte. Mice were kille t or, fe or fste for h (n= ). Vlues were normlise to Gph n re presente reltive to fe control, set () t. A-li, -liitum-fe gluconeogenic genes emonstrte tht mrna levels of Pck n glucose -phosphtse (Gpse, lso known s Gpc) s well s protein levels of phosphorylte camp responsive element ining protein (CREB) n phosphoenolpyruvte croxykinse, cytosolic (PCK) were ecrese y knockown roun, ut not (Fig. g). We further confirme these results y elivering Rpi vi n enossocite virus (Ri), which cuse very mil stining on the immunolot (ESM Fig. ). To explore the possiility tht intrcellulr heptic myeimplicteintheregultionofglucosehomeostsis, we prepre n enovirus expressing Rp with the eletion of secretory signl peptie (A-Rp-Δ). Injection of this virus significntly increse resting loo glucose levels in mice roun n, ut h little effect uring the rest of the y (ESM Fig. 5). Consistently, glucose tolernce ws impire in mice expressing heptic A-Rp-Δ, compre with those injecte with control enovirus, t roun (ESM Fig. 5). In prllel with these results, the mrna levels of gluconeogenic genes Pck, Pgcα (lso known s Pprgc) nfp were lso significntly increse in the livers of A-Rp-Δ-injecte mice t (ESM Fig. 5c f). Menwhile, protein levels of PCK n phosphorylte CREB were increse y A-Rp-Δ in the liver without ffecting the totl level of CREB protein t (ESM Fig. 5g). Importntly, injection of A-Rp-Δletosignificnt ccumultion of Rp-Δ within the liver, ut h little effect on plsm levels (ESM Fig. 5g). knockown in the liver improves whole-oy insulin sensitivity As heptic prticiptes in the regultion of insulin sensitivity, we expecte to lso e involve in glucose metolism. In line with the effects of Rpi(Fig. ), the injection of -knockown enovirus (Dpi) not only lowere loo glucose levels uring the ytime in mice fe liitum ut lso meliorte insulin sensitivity roun ut not (Fig. c). Consistently, Dpi significntly reuce the protein n mrna levels of gluconeogenic genes in livers from h fste mice n mice fe liitum collecte roun ut, in contrst, h little effects in those roun (Fig. h). The involvement of in BMAL/ regultion of glucose homeostsis Bse on our finings, we speculte tht is involve in -regulte glucose metolism. Inee, trnsuction of enovirus significntly increse the mounts of PCK protein in primry heptocytes erive from mice, ut not in those from Rp-knockout mice (Fig. 5). In ition, enovirl expression of Rp restore the reuction in PCK protein y A-Dpi in primry heptocytes (Fig. 5). These oservtions prompte us to exmine the role of in meitting the effects of BMAL on glucose metolism. Consistent with our previous stuy [], L-Bml / mice h enhnce glucose tolernce compre with littermtes uring the ytime, together with ecrese protein levels of GPse, PCK n phosphorylte CREB. However, heptic expression of Rp y enovirl

6 Dietologi () 59:5 59 c , Gpse mrna (fol) e f g h Pck mrna (fol) Rp mrna (fol) Dp mrna (fol).5 PCK A-li Fst h Dpi Dpi A-li Fst h Dpi Dpi Plsm Fig. Temporl effects of heptic knockown on glucose metolism. mice were injecte with enovirus expressing Dp RNAi (Dpi) or control. () Resting glucose levels were mesure roun the s inicte (n= ). () GTT n (c) ITT were performe roun n (n= ). Mice were fste for h efore GTT n h efore ITT. AUCs were mesure. ( c) Blckimons n rs, ; white circles n rs, Dpi; p<.5n p<.; onewy ANOVA. ( g) Quntittive PCR nlysis of reltive Gpse, Pck Rp n Dp mrna levels in the liver. White rs, -liitum-fe ; htche rs, -liitum-fe Dpi; grey rs, fste ; lck rs, fste Dpi; p<.5 n p<. with Stuent s t test. (h) Immunolotting of the liver homogentes or plsm with the ntioies inicte. Mice were kille t or, fe or fste for h (n=). Vlues were normlise to Gph n re presente reltive to fe control, set () t. Dt re presente s men±sem. A-li, -liitum-fe elivery reverse such eneficil effects of L-Bml eficiency in mice (Fig. 5c e). Discussion Previous stuies hve revele tht sustntil frction ( %) of ll liver mrna is expresse in rhythmic fshion, n mny of the proteins re involve in glucose metolism []. In this stuy, we foun tht oscilltes in circin mnner throughout h light/rk cycles, not only in the liver n plsm, ut lso in the white ipose tissue. We lso foun tht this iurnl vrition is mpene in liver-specific Bml-knockout mice. These results inicte criticl role of BMAL in the regultion of heptic Rp trnscription, which etermines its rhythm in the plsm. By using ChIP nlysis n enovirusmeite RNA interference, we provie mechnism through which BMAL rives the circin expression of heptic vi. The liver-enriche trnscription fctor oscilltes in ily mnner uner the control of BMAL [, ]. In line with previous stuies [, ], mrna levels of Dp pek roun (ESM Fig. ), which is comptile with the ccumultion of Rp trnscripts (Fig. ). Our results emonstrte tht is novel trnscriptionl ctivtor of in the liver. Interestingly, our t show tht the rhythm of plsm isplys n h phse vnce compre with its heptic expression (Fig. c). Bse on the importnt role of iet in retinol supply, we speculte tht this phse vnce coul e ue to ietry retinol uptke control y feeing cycle. In contrst to the role of in the trnsport of heptic retinol to plsm uring resting/fsting perios (ytime for mice), postprnil retinol is minly moilise to peripherl tissues vi chylomicrons. Therefore, the oscilltion of serum peks in the ytime n reches nir fter eting (night-time), wheres heptic oscilltes with reverse phse, regulte y the circin clock, which les to the pprent h phse lg etween these two rhythms. Although circulting levels re consistently correlte with insulin resistnce in mice [, ], clinicl stuies in humn hve not reche consensus on this issue [ ]. We hve emonstrte tht isplys n oscilltory pttern in the plsm (Fig. c), which provies n lterntive interprettion for the propose correltion. As the mount of in the loo chnges within couple of hours, if the smpling time intervl is too long or the smpling time points re ifferent, the results coul e conflicting, which is further supporte y the fct tht resting glucose levels re not ltere in -knockown mice roun n (Fig. ). Menwhile, insulin sensitivity is increse in -knockown mice t, ut remins unchnge t, which strongly implies n unerlying circin process in the regultion of glucose metolism y (Fig., c). Together, our stuies emphsise the nee to pply strict control to the timing of loo smpling n to consier circin effects when interpreting results. Compre with ipose tissue, the liver is the mjor site for the synthesis n secretion of [], s one heptocyte expresses out five times s much Rp mrna s n ipocyte uner either len [5] oroese[] conitions.

7 Dietologi () 59:5 c PCK F- HSP9 PCK β-actin f Rp -/- F- F- Dpi Dpi DBS CLOCK AUC (mol/l min) BMAL E-ox DBS Dp Rp e Reltive PCK protein levels (fol) Reltive PCK protein levels (fol) PCK GPASE P-CREB CREB Tuulin Menwhile, severl stuies hve foun no correltion etween ipose Rp mrna expression n serum protein level [9,, 7, ]. Moreover, Rp mrna mounts re selectively Rp -/- Control Insulin resistnce Glucose intolernce A-Rp L-Bml -/- L-Bml -/- Plsm Fig. 5 Heptic is involve in BMAL/ regultion of glucose metolism. Immunolotting, with the ntioies inicte, of: () or Rp-null mice (white rs, ; lck rs, ); () mouse primry heptocytes infecte with inicte enoviruses (white rs, ; lck rs, Dpi). Heptocytes were synchronise y exmethsone ( μmol/l) for h. Reltive protein levels of PCK re presente. Vlues were normlise to HSP9 or β-ctin n re presente reltive to control, set t ; p<.5 with Stuent s t test. Glucose tolernce of n L-Bml / mice efore (c) nfter() the injection of A-Rp (n=7 ) t. AUCs were mesure. Blck imons n rs, ; white circles n rs, L-Bml / ;p<.5np<. with one-wy ANOVA. (e) Immunolotting nlysis in the liver homogentes or plsm of n L-Bml / mice, with or without injection of A-Rp (n=). Mice were fe liitum n were kille t. (f) Schemtic igrm epicting the circin regultion of y BMAL through, which contriutes to insulin resistnce n glucose intolernce in mice. Dt re presente s men±sem Plsm ecrese in the liver, ut not in the white ipose tissue, of insulin-sensitive L-Bml / mice (Fig. n ESM Fig. ). Inee, ccumultion of non-secretory in the liver impirs glucose tolernce y inucing gluconeogenic genes (ESM Fig. 5 g), which suggests tht the metolic ctions of enogenous within the liver lso contriute to the slutry phenotype of -knockown mice. Together, our finings highlight the importnce of the heptic for systemic glucose metolism. Previous stuies hve reporte tht mice exhiit iurnl vritions in insulin sensitivity: the highest insulin sensitivity of mice ws foun uring the night n the lowest ws foun uring the ytime [9, ]. Mice with n L-Bml / genotype exhiit time-of-y-epenent insulin-sensitive phenotype []. While it hs een shown tht Glut (lso known s Slc) hs lost its circin expression in L-Bml / mice, the mechnism y which BMAL exerts its regultory function remins lrgely unknown. Our t support the hypothesis tht the insulin hypersensitivity of L-Bml / mice coul e ue to eficit of (Fig. 5c e). Although hs een reporte to e potentilly involve in the regultion of cholesterol metolism [], its role in glucose homeostsis hs not een investigte. In our stuy, we emonstrte tht oth n re require for the norml circin chnges in insulin sensitivity (Figs n ). The results of ecrese heptic n circulting expression in -knockown mice, long with the similrity in phenotypes of - n -eficient mice, suggest tht my exert its metolic ction through n -epenent mechnism. Inee, the ility of to inuce PCK expression is compromise y eficiency n the effect of knockown on PCK protein is olishe y enoviruses (Fig. 5, ). Furthermore, heptic overexpression of reverses the insulin-sensitive phenotype of L-Bml / mice n restores the expression of gluconeogenic genes (Fig. 5c e), which strongly implictes key role of in the mechnism y which the circin clock mintins glucose homeostsis (Fig. 5f). Tken together, our results ientify Rp s novel gene uner circin clock control n provie mechnistic insight into its circin regultion. Furthermore, our results support the prominent role of heptic in the mintennce of metolic control, n confirm time-epenent ssocition etween n insulin resistnce in mice. Importntly, the time-of-y-epenent effect of on insulin sensitivity is of potentil importnce in the clinicl prognosis of iniviuls with type ietes. Acknowlegements We thnk Y. Liu (College of Life Sciences, Wuhn University, Wuhn, Chin) for generously proviing plsm ntioy. We thnk Z. Qin, F. Dng, Q. Xu n R. Wu (Key Lortory of Nutrition n Metolism, Institute for Nutritionl Sciences, Shnghi Institutes for Biologicl Sciences, Chinese Acemy of Sciences, Shnghi, Chin) for ssisting with experiments.

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