Light at night acutely impairs glucose tolerance in a time-, intensity- and wavelength-dependent manner in rats

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1 Dietologi (21) :1 1 DOI 1.1/s y ARTILE Light t night utely impirs gluose tolerne in time-, intensity- nd wvelength-dependent mnner in rts Anne-Loes Opperhuizen 1,2 & Dirk J. Stenvers 2, & Remi D. Jnsen 1 & Ewout Foppen 2, & Eri Fliers & Andries Klseek 1,2, Reeived: 12 Deemer 21 /Aepted: 2 Mrh 21 /Pulished online: April 21 # The Author(s) 21. This rtile is pulished with open ess t Springerlink.om Astrt Aims/hypothesis Exposure to light t night () hs inresed drmtilly in reent dedes. Animl studies hve shown tht hroni dim indued oesity nd gluose intolerne. Furthermore, severl studies in humns hve demonstrted tht hroni exposure to rtifiil my hve dverse helth effets with n inresed risk of metoli disorders, inluding type 2 dietes. It is well-known tht ute exposure to ffets iologil lok funtion, hormone seretion nd the tivity of the utonomi nervous system, ut dt on the effets of on gluose homeostsis re lking. This study imed to investigte the ute effets of on gluose metolism. Methods Mle Wistr rts were sujeted to i.v. gluose or insulin tolerne tests while exposed to 2 h of in the erly or lte drk phse. In susequent experiments, different light intensities nd wvelengths were used. Results exposure erly in the drk phse t ZT1 used inresed gluose responses during the first 2 min fter gluose infusion (p <.1), wheres exposure t the end of the drk phse, t ZT21, used inresed insulin responses during the first 1 min (p <.1), inditing tht Anne-Loes Opperhuizen.opperhuizen@nin.knw.nl immeditely indues gluose intolerne in rts. Susequent experiments demonstrted tht the effet of ws oth intensity- nd wvelength-dependent. White light of nd 1 lx indued greter gluose responses thn nd 2 lx, wheres ll intensities other thn lx redued loomotor tivity. Green light indued gluose intolerne, ut red nd lue light did not, suggesting the involvement of speifi retin rin pthwy. onlusions/interprettion Together, these dt show tht exposure to hs ute dverse effets on gluose metolism in time-, intensity- nd wvelength-dependent mnner. Keywords Biologil lok. Gluose intolerne. Green light. Insulin sensitivity. Light t night. Rt. Rodent Arevitions ANS Autonomi nervous system iprg Intrinsilly photosensitive retinl gnglion ell IVITT Intrvenous insulin tolerne test Light t night LEDs Light-emitting diodes SN Suprhismti nuleus ZT Zeitgeer time 1 2 Hypothlmi Integrtion Mehnisms, Netherlnds Institute for Neurosiene (NIN), Meiergdreef, 11 BA Amsterdm, the Netherlnds Lortory of Endorinology, Deprtment of linil hemistry, Ademi Medil entre (AM) University of Amsterdm, Amsterdm, the Netherlnds Deprtment of Endorinology nd Metolism, Ademi Medil enter (AM) University of Amsterdm, Amsterdm, the Netherlnds Introdution Eletri lighting is widely used round the world, ut possile hrmful effets of the presene of light 2 h dy dys week hve eome pprent only reently. In prtiulr, the use of light t night () is onsidered potentilly disruptive, s orgnisms re not physiologilly prepred to del with light signls during the night. Light is ught y the eyes nd trnsformed into eletril signls y retinl reeptors nd

2 1 Dietologi (21) :1 1 onduted through the opti nerves to the rin res importnt for vision, utonomi funtions nd irdin timing. Historilly, sunlight would e minly responsile for these funtions, ut rtifiil light with the pproprite wvelength nd intensity my mimi the effets of sunlight. Exposure to hs een orrelted with n inresed risk of developing oesity [1, 2], dietes [] nd dyslipidemi [2] in humns. In ddition, rodent studies showed tht hroni exposure to redues the mplitude of the sleep/wke rhythm [ ], inreses ody weight nd dereses gluose tolerne [, ]. Aute exposure to hs een shown to lter the profiles of the hormones meltonin nd ortiosterone [, 8]. Moreover, lters lok gene expression in the hypothlmi irdin lok [9], nd ffets gene expression in peripherl orgns [, 1]. Light is the most importnt ue for the irdin lok to synhronise its endogenously generted rhythmi tivity with the environmentl light/drk yle. The lok, loted in the suprhismti nuleus (SN), trnsmits timing informtion to downstrem trgets vi hormones nd the utonomi nervous system (ANS) to optimlly prepre the orgnism for regulr dily hnges. For exmple, the SN diretly ontrols gluose homeostsis, inluding sl gluose levels nd gluose tolerne [11, 12], t lest prtly through the ANS [1]. Visul nd non-visul effets of light re medited initilly y retinl ells. Intense reserh into the non-visul effets of light reveled the existene of novel retinl photoreeptor, the intrinsilly photosensitive retinl gnglion ells (iprgs), whih express the photopigment melnopsin [1, 1]. Knowledge of rods nd ones is well estlished, nd these ells re still onsidered to e the most importnt photoreeptors for vision, with rods eing dominntly tive in dim light nd ones in right light onditions. The iprgs provide the mjority of retinl input to non-visul rin strutures nd dominntly innervte the SN, providing informtion essentil for irdin physiology [1]. However, the visul nd non-visul pthwys re not ompletely seprte entities, s iprgs lso reeive indiret input from rods nd ones [1 1]. The orreltion etween nd metoli disorders together with the power of light to ontrol the SN nd downstrem trgets led to the development of hypothesis tht ffets gluose homeostsis. The urrent study tested this hypothesis y sujeting Wistr rts to gluose nd insulin tolerne tests during ute t the eginning nd end of the drk phse. As seondry ojetive, we investigted gluose intolerne indued y ute exposure in the erly drk phse s funtion of light intensity nd wvelength. Mterils nd methods Animls nd housing Mle Wistr rts (hrles River Breeding Lortories, Sulzfeld, Germny) weighing g were mintined under ontrolled 12/12 light/drk yle (lights on : hours, Zeitgeer Time (ZT); lights off 19: hours, ZT12), with d liitum ess to wter nd regulr how diet (Tekld Glol Diet, Hrln, Horst, the Netherlnds), unless stted otherwise. Red dim light (mx. photopi lux [lx]) ws present in the room during the drk phse, wheres mixed white fluoresent light (mx. 1 lx) ws present during the light phse. All experimentl proedures were performed in ordne with the ounil Diretive 21/EU of the Europen Prliment nd the ounil of 22 Septemer 21 on protetion of nimls used for sientifi purposes. All experimentl proedures were pproved y the Animl Ethis ommittee of the Royl Duth Ademy of Arts nd Sienes (KNAW, Amsterdm, the Netherlnds) nd in ordne with the guidelines on niml experimenttion of the Netherlnds Institute for Neurosiene. Experimentl design After dpttion to the niml fility, ll rts, exept the experimentl groups for loomotor tivity reordings, were nesthetised with 8 mg/kg ketmine (Eurovet Animl Helth, Bldel, the Netherlnds), 8 mg/kg Rompun (xylxine, Byer Helth re, Mijdreht, the Netherlnds) nd.1 mg/kg tropine (Phrmhemi, Hrlem, the Netherlnds). Susequently, n intr-tril silione theter ws implnted unilterlly in the jugulr vein nd fixed on the skull [12]. Animls were left to reover for t lest 1 week efore the strt of experiments. To study whether ffets gluose or insulin tolerne, nimls were exposed to 2 h of light during whih n IVGTT or intrvenous insulin tolerne test (IVITT) [12] ws performed. Food ws removed 2. h prior to the tests. In rndomly ssigned rossover design, ll nimls inluded in the finl dt sets were tested twie, one under ontrol (i.e. dim red light) nd one under experimentl (i.e. ) onditions, with 1 week reovery period etween. Animls with inomplete reovery of ody weight or lokge of the theter were exluded from the experiment. Experiment 1 Animls were rndomly ssigned to one of two groups to study the effets of (white light ~12 ± 2 lx) on gluose tolerne: group 1A (n = 11) where under onditions the light ws turned on from ZT1 1 nd the IVGTT strted t ZT1, nd under ontrol onditions the IVGTT strted t ZT1; nd group 1B (n = 1) where under onditions the light ws turned on from ZT2 22 nd the IVGTT strted t ZT21, nd under ontrol onditions the IVGTT strted t ZT21. Two dditionl groups of nimls were used to study insulin tolerne using n IVITT under the sme light onditions s desried for groups 1A nd 1B. Group 1 (n = 1) ws exposed to 2 h from ZT1 1 nd IVITT strted t ZT1, nd group 1D (n = ) ws exposed to 2 h from ZT2 22 nd IVITT strted t ZT21.

3 Dietologi (21) :1 1 1 Experiment 2 Four groups were inluded to study the effet of different light intensities. The onditions were similr to experiment 1A: mixed white from ZT1 1 nd n IVGTT strted t ZT1. Animls were rndomly ssigned to one of four groups: lx (n =1),2lx(n = ), lx (n =9)or 1 lx (n =9). To study the effets of on loomotor tivity, four dditionl groups of nimls were solitry housed in trnsprent ges to reord loomotor tivity with pressure pltes [12]. Eh mesurement inluded 8 h ontinuous reording with min intervls. In the first 2 h, loomotion ws mesured while nimls were exposed to the norml (12/12) lighting shedule. In the seond 2 h reording period, nimls were exposed to from ZT1 1. exposure during this 2 h period ws t lx (n = ), 2 lx (n = ), lx (n =)or 1 lx (n =). Experiment Four groups were inluded to study the effets of wvelength. The onditions were similr to experiment 1A. Animls were rndomly ssigned to one of four groups nd exposed to white light rnging from nm (n = 9), lue light with pek t nm (n = 9), green light with pek t 2 nm (n = 8) or red light with pek t nm (n =1). Four dditionl groups of nimls were inluded s desried in experiment 2 to study the effets of wvelength on loomotor tivity. exposure ws the sme s for the IVGTTs: white (n = 8), lue (n = 9), green (n =9)orred (n =9). Light exposure White light exposure during experiments 1 nd 2 ws done with SMD28 light-emitting diodes (LEDs; Wtshome). Light exposure of different wvelengths in experiment ws done with SMD LEDs (Wtshome). Adhesive strips of LEDs were tthed to in-house-uilt plsti wlls surrounding the niml ges with horizontl light exposure t eye level of the nimls. LEDs were ontrolled y softwre uilt in-house. The illuminne of the light for the differentonditionsinexperimentwsdjustedsuhtht photon flux nd irrdine were similr etween onditions (see Tle 1 for irrdine spetrum [18]). Plsm mesurements Blood smples were ooled fter olletion, susequently entrifuged (, 199 g, 1 min) nd plsm ws stored t 2 until further nlysis. Plsm gluose onentrtions were determined immeditely fter lood smpling with lood gluose monitoring system (FreeStyle Freedom-Lite, Aott Dietes re, Almed, A, USA). Rdioimmunossys were used ording to the mnufturer s protool to mesure plsm insulin onentrtions (Millipore, St hrles, MO, USA) nd plsm ortiosterone onentrtions (MP Biomedils, Snt An, A, USA). Smples with inomplete duplites due to tehnil resons were exluded from the dt. Sttistil nlysis All dt re expressed s mens ± SEM. Pired t tests were used to detet group differenes in seline onentrtions for gluose (experiments 1, 2 nd ), insulin (experiments 1 nd ) nd ortiosterone (experiments 1 nd ). Pired t tests were lso used to detet group differenes in loomotor tivity in experiments 2 nd. A repeted mesures two-wy ANOVA ws used to test for effets of tretment (ontrol or ), time or intertion (tretment time) on the responses of gluose (experiments 1, 2 nd ), insulin (experiments 1 nd ) nd ortiosterone (experiments 1 nd ). If tretment or intertion effet ws found, post ho Sidks multiple omprisons tests were used to determine differenes etween light onditions t individul time points. The net AU ws determined from min using the trpezoid rule. Delt vlues re lulted y sutrting the seline (t = min) from eh time smple (t =,1,2,nd min) rw dt vlue. Pired t tests were used to detet group differenes in AU for gluose (experiments 1, 2 nd ), insulin (experiments 1 nd ) nd ortiosterone (experiments 1 nd ). All sttistil nlyses were performed with GrphPd Prism version.1 for Windows (GrphPd Softwre, L Joll, A, USA) using signifine level of p <.. Tle 1 Spetrl sensitivity of light onditions used in experiment Retinl photopigment omplement White Blue Green Red S-one (rn s [λ]) Melnopsin (rn z [λ]) Rod (rn r [λ]) M-one (rn m [λ]) Irrdine (μw/m 2 ) Photon flux (1 m 2 s 1 ) All dt sed on the rodent-toolox provided y Lus et l [18] The upper rows represent weighted ontriution of rodent retinl photopigments (S-one, iprg [melnopsin], rod, M-one) in α-opi rodent lux. The lower rows represent unweighted hrteristis of the LEDs (irrdine, photon flux)

4 1 Dietologi (21) :1 1 Results Experiment 1 Effets of ute exposure to on gluose homeostsis were tested y exposing rts to 2 h light t the eginning (ZT1 1)orend(ZT2 22) of the drk phse. In the erly drk phse (ZT1), indued inresed plsm gluose levels fter gluose infusion (Fig. 1, ). No signifint differenes were oserved for insulin (Fig. 1e, f). In the lte drk phse (ZT21), used trend towrds inresed plsm gluose (Fig. 1, d). In ontrst to ZT1, insulin plsm levels were inresed y t ZT21 fter gluose infusion (Fig. 1g), resulting in trend towrds higher AU for insulin (Fig. 1h). ortiosterone responses were unffeted y t ZT1 or ZT21 (Tle 2). Tolerne tests were initited t the strt of the seond hour of the light exposure; therefore, seline smples (t = min) were otined fter 1 h light exposure (oth ZT1 nd ZT21). Bseline gluose onentrtions were unffeted y 1 h t oth time points (Fig. 2). Bseline insulin onentrtions tended to e lower oth t ZT1 nd ZT21 (Fig. 2). Bseline levels of ortiosterone were slightly redued y t ZT1 nd showed trend towrds deresed levels t ZT21 (Fig. 2). To investigte whether ffets insulin sensitivity, nimls were exposed to n IVITT. did not indue hnges in plsm gluose onentrtions fter insulin infusion t ZT1 (Fig., ) or ZT21(Fig. d, e). Also ortiosterone responses were not different etween ontrol nd onditions. Bseline onentrtions of gluose (ZT1 Fig., Fig. 1 dereses gluose tolerne t ZT1 nd inreses insulin responses t ZT21. from ZT1 1 deresed gluose tolerne with higher gluose onentrtions (, ) nd unhnged insulin responses (e, f). from ZT2 22 did not ffet plsm gluose onentrtions (AU p =.8)(, d), ut inresed insulin onentrtions (AU p =.9) (g, h). AU figures show the group men on the left nd individul nimls on the right. ontrol (), solid lines, lk olumns nd symols;, dshed lines, white olumns nd symols. p <.,p <.1, p < Time (min) Time (min) AU gluose (mmol/l min) d AU gluose (mmol/l min) AU gluose (mmol/l min) AU gluose (mmol/l min) e Δ insulin (pmol/l) Δ insulin (pmol/l) g Time (min) Time (min) f AU insulin (pmol/l min) h AU insulin (pmol/l min) 8,,, 2, 8,,, 2, AU insulin (pmol/l min) AU insulin (pmol/l min) 8,,, 2, 8,,, 2,

5 Dietologi (21) :1 1 1 Tle 2 Sttistil nlyses of effets of light on gluose, insulin nd ortiosterone responses during gluose nd insulin tolerne tests in experiment 1 Experiment Vrile p for tretment p for time p for intertion p for AU IVGTT ZT1 Gluose.2 <.1 <.1. Insulin.9 < ortiosterone ZT21 Gluose.9 <.1..8 Insulin.1 <.1..8 ortiosterone IVITT ZT1 Gluose.1 < ortiosterone.2 < ZT21 Gluose.81 < ortiosterone Tretment (light ondition), time (smple time) nd intertion effets were determined using repeted mesures two-wy ANOVA Gluose, insulin nd ortiosterone responses re reported for the IVGTT t ZT1 nd ZT21. Gluose nd ortiosterone responses re reported for the IVITT t ZT1 nd ZT21 The AU olumn ontins results of pired t test on net AU urve ZT21 Fig. f) nd ortiosterone (ZT1: 1 ± 9 in ontrol vs ± 19 in, p =.22; ZT21: 1 ± 1 vs 1 ± 2, p =.88) were unffeted y t oth time points. The sttistil nlyses of experiment 1 re reported in Tle 2. Experiment 2 The effets of on gluose tolerne were lrgest in the erly drk phse (t ZT1) nd therefore this time point ws hosen to further investigte the hrteristis of light-indued gluose intolerne. Four different light intensities were used to test whether the effets of light on gluose tolerne depend on intensity. Signifintly higher peks were found fter gluose infusions in nd 1 lx onditions when ompred with nd 2 lx (time p <.1; tretment [intensity] p =.1; intertion [intensity time] p =.). A signifint tretment effet ws only oserved in the nd 1 lx onditions with higher gluose levels in onditions when ompred with ontrol (Fig. d). AU ws signifintly higher with lx nd tended to e t 1 lx. Bseline levels of gluose were unffeted y, 2 or 1 lx, nd slightly redued with lx (Fig. e h). Loomotor reordings during nd ontrol onditions showed deresed loomotion with 2, nd 1 lx. The derese ws signifint for 2 nd 1 lx (2 lx p =.1;1lxp =.), with similr trend for lx (p =.2). Loomotor tivity in nimls exposed to lx ws unffeted (p =.91, Fig. i l). The sttistil nlyses of experiment 2 re reported in Tle. Experiment To study whether -indued gluose intolerne ws wvelength-dependent, four different wvelengths were used: short (lue), middle (green) nd long (red), s well s rod-spetrum white light inluding ll wvelengths (Tle 1). As reported for experiments 1 nd 2, white indued gluose intolerne during 2 h exposure strting t ZT1 (Fig., ). Inresed gluose onentrtions in the ondition were lso oserved in green light (Fig. i, j),utnot in lue (Fig. e, f) or red light (Fig. m, n). Insulin responses were not ffeted y wvelength (Fig. o). ortiosterone responses were unffeted y lue, green or red light, ut were ZT1 ZT21 1 Fig. 2 dereses seline ortiosterone t ZT1 ut does not ffet seline gluose nd insulin onentrtions. No signifint differenes were oserved in seline gluose () nd insulin onentrtions () Insulin (pmol/l) 2 ortiosterone (ng/ml) ZT1 ZT21 ZT1 ZT etween ontrol (lk symols) nd (white symols) onditions during experiment 1. signifintly deresed sl ortiosterone onentrtions t ZT1, ut not t ZT21 (). p <.

6 18 Dietologi (21) :1 1 Fig. Blood gluose onentrtions re unffeted y during IVITT t ZT1 nd ZT21. did not lter the derese in lood gluose fter insulin infusion t ZT1 (, ) or ZT21 (d, e). Bseline onentrtions were not ffeted y t ZT1 () orzt21(f) when ompred with ontrol onditions. ontrol (), solid lines, lk olumns nd symols;, dshed lines, white olumns nd symols ZT21 ZT1 d. Time (min) Time (min) AU gluose (mmol/l min) e AU gluose (mmol/l min) f signifintly inresed y white light exposure. Loomotor tivity reordings demonstrted tht white (p =.2), lue (p =.19) nd green (p =.28), ut not red (p =.812), light deresed loomotion ompred with the ontrol drk onditions (Fig. d p). Bseline onentrtions of gluose, insulin nd ortiosterone were unffeted y ny of the light onditions (Fig. ). The sttistil nlyses of experiment re reported in Tle. e i Loomotor tivity (AU) Time (min) f Time (min) Time (min) d Time (min) lx 2 lx lx 1 lx lx j Loomotor tivity (AU) lx Fig. The effets of on gluose tolerne nd loomotor tivity re intensity-dependent. with intensity of lx () did not indue hnges in lood gluose onentrtions, ut 2 lx (), lx () nd 1 lx (d) did. Bseline gluose levels were unffeted in lx (e), 2 lx (f) or 1 lx (h) onditions, ut slightly deresed y of lx (g). g k Loomotor tivity (AU) lx h l Loomotor tivity (AU) lx Totl loomotor tivity levels reorded from ZT1 1 were redued during light onditions ompred with ontrol onditions for 2 lx (j), lx (p =.)(k) nd 1 lx (l), ut not lx (i). ontrol (), solid lines, lk olumns nd symols;, dshed lines, white olumns nd symols. p <.,p <.1, p <.1

7 Dietologi (21) : Tle Sttistil nlyses of effets of light on gluose responses during gluose tolerne tests in experiment 2 Intensity Vrile p for tretment p for time p for intertion p for AU lx Gluose.8 < lx Gluose.2 < lx Gluose.1 <.1 < lx Gluose. <.1..1 Tretment (intensity), time (smple time) nd intertion effets were determined using repeted mesures twowy ANOVA Gluose responses re reported for the IVGTT during four light intensities The AU olumn ontins results of pired t test on the net AU urve. The threshold for signifine is p <. Disussion We show tht exposure to uses ute gluose intolerne in rts nd tht this effet is dependent on the time of dy, intensity nd wvelength of the light exposure. The indued gluose intolerne t the strt of the drk period ws refleted y inresed plsm gluose levels, wheres -indued gluose intolerne t the end of the drk period ws minly refleted y inresed plsm insulin. Surprisingly, green, ut not lue, light est mimiked the effets of white light. These results suggest n importnt role for middle-wvelength ones in the -indued effets on gluose intolerne. Most non-visul light responses, suh s the pupillry light response, meltonin inhiition nd modultion of hert rte, re exerted vi the ANS. Animl studies hve shown tht utely inreses symptheti tivity nd dereses prsymptheti tivity of the utonomi nerves innervting peripherl orgns, inluding the liver nd pnres [, 19, 2]. Moreover, denervtion of trget orgns, suh s the liver [1] nd drenl glnd [], prevented -indued hnges in gene expression. Here, we demonstrted tht erly inresed gluose levels without n ompnying inrese in insulin response. In ontrst, lte inresed the insulin response with only smll effets on the gluose response. These dt suggest tht my ffet gluose metolism y severl prllel mehnisms. my hve stimulted hepti gluose prodution, whih would explin the hyperglyemi nd is ongruent with the reported upregultion of Pepk in the liver y [1]. Stimultion of gluose prodution n led to hyperglyemi if the insulin response is indequte. The disrepny etween the dequte insulin response t ZT21 nd indequte response t ZT1 indites tht the oserved gluose intolerne t ZT1 is proly due to redued et ell sensitivity or n inhiition of insulin relese. The inhiitory effet on insulin relese seems to e refleted y the redued seline insulin onentrtion t ZT1 nd ZT21. Suh n inhiitory effet is in line with the previously reported lightindued redution of gluose-stimulted insulin seretion [21] nd the light-indued deresed prsymptheti nd inresed symptheti input to the pnres [2]. Moreover, in reent study we found inresed fsting nd postprndil gluose levels s well s inresed symptheti tivity in individuls with type 2 dietes during erly morning exposure to right light [22]. Gluose intolerne ould lso hve een used y impired insulin-independent nd/or insulin-dependent gluose uptke y metoli tissues. Little is known out the role of the ANS in gluose uptke, lthough it hs een suggested tht insulin-independent gluose uptke in dipose tissues nd skeletl musle depends on symptheti innervtion [2 2]. Whether light exposure stimultes symptheti or prsymptheti innervtion to musles in similr wy s to the liver nd pnres [19, 2] hs not een studied, nd whether this would result in n inrese or derese in gluose uptke remins to e determined. However, the IVITT experiments showed tht did not redue whole-ody insulin sensitivity. The disrepny etween insulin responses t the eginning nd end of the drk period suggests tht the SN my modulte the light signl towrds the pnres in timedependent mnner. Indeed, mny other SN-medited effets of light hve een shown to differ ross the dy [1, 2], nd tring studies hve shown neurl onnetions etween the SN nd pnres [28]. Hypothetilly, light-indued SN stimultion my lso ffet musle funtion in gluose homeostsis, s it ws reported tht dily rhythm exists in gluose uptke y musle tissue in vitro [29], nd disruption of the intrinsi musle lok leds to the disturne of gluose metolism [, 1]. One of the est-studied effets of is the suppression of noturnl meltonin relese y the pinel glnd. Experimentl [2, ] nd geneti studies [ ]hvessoited dysfuntion of the meltonin system with gluose metolism. Green nd lue light exposure hve oth een shown to suppress meltonin seretion [, 8]. However, the distint gluose responses to with lue nd green light in this study mke it unlikely tht meltonin suppression is mjor ontriutor to the oserved gluose intolerne. Melnopsin, the photosensitive pigment in iprgs, is most sensitive to short-wvelength light (~λ nm, the lue

8 1 Dietologi (21) : AU gluose (mmol/l min) 1 1 AU insulin (pmol/l min), 2, 1, d % 2 h loomotor tivity e f AU gluose (mmol/l min) 1 1 g AU insulin (pmol/l min), 2, 1, h % 2 h loomotor tivity i j AU gluose (mmol/l min) 1 1 k AU insulin (pmol/l min), 2, 1, l % 2 h loomotor tivity m Time (min) Fig. The effets of on gluose tolerne nd loomotor tivity depend on wvelength. Two hours of white inresed gluose onentrtions (, ), did not indue hnges in the insulin response () nd deresed totl loomotor tivity (d). Blue light did not ffet gluose onentrtions (e, f) or the insulin response (g), ut deresed totl loomotor tivity (h). Green light exposure inresed gluose onentrtions n AU gluose (mmol/l min) 1 1 o AU insulin (pmol/l min), 2, 1, (i, j), did not ffet the insulin response (k) nd deresed totl loomotor tivity (l). Red light showed no effets on gluose onentrtion (m, n), the insulin response (o) or totl loomotor tivity (p). ontrol (), solid lines, lk olumns nd symols;, dshed lines, white/oloured olumns nd symols. p <.,p <.1, p <.1 p % 2 h loomotor tivity spetrum) nd is responsile for phse-shifting [1], meltonin suppression [9] nd rousl []. Our dt, surprisingly, showed no effet of lue light on gluose metolism. In ontrst, green light lerly indued gluose intolerne. Therefore, our dt do not support diret role for the melnopsin pthwy in the gluoregultory effets of light. The humn retin ontins three types of ones: short (S-), middle (M-) nd long (L-)ones, with M-ones eing speifilly sensitive to the green spetrum of light (λ2 nm). Dt re limited on the physiologil effets of green light nd the role of M-ones, esides the suppression of meltonin seretion [, 8] nd involvement in irdin lok entrinment [1]. A reent study in mie [] desried distint melnopsin-dependent effets of lue nd green light on sleep,

9 Dietologi (21) : White Blue Green Red Insulin (pmol/l) 1 1 White Blue Fig. Bseline onentrtions of gluose, insulin nd ortiosterone re unffeted y t different wvelengths. No signifint differenes were oserved in seline gluose (), insulin () nd ortiosterone () onentrtions etween ontrol (lk symols) nd onditions (white symols) Green Red ortiosterone (ng/ml) 2 1 White Blue Green Red rousl nd ortiosterone relese. The uthors proposed tht lue light stimultes M1-type iprgs projeting to the SN nd therey ts on rousl nd the ANS, wheres green light stimultes different M-type of iprg tht projets to the ventrolterl preopti re nd ffets sleep. In line with this model, the effets of light on gluose metolism in our study my e exerted vi non-m1 iprg sutype proly projeting preferentilly to non-sn trget res. Indeed, light-evoked effets on hert rte vriility [2] nd drenl funtion [] were lso shown to e independent of iprgs innervting the SN. Noturnl rodents lk L-ones (λ mx nm) nd therefore do not show irdin, ehviourl or metoli effets in response to red light [, 8, ]. Our study is ongruent with this s low illuminne ( lx) nd red light, min stimultors for L-ones, did not indue gluose intolerne. Furthermore, it is unlikely tht S-ones ply signifint role s none of our light soures stimulted these photoreeptors (Tle 1). In ddition to M-ones, rod photoreeptors lso respond to green light nd, therefore, mediting role of rods in the effets of light on gluose tolerne nnot e exluded. However, the lue nd green LEDs used in our study use ner-equl rod stimultion, wheres M-one stimultion is higher with green light ompred with lue light; therefore, we rgue tht M-ones re more importnt thn rods in mediting the effets of on gluose metolism. The inhiitory effets of on ehviourl tivity in noturnl nimls re well-known nd often reported s msking []. Inhiition of loomotor tivity my result in redued gluose uptke y musle tissue; hypothetilly explining the oserved gluose intolerne in this study. However, in view of the distint responses of loomotion nd gluose tolerne to lue light nd low intensity white light, it is unlikely tht the oserved effets on gluose metolism re medited vi this pthwy. Due to the widespred vilility of rtifiil light, humns nd nimls round the world re inresingly exposed to nd knowledge on the metoli effets of is thus ruil. Disturne of the irdin orgnised physiology y is thought to led to dverse metoli Tle Sttistil nlyses of effets of light on gluose, insulin nd ortiosterone responses during gluose tolerne tests in experiment Wvelength Vrile p for tretment p for time p for intertion p for AU White Gluose.2 < Insulin.1 < ortiosterone.1 < Blue Gluose.9 < Insulin.9 < ortiosterone Green Gluose.21 < Insulin.28 < ortiosterone Red Gluose.21 < Insulin. <.1.. ortiosterone Tretment (wvelength), time (smple time) nd intertion effets were determined using repeted mesures twowy ANOVA Gluose, insulin nd ortiosterone responses re reported for the gluose tolerne test in onditions with four different wvelength spetr The AU olumn ontins results of pired t test on the net AU urve

10 12 Dietologi (21) :1 1 helth onditions in long-term studies []. Our study shows tht utely ffets the importnt homeostti proess of gluose tolerne. The effets on gluose nd insulin suggest tht light utely ffets the ody t multiple levels downstrem of the retin nd hypothlmus. The noturnlity of rts nd the diurnlity of humns, s well s the differenes in retinl sensitivity, should e tken into onsidertion when ttempting to trnslte the findings in this study from niml to humn. Results of three reent humn studies (two performed during dytime nd one t nighttime) re niely in line with our urrent findings in demonstrting the potentil of ute light exposure to ffet gluose metolism [22,, 8]. Therefore, hroni nd ute light exposure now hve een demonstrted to ffet gluose metolism in oth rodent [] nd humn studies [1, 22,, 8]. hroni nd ute exposure re oth representtive onditions for humn shift workers [, 9], s well s for the glol ommunity exposed to inresing light pollution nd the growing use of light-emitting produts. Our study inreses the knowledge of light effets on physiology, whih my ontriute to etter understnding of the orreltion etween, shift work nd metoli disorders inluding dietes. In onlusion, we showed in rts tht noturnl light exposure utely indued gluose intolerne, possily vi redued et ell sensitivity. This effet depended on the time of dy, pointing towrds n intertion with irdin physiology. The effet of light on gluose tolerne ws lso dependent on intensity nd wvelength, suggesting mediting role of non-m1 iprgs nd M-one photoreeptors. Our study wrrnts ttention for the potentilly hzrdous tions of light on helth in order to ontrol the regultion of design nd use of light-emitting produts, long with dvie for nd protetion of frequently exposed individuls. Dt vilility The dt tht support the findings of this study re ville from the orresponding uthor upon resonle request. Funding This study is funded y STW grnt OnTime (projet 1219). DJS is supported y ZonMW (AGIKO 9292). Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Author ontriutions All uthors provided sustntil ontriutions to oneption nd design (ALO, DJS, AK), quisition of dt (ALO, RDJ, EFo), or nlysis nd interprettion of dt (ALO, EFl, AK). Authors ontriuted to drfting the rtile (ALO, AK) or revising it (ALO, DJS, RDJ, EFo, EFl) ritilly for importnt intelletul ontent. All uthors pproved the finl version of this mnusript. AK is the gurntor of this work. 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