ACTA BIOLOGICA SLOVENICA LJUBLJANA 2013 Vol. 56, [t. 1: 29–43 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2013 Vol. 56, [t. 1: 29–43 Circadian rhythms and theirrolein living organisms Cirkadiani ritmi in njihova vloga v živih organizmih Rok Košir Center za funkcijsko genomiko in biocipe, Zaloška cesta 4, 1000 Ljubljana DiaGenomi d.o.o., Cešnjica 3, 1261 Ljubljana-Dobrunje correspondence: rok.kosir@mf.uni-lj.si Abstract: Numerous physiological processes in organisms as diverse as bacteria and man are regulated by a small molecular clock termed the circadian clock. It is present in virtually all cells of the body and enables various physiological processes to occur at specific times of the day and with a period of about 24 hours. It was not until recent years that the role of the circadian clock has become evident for normal physiology of humans as well as other mammals. Disruption of the normal circadian rhythms canlead to anumberofmetabolicdisorders characteristicof modern lifestyle including diabetes, obesity and cancer. It is the aim of this review to give the reader a general overview of what circadian rhythms are, how they look at the molecular level and why they can influence various metabolic processes in the way they do. Keywords: Biological rhythms, circadian rhythms, chronobiology, circadian clock Izvlecek: Številne fiziološke procese v raznolikih organizmih uravnava majhna molekularnaura,kijoimenujemocirkadianaura.Nahajasevskorajvsehcelicahtelesa in omogoca, da razlicni procesi v telesu potekajo ob dolocenih delih dneva ter da se le ti ponovijo v periodi 24 ur. Šele v zadnjih nekaj letih je pomen cirkadiane ure postal jasen tudi za pravilno homeostazo telesa, tako cloveka, kakor tudi drugih sesalcev. Motnje normalnega cirkadianega ritma lahko vodijo v razvoj metabolnih motenj, kot sta diabetes in prekomerna telesna teža, znacilnih za sodoben nacin življenja. Namen preglednega clanka je bralcu predstaviti osnove cirkadianih ritmov, njihove lastnosti na molekularnem nivoju ter njihovo prepletenost s procesi metabolizma. Kljucne besede: Biološki ritmi, cirkadiani ritmi, kronobiologija, cirkadiana ura Biological rhythms rhythms are an integral part of life. We are all awareofourheartbeat,yetwealmostnevertruly What would life on Earth look like if there comprehend it as a biological rhythm, despite werenobiologicalrhythms?Thismightseemlike the fact that the absence or perturbation of its an irrelevant question since obviously biological rhythm is used in everyday medical practice to rhythms are not that important, or are they? If distinguish between life and death or illness and you look at various processes occurring in liv-health. This is one simple example among many ing organisms it becomes evident that biological that shows how biological rhythms are not only Acta Biologica Slovenica, 56 (1), 2013 Figure 1: Characteristics of biological and circadian rhythms. A– An example of a circadian rhythm of hormone concentration in blood is shown. The difference between the maximum (peak) and minimum (trough) concentration is the amplitude. Period equals the interval between two time points. B – Endogenous rhythmspersistwithnodampeninginconstantconditionssuchascompletedarkness.Contrary,exogenous rhythms dampen when put in constant conditions. C – Resetting of the circadian rhythm. Exposure to light (black arrow) can shift the rhythm either back (delay) or ahead (advance) depending when during the cycle it is presented. Normal rhythm is depicted by a full gray line. D – temperature compensation. If circadian clocks were not temperature compensated they would run faster at higher temperatures (high T, period < 24h) and slower in lower temperatures (low T, period > 24h) comparedto normal conditions. Slika 1: Lastnosti bioloških in cirkadianih ritmov. A– primer prikazuje cirkadiani ritem koncentracije hormona v krvi.Razlikamednajvecjokoncentracijo(vrh)innajnižjokoncentracijo(dno)jeenakaamplitudi.Perioda je enaka intervalu med dvema tockama. B – endogeni ritmi ohranjajo amplitudo tudi v primeru konstantnih pogojev, kot je popolna tema. V nasprotju pa zacne amplituda pri eksogenih ritmih v konstantnih pogojihpocasiupadati.C–ponastavitevcirkadianegaritma.Svetlobnipulz(crnapušcica)lahkopovzroci premikfazecirkadianegaritmanazaj(zamuda)alinaprej(napredovanje)vodvistnostiodtegakdajvfazi cikla je bil pulz prisoten. Normalni ritem je prikazan s polno sivo crto. D – Temperaturna kompenzacija cirkadianih ritmov omogoca, da le-ti v primeru visokih temperature ne potekajo hitreje (perioda < 24h) in v primeru nizkih temperature pocasneje (perioda > 24 h), kot pri normalni temperaturi. important for the survival of an organism itself women. In male subjects on the other hand the but also for the survival of species and the eco-presence of such rhythms is still controversial. system in general. Very few studies have been conducted on man, Biological rhythms are defined as biological mainly due to the lack of a distinct marker, such events or functions that reoccur in a repeated order as monthly bleeding in women. The results and with a repeated interval (period) between although statistically significant have indicated occurrences (Aschoff 1981) and can be divided several variables to have an infradian period, into three classes based on the duration of the amongthembodyweight,gripstrength,estrogen phase (Fig. 1A). While the majorityof efforts in and testosterone production, sexual activity and both early studies as well as in recent years have mood. The small number of subjects on which been focused on circadian rhythms with a period the studies have been performed necessitates of about 24 h, rhythms having longer or shorter additional research to confirm published results periods are also important. Infradian rhythms (Koukkari and Sothern 2006).Ultradianrhythms are rhythms with periods longer than 28 hours. on the other end have periods shorter than 20 Awell-known example is the menstrual cycle in hours as shown in the example of the heart beat Košir: Circadian rhythms Table 1: Examples of biological rhythms. The table shows examples of different biological rhythms belonging to three classes (ultradian, circadian and infradian), defined by the length of the period (based on Koukkari and Sothern (2006)). Tabela 1:Primeri bioloških ritmov.Tabela prikazuje primere razlicnih bioloških ritmov iz vseh treh razredov (ultradiani, cirkadiani in infradiani), ki jih definira dolžina periode. Povzeto po Koukkari in Sothern (2006). Time Period Variable Organism Source Seconds < 1s EEG activity (delta Human (Homo sapiens) (Kripke 1972) frequency) < 1s ECG (depolarization of heart Human (Homo sapiens) (Koukkari and Sothern ventricles) 2006) Minutes 2–4 min Leaflet movement Telegraph plant (Desmodium (Koukkari et al. 1985) gyrans) 15 min Cortisol secretion Horse (Equus caballus) (Drake and Evans 1978) 30 min Transpiration Ota (Avena sativa) (Johnsson 1973) 90–100 min REM-NREM sleep Human (Homo sapiens) (Aserinsky and Kleitman 1953) Hours 4 h Enzyme activity Euglena (Euglena gracilis) (Balzer et al. 1989) 12 h Amylase activity Alfalfa (Medicago sativa) (Henson et al. 1986) Day 24 h Body temperature Human (Homo sapiens) (Aschoff et al. 1972) 24 h Sleep-wakefulness Human (Homo sapiens) (Kleitman 1963) 24 h Leaf movements Alibizzia (Alibizzia (Koukkari et al. 1974) julibrissin) 24 h Activity Mouse (Mus musculus) (Decoursey 1960) Week 7 days Oviposition (egg laying) Spring Tail (Folsomia (Chiba et al. 1973) candida) 7 days Organ transplant Human (Homo sapiens) (DeVecchi et al. 1981) 7 days Imbibition of seeds Bean (Phaseolus vulgaris) (Spruyt et al. 1987) Month 27–34 days Menstrual cycle Human (Homo sapiens) (Presser 1974) 6 months Ulcer perforation Human (Homo sapiens) (Svanes et al. 1998) Year 1 year Seed germination Pole bean (Phaseolus (Spruyt et al. 1988) vulgaris) 1 year Migration Willow warbler (and others) (Gwinner 1977) (Phylloscopus trochilus) 1 year Hibernation Golden-mantled ground (Pengelley and Fisher squirrel (Citellus lateralis) 1963) 1 year Gonadal weight Purple sea urchin (Halberg et al. 1987) (Strongylocentrotus purpuratus) 8–10 years Population Ruffed Grouse (Bonasa (Gullion 1982) umbellus) 100–200 Flowering Chinese bamboo (Janzen 1976) years (Phyllostachys bamusoides) above. In humans several ultradian rhythms are known both in males and females. Among them is the cycling of the human brain between REM and non-REM sleep (Kishi et al. 2011), regulation of body temperature (Lindsley et al. 1999), hormone release (Ho et al. 1988, Saad et al. 1998, Simon and Brandenberger 2002) and bowel ac tion (Moore 1992). Some examples of different biologicalrhythmsareshowninTable1.Another importantaspectofbiologicalrhythmsiswhether they are endogenous or exogenous. Exogenous rhythms are simply responses of the organism to external cyclic stimuli, whereas endogenous rhythms are a product of the organism itself and Acta Biologica Slovenica, 56 (1), 2013 are self-sustained (Fig. 1B) (Aschoff 1981). This reviewisintendedtointroducethebasicprinciples of circadian rhythms, their molecular structure and their role in normal physiology. Circadian rhythms Although the first mention of daily rhythms dates all the way back to 4th century BC, when Androsthenes,ahistorianofAlexandertheGreat, describeddiurnalmovementsofleavesofseveral trees,theFrenchastronomerJeanJacquesOrtous de Mairan, is regarded as the discoverer of circadian rhythms. In 1729 he was the first to describe the daily opening and closing of leaves of the mimosa plant (Mimosa pudica) even when put in complete darkness (Devlin 2002). However it was not until the 1950s that the field of circadian biologybegantodevelopwithstudiesonfruitflies andhumansdonebyColinPittendrighandJürgen Aschoff respectively (Vitaterna et al. 2001). Asmentionedcircadianrhythmsarebiological rhythms with a period of about 24 hours, which is implied by the term circadian derived from the Latin circa,meaning“aroundorapproximately“, and diem,meaning“day”.Inorderforabiological rhythm to be classified as circadian four criteria need to be meet (Vitaterna et al. 2001). First the biological process or function needs to repeat itself with a period of approximately 24 hours. Secondly, the rhythm has to have a characteristic of an endogenous cycle, meaning that it has to continue with a period of close to 24h even in constant conditions devoid of any external time- giving cues (Fig. 1B). Thirdly, the rhythm needs to maintain its period over a range of different temperatures, called temperature compensation. Temperature compensation is important because without it the clock would run faster at higher temperatures compared to lower temperatures due to higher thermal energy of molecular processes (Fig. 1D). Lastly, the rhythm has to have theabilitytoadapttochangesintheenvironment andsynchronizeitselftonewconditions(Fig.1C). This process called entrainment is achieved with the help of external time cues (Zeitgebers), the main one being the light-dark cycle produced by Earth’s rotation around its axis (Vitaterna et al. 2001). Pittendrigh discovered that animals will respond differently to light depending on the phase of the cycle they are at (Pittendrigh 1960). For instance,ifanimalsareexposedtolightintheearly part of their normal night, they will respond with Figure 2: Phase response curve (PRC). Aphase response curve shows in what way (advance or delay) the phase of a circadian function (e.g. locomotor activity) will respond, when an external stimuli is given at different times of the circadian cycle. The x-axis represent the time of day, the y-axis shows the amount of phase shift in hours. Light pulse A– (subjectiveday) won’t have any effect on the phase of the circadian function; light pulse B – (beginning of subjective night) will induce a phase delay in the circadian function (also see Fig. 1C); light pulse C – (end of subjective night) will induce a phase advance in the circadian function (also see Fig. 1C). Slika 2: Krivulja faznega odziva (KFO). Krivulja faznega odziva nam pove v katero smer (zamuda ali napredovanje) sebopremaknilafazacirkadianegaprocesa(npr.lokomotornaaktivnost),kotposledicaodgovora na zunanji dražljajev (svetloba), ki ga dajemo ob razlicnih casih dneva. X os predstavlja cas dneva, y os predstavlja velikost zamika faze naprej ali nazaj v urah. Svetlobni signal A– (subjektivni dan) ne bo imel vpliva na fazo cirkadianega procesa; svetlobni signal B – (pricetek noci) bo povzrocil zamik faze cirkadianega procesa (glej tudi Sl. 1C); svetobni signal C – (konec noci) bo povzrocil napredovanje faze cirkadianega procesa (glej tudi Sl. 1C). Košir: Circadian rhythms Table 2: Examples of circadian rhythms. Some examples of circadian rhythms present in a variety of organisms ranging from bacteria to humans. Tabela 2:Primeri cirkadianih ritmov. Nekateri primeri cirkadianih ritmov prisotni pri razlicnih organizmih od bakterij do cloveka. Domain Process Organism Source Archea Oxygen-dependent metabolism Halobacterium salinarum (Whitehead et al. 2009) Bacteria Cyclic surface variations during growth N.D. Rhythms of nitrogen fixation Pseudomonas putida Thermosynechococcus elongatus Synechococcus sp. RF-1 (Soriano et al. 2010) (Onai et al. 2004) Fungi Plants Growth patterns Several physiological processes Leaf movement rhythm, germination, growth, enzyme activity, stomatal movement and gas exchange, photosynthetic activity, flower opening, and fragrance emission Neurospora crassa Chlamydomonas reinhardtii Mimosa pudica, Arabidopsis thaliana, bean (Phaseolus vulgaris), chestnut (Castanea sativa) , pea (Pisum sativum), soybean (Glycine max), tail (Brassica rapa), tomato (Solanum lycopersicum), poplar (Populus spp.)*, papaya (Carica papaya)*, grape (Vitis vinifera)* (Pittendrigh et al. 1959) (Mittag et al. 2005), (McClung 2013), (McClung 2011) Animals Time of Eclosion, foraging and mating activities visit flowers to collect pollen and nectar in a rhythmic manner Timing of their mating flights Timing of migratory flights Preparation for hibernation Diving timing Locomotor activity Body temperature and locomotor activity fruit fly (Drosophila melanogaster) (Panda et al. 2002) honeybee (Apis mellifera) (Moore et al. 1998) ant (Camponotus compressus) (Sharma et al. 2004) Monarch butterflies (Danaus (Froy et al. 2003) plexippus) Golden-mantled ground squirrel (Dunlap et al. 2004) (Callospermophilus lateralis) loggerhead turtle (Caretta caretta) (Oishi et al. 2010) Japanese grass lizard (Takydromus (Oishi et al. 2010) tachydromoides) Iguana iguana (Oishi et al. 2010) Diurnal rhythms in hypothalamic/ pituitary AVT synthesis and secretion Body temperature, blood pressure, metabolism, hormone synthesis etc. Body temperature, blood pressure, metabolism, hormone synthesis etc. Oncorhynchus mykiss (rainbow (Rodriguez-Illamola trout) et al. 2011) Mouse (Mus musculus) (Green et al. 2008; Tzameli 2012) Human (Homo sapiens sapiens) (Green et al. 2008; Tzameli 2012) *– clock genes have been found by genome wide analysis however functional assesments of the clock are still missing (McClung 2013). a phase delay, whereas they will respond with a zeitgeberataspecifictimecanbestudiedwiththe phase advance when they are exposed to light in help of phase response curves. Aphase response the later part of their normal night (Fig. 1C, Fig. curve is constructed by determining whether a 2). The exact way an animal will respond to a phase advance or delay of a certain circadian Acta Biologica Slovenica, 56 (1), 2013 variable (e.g. locomotoric activity) is produced whenthesamezeitgeberisgivenatdifferenttimes of the circadian cycle (Fig. 2) (Pittendrigh 1960, Golombek and Rosenstein 2010). The importance of entrainment may not seem obvious at first, but simple mathematics shows how quickly a species can come out of synch with the day-night cycle if the phase of the rhythmchangesbyjustafraction.Let’sassumea mouse’s endogenous period would be a mere 10 minutes longer tha.n 24 h. With no entrainment to external conditions, it would takeonly 6 days for the mouse to be 1h in advance of the normal day night cycle and in just a matter of 2 months it would become a diurnal instead of a nocturnal animal. This would have a significant negative impact on the fitness of an individual that would substantially reduce its success of survival and reproduction. For this reason if a mouse’s active nightperiodistoolongandextendsintomorning hours, the light will trigger a phase advance (Fig. 2). As a consequence the active period will begin sooner in the coming day and also end before the morning, entraining the internal mouse clock to the environmental conditions. In spite of these four restrictions a large fraction of today’s organisms, ranging from bacteria to humans, display a clear circadian rhythm in various physiological and behavioral processes (some are listed in Table 2). Due to its almost ubiquitouspresence,thecircadianrhythmclearly has an evolutionary advantage. Anticipation of dailychanges intheenvironmentbyan organism ratherthanjustreactingtothemseemstobeoneof the main ones (Ramsey et al. 2007). At least two studies in cyanobacteria and D. melanogaster have shown that wild-type strains are more successful in survival compared to their mutant ones when grown in the same test tube (Johnson et al. 1998, Klarsfeld and Rouyer 1998). Genetics of the clock Despite the discovery of the double helix in 1953 and the development of various genetic and molecular biology techniques thereafter, the first two decades of circadian rhythm research were devoted mainly to understanding the basic principles (Pittendrigh et al. 1959, Pittendrigh 1960) including resetting of the rhythm by light pulses (Bruce et al. 1960), construction of phase response curves (Aschoff 1965), temperature compensation (Zimmerman et al. 1968) etc. It was not until 1971 that the era of clock genetics began, when Ron Konopka and Seymour Benzer first described the existence of the period (per) locus in Drosophilla melanogaster.Usinggenetic screens of mutated fruit flies they discovered 3 mutants which significantly changed their 24h rhythm of both eclosion and locomotor activity: long period (28h rhythm), short period (19h rhythm) and arrhythmic (no rhythm) (Konopka and Benzer 1971). By the beginning of the 21th century, similar genetic screens were used in various model organisms to discover other clock related genes including: per and timeless (tim) in D. melanogaster; white collar 1 and 2 (wc1 and wc2) and frequency (frq) in N. crassa; timing of crab (TOC1) in A. thaliana and Clock, Bmal1, Per1, Per2, Cry1 and Cry2 in mice (Takahashi 2004, Zhang and Kay 2010). Regardless of the fact that different organisms use different sets of genes the basic molecular mechanism behind all circadian clocks seems to be the same and can be described by a simple transcription-translation feedback loop (Roenneberg and Merrow 2002). Whilethetranscription-translationfeedbackloop remains at the core of the circadian clock, the use of novel high-throughput technologies in the last decadeshowedthattheclockis notasimpleloop but is composed of multiple networks operating on different levels (Zhang and Kay 2010).Itisnot within the scope of this review to present any detailsaboutthemolecularcomponentsofcircadian clocks in various organisms. However, since the basic principle of how molecular clocks work is similarinallspecies,wewilltakeacloserlookat the molecular clock of mammals (Fig. 3). Thetranscription-translationfeedbackloopof mammalsiscomposedofapositive,representedby Clock and Bmal1 andanegativelimb,represented by Per1, Per2, Cry1 and Cry2. During the day, CLOCKandBMAL1proteinsformaheterodimer thatactsasatranscriptionalfactor,bindingtoE-box promoter regions of various genes, including Pers and Crys, and activating their transcription. The resulting PER and CRYproteins heterodimerize and translocate back to the nucleus where they inhibit the transcriptional activity of the CLOCK/ Košir: Circadian rhythms Figure 3: Molecular organization of circadian rhythms. Although different model organisms have different clock components the overall architecture of the transcriptional translational feedback loop is similar. The CLOCK/BMAL1heterodimerpresentsthepositivelimbandactivatestranscriptionofvariouscoreclock andclockoutputgenes.PERandCRYproteinsrepresentthenegativelimbthatinhibitsCLOCK/BMAL1 transcriptional activation. PER and CRYdegradation leads to a new round of CLOCK/BMAL1 initiated activation. Various other loops (D-box and RRE) can influence the core clock mechanism. In addition DNAmethylation and chromatin modifications influence various components of the clock mechanism. Clockcontrolledgenes(CCG)regulate circadianphysiological processesandcanalsofeedbackinformation to the core clock mechanism. Slika 3: Molekularna osnova cirkadianih ritmov. Kljub razlikam v sestavi genov in proteinov, ki tvorijo mole- kularno osnovo cirkadianih ur pri razlicnih organizmih, pa je arhitektura transkripcijsko translacijske povratne zanke pri vseh podobna. Heterodimer CLOCK/BMAL1 aktivira izražanje genov centralne cirkadiane ure in output genov. Proteina PER in CRYpredstavljata negativno povratno zanko, ki inhibira transkripcijsko aktivnost heterodimera CLOCK/BMAL1. Proteolitska razgradnja PER in CRY proteinov povzroci ponovno aktivacijo transkripcije preko heterodimera CLOCK/BMAL1. Poleg opisanih, obstajajo še druge zanke, kot sta D-box in E-box zanka, ki lahko vplivajo na mehanizem centralne ure. Mehanizem centralne cirkadiane ure je podvržen tudi regulaciji preko DNAmetilacije in modifikacije kromatina. CCG (Clock controled genes): geni, ki jih uravnava cirkadiana ura, omogocajo cirkadiano izražanje fizioloških procesov, hkrati pa lahko tudi posredujejo informacije nazaj k cirkadiani uri. BMAL1 heterodimer. During the night however, responseelement(RRE)loop,whichiscomposed the PER/CRYheterodimer is degraded enabling ofproteinsbelongingtothenuclearreceptorfamily a new round of transcription by the CLOCK/ of transcriptional factors. By binding to the RRE BMAL1 dimer to start. This whole process takes element inpromoterregionsof Bmal1 proteins such about 24h to complete (Ko and Takahashi 2006). as Rora, Rorß and Ror. or Rev-erba and Rev-erbß In addition to the core loop other loops exist that activate or repress its transcription respectively interact with the core loop. One such is the REV-(Preitner et al. 2002). Similar to the RRE, the D Acta Biologica Slovenica, 56 (1), 2013 box loop represents the third feedback loop and is generated by transcription factors D-box binding protein(DBP),thyrotrophembryonicfactor(TEF) and hepatic leukemia factor (HLF) as activators and E4 promoter-binding protein 4 (E4BP4) as a repressor (Fig. 3)(Takahashi et al. 2008). These additionalloopsareimportantbecausetheyprovide (1)robustnessoftheclock,(2)enabletheclockto receiveentrainmentsignalsfromvarioussources and(3)provideseveraldifferentclockoutputways (Zhang and Kay 2010). Regardless of the complexity of the loops mentioned above the circadian rhythm in cells is also controlled by other means. In mammals posttranslational modifications (PTM) play an importantrolebymodulatingproteinhalf-lifeand their subcellular location. All of the core clock proteinsinmammals(CLOCK,BMAL1,PERsand CRYs)areknowntobemodifiedbyoneorseveral modifications including phosphorylation (all), acetylation(BMAL1,PER2),ubiquitination(all) and sumoylation (BMAL1) (Bellet and SassoneCorsi2010). PTMarealsoimportantforepigenetic controloftheclocknexttoDNAmethylationand miRNA.Severalstudieshaveshownthatchromatin remodelingisinvolvedinexpressionofcircadian genes as well as that chromatin modifications appear to follow a circadian pattern at different clock controlled genes (CCG) (Curtis et al. 2004, Doi et al. 2006, Bellet and Sassone-Corsi 2010). It is evident that the control regulation of circadian clocks in cells is a complexprocess in- volvingdifferentlevelsofregulationrangingfrom transcriptional control all the way to epigenetic modifications. Likewise, because of the interaction between different molecular loops that feed informationintothecorecircadianloop,theclock is well integrated with other physiological processesandviceversa. Theexactinterplaybetween the clock and cell physiology and metabolism is still a matter of research, but much has been learned in recent years. Interplaybetween circadianrhythms andmetabolism? In multicellular organisms such as mammals light cannot reach every cell in the body and therefore cannot synchronize the clock in these cellsdirectly.Forthisreasonthecircadiansystem evolvedahierarchicalstructureinwhichamaster clockresidinginthesuprachiasmaticnuclei(SCN) ofthehypothalamussynchronizesperipheralclocks in various tissues such as liver, adipose tissue, heart, intestine and adrenal gland (Fig.4). The SCN receives light signals from the retina through the retinohypotalamic tract and thereby synchronizes its internal clock to the outside world (Reppert and Weaver 2002). It is responsiblefordrivingvariousbehaviorrhythms (e.g.locomotoractivity)aswellassynchronizing circadian clocks in peripheral tissues, with the help of neural and humoral signals, to maintain proper phase relationships and prevent clocks in these tissues from dampening out (Dickmeis 2009). While the SCN is primarily entrained by light, peripheral tissues can in addition to SCN signals, also be entrained to various other stimuli among which feeding is the dominant zeitgeber (Damiola et al. 2000). There has been a lot of debate in recent years of whether the SCN can also be entrained by temperature fluctuations or not. While some publications have shown this to be true (Ruby et al. 1999, Herzog and Huckfeldt 2003) other have proven the opposite (Buhr et al. 2010).Whathasbeenshowbyallisthefactthata singleSCNneuroncanbeaffectedbytemperature fluctuations,howeverfortheSCNasawholethis has not yet been proven and is still a matter of further research. Itwasnotuntilonlyrecentlythattheinfluence ofcircadianclocksonmetabolismbecameevident in mammals. With the use of DNAmicroarrays it was shown that between 5 % and 20 % of all transcripts in a particular tissue have circadian profiles of expression. Different tissues showed only limited overlap between rhythmic genes, suggesting that the expression is regulated in a tissue specific manner (Akhtar et al. 2002, Durgan et al. 2006, Zvonicet al. 2006, Kosir et al. 2012). Amonggenesshowntohaverhythmicexpression were transcripts involved in gluconeogenesis, gly- colysis,lipidandcholesterolmetabolism,steroid hormone synthesis and xenobiotic metabolism (Green et al. 2008, Acimovic et al. 2011, Zmrzljak and Rozman 2012, Kosir et al. 2013). It has also been discovered that different hormones regulatingmetabolisminmammals includingglucagon, insulin, leptin, adiponectin and corticosterone Košir: Circadian rhythms Figure 4: Anatomicalorganizationofcircadianrhythmsinmammals.Inmammalsthecircadiansystemiscomposed of a master oscillator located in the suprachiasmatic nucleus and is synchronized to the outside world by lightpulsesthatreachitfromtheretinathroughtheretinohypotalamictract.TheSCNcontrolsperipheral clocks in tissues through various humoral and neural signals. In addition to SCN signals, food can also entrain some peripheral tissues especially liver. Adrenal glands excrete glucocorticoids in a circadian fashion that can also influence expression of genes in other tissues. Slika 4: Anatomska struktura cirkadianih ritmov pri sesalcih. Pri sesalcih je cirkadiani sistem zgrajen iz glavne cirkadiane ure, ki se nahaja v suprahiazmaticnem jedru (SCN) v hipotalamusu, ter perifernih cirkadianih ur, ki se nahajajo v razlicnih organih. SCN se vsakodnevno sinhronizira z zunanjimi pogoji svetlobe in teme, preko retine in retinohipotalamicnega trakta. V nadaljevanju SCN preko živcnih ali hormonskih poti sinhronizira periferne cirkadiane ure. Periferne cirkadiane ure se lahko sinhronizirajo tudi z drugimi signali, neodvisno od SCN, kot je npr. hrana. Nadledvicna žleza izloca tudi glukokortikoide, ki prav tako vpliva na izražanje genov v nekaterih perifernih tkivih. Koncentracija glukokortikoidov, kot je kortizol ali kortikosteron je v plazmi cirkadiana. show circadian oscillations (Froy 2011). These examples clearly show a direct influence of the circadian clock on various metabolic processes, but there are several key metabolic factors which can also influence the core clock mechanism. We previously already mentioned REV-ERBa and RORa, that regulate the expression of Bmal1 but are also important in adipocyte differentiation and regulation of lipogenesis respectively (Froy 2011). PPARa, another member of the nuclear receptor family, is important in lipidand glucose metabolism. It shows circadian rhythmicity but also activates the transcription of Bmal1,indicating yet another feedback loop of the clock (Canaple et al. 2006). Other molecules such as AMPK (AMP-activatedproteinkinase),PGC-1a(PPARg co-activator 1a) and SIRT1 (sirtuin I) have also been implicated in the regulation of clock genes either directly through transcription (PGC-1a) or indirectly through phosphorylation (AMPK) and deacetylation (SIRT1) (Canto and Auwerx 2009). Theimportanceofanintactcircadianclockfor normalhomeostasisandmetabolismhasbeenwell established and it has been show that disruption ofcircadianrhythmsmayleadtodevelopmentof various forms of metabolic syndrome (Green et al. 2008, Froy 2011, Naik et al. 2013). The most compellingevidencecomes frommousemodels. Here both obesity and metabolic syndrome have beendiscoveredinmicecarryingmutationsincore clock genes. For example Bmal1 knock-out mice arecompletelyarrhythmicandhavedisruptionsin rhythmic levels of glucose and triglycerides. To see whether these disruptions are a consequence of the lossof rhythmicity of the SCNor of peripheraloscillators, Bmal1 liverandpancreasspecific knock-out mice we generated. Despite normal locomotorrhythmbothtissuespecificknock-outs Acta Biologica Slovenica, 56 (1), 2013 Table 3: Mouse experimental models. Examples of metabolic defects in mice with mutations or gene knock-outs of clock genes. Based on Froy (2011) and Sahar and Sassone-Corsi (2012). Tabela 3:Eksperimentalni mišji modeli. Primeri metabolnih motenj, ki se pojavijo pri miših z mutacijami ali izbitimi geni cirkadiane ure. Povzeto po Froy (2011) ter Sahar in Sassone-Corsi (2012). Genemutatedorknocked-out MetabolicConsequence Clock Hyperlipidemia, hyperleptinemia, hypoinsulinemic and hyperglycemia Bmal 1 Abolished oscillations in plasma glucose and triglycerides impaired gluconeogenesis, hyperleptinemia, glucose intolerance, and dyslipidemia Per 1 Increased urinary sodium excretion Per 2 Altered lipid metabolism, lower body weight Cry 1 and Cry 2 Hyperglycemia Salt-sensitive hypertension Reverba Increased serum triglycerides Rora Reduced plasma triglycerides and HDL Enhanced atherosclerosis Pgc-1a Increased sensitivity to insulin Altered thermogenesis Nocturnin Resistant to diet-induced obesity Altered lipid metabolism displayed disturbances in blood glucose levels (Sahar and Sassone-Corsi 2012).Severalothermouse models with mutations or deletions of core clock genes have been generated that display perturbation to normal metabolism (Tab. 3). In addition tomousegeneticmodelsepidemiologicalstudies on humans have identified a correlation between shift work and metabolic disorders. Humans that were active and eating during normal night were shown to have decreased leptin (adipose tissue specifichormonethatpromotessatiety)levelsand increasedinsulinandglucoselevels.Leptinlevels were also found to be reduced in healthy patients that were subjected to only 4 hours of sleep in six consecutive nights (Spiegel et al. 2004). It is interestingtonotethatinthesametimeperiodthat we have seen an increase in metabolic diseases and obesity we have also seen a decrease in the quality and duration of sleep. Low quality and duration of sleep and disruptions of the normal circadian rhythm can also be related to another problem facing modern societies: light pollution. Light pollution is defined as artificial light (usually over illuminated streets, buildings, commercial ads etc.) present during the otherwise dark night. The effects of light pollution on various animal species have been well established unfortunately less research has been done on human subjects. Nevertheless a study doneinIsraelcomparedthelevelofartificiallight at night and occurrence of breast cancer in 147 communities and discovered that women living inareaswithhighnightlighthadagreaterchance for developing breast cancer (Kloog et al. 2011). Several studies have shown that the production of the night hormone melatonin, by the pineal gland, is abruptly terminated when individuals are exposed to light during the night faze. Since melatonin is known for helping to regulate the body’s biologic clock, it might be an important link between the disrupted circadianclock of the body and light pollution (Chepesiuk 2009). Conclusion The presence of circadian rhythms in almost allorganismsrangingfrombacteriaandunicellular eukaryotes to multicellular organisms including humans clearly shows their importance and evolutionaryadvantage. Whilealothasbeenlearned in the six decades of circadian rhythm research it is only in the last few years that we began to appreciatetheirimportanceinhumanhealth.The alarming increase in the rate of hypertension, obesity, metabolic syndrome and cancer worldwide, especially in developed and developing countries, could well be related to a disrupted Košir: Circadian rhythms circadianrhythmcausedbylifestylechanges.For thisreasonmuchresearchisneededtocompletely understandtheintricaterelationshipsbetweenthe circadian clock and metabolism as well as the circadian clock and cancer to eventually be able to reset the inner clock and prevent metabolic or cancer disorders from developing. Povzetek Cirkadiani ritmi so biološki ritmi, ki se ponavljajo s periodo okoli 24h in predstavljajo pomembno evolucijsko adaptacijo organizmov na ciklicne spremembe v okolju, ki so posledica vrtenja Zemlje okoli svoje osi. Najdemo jih v skorajdavsehorganizmihodbakterijpavsedoljudi, kjer uravnavajo številne fiziološke in metabolne procese.Zacetkiobširnejšihraziskavcirkadianih ritmov segajo v 50. leta 20. stoletja, ko sta Colin Pittendrigh in Jürgen Aschoff predvsem z opazovanjem sprememb obnašanja živali razkrila osnovneznacilnosticirkadianihritmovinnjihove lastnosti. Moderna doba raziskav cirkadianih ritmov,kijevkljucevalatudimolekularneosnove ritma, pa se je pricela šele v 70 letih20. stoletja. Vtemcasu sta Ron Konopka in Seymour Benzer odkrila prve mutante lokusa period pri vinski mušici (D. melanogaster), ki so povzrocile spremenjen cirkadiani ritem lokomotorne aktivnosti mušic. Kmalu so z uporabo razlicnih modelnih organizmov kot so N. crassa, D. melanogaster, M. musculus in še nekaterih drugih odkrili, da je osnovni molekularni mehanizem cirkadiane ure pri vseh organizmih zelo podoben. Osnova ritma References je transkirpcijsko translacijska povratna zanka, ki je npr. pri sesalcih sestavljena iz aktivatorjev, kot sta Clock in Bmal1, ter represorjev, kot so družina genov period in kriptokrom. Proteina CLOCK in BMAL1 v heterodimeru delujeta kot transkripcijska faktorja, saj aktivirata izražanje represorjev kot tudi številnih drugih genov uravnavanih s cirkadiano uro. Proteini PER in CRY pa delujejo tako, da preprecijo traskripcijsko aktivnost heterodimera CLOCK/BMAL1 in ustavijo transkripcijo tako sebe kot drugih genov. Po dolocenem casu se proteini PER in CRYrazgradijointakoomogocijo,daseaktivacija transkripcijesCLOCK/BMAL1ponovnopricne. Celoten cikel traja približno 24 ur da se ponovi. Predvsem pri višjih organizmih, kot so sesalci in clovek, v zadnjih nekaj letih prihaja vedno bolj do izraza prepletenost cirkadiane ure in razlicnih fizioloških procesov ter metabolizma. Postalo je jasno,dalahkoporušencirkadianiritempovzroci nastanek razlicnih metabolnih motenj, kot sta diabetes in prekomerna teža. Nadaljne raziskave bodopripomoglekboljšemurazumevanjuprepleta med cirkadiano uro in metabolizmom ter morda v prihodnosti omogocile izdelavo režima, s katerimbomovzpostavilinormalnodelovanje, sicer porušene cirkadiane ure pri številnih bolnikih, in tako pripomogli k njihovemu zdravljenju. Acknowledgement This study was supported by the Slovenian Research Agency program, project J7-4053 and by funds from Diagenomi, d.o.o. Acimovic, J., Kosir, R., Kastelec, D., Perse, M., Majdic, G., Rozman, D., Kosmelj, K.Golicnik, M., 2011. Circadian rhythm of cholesterol synthesis in mouse liver: a statistical analysis of the post- squalene metabolites in wild-type and Crem-knock-out mice. Biochem Biophys Res Commun, 408 (4), 635–641. Akhtar, R. A., Reddy, A. B., Maywood, E. S., Clayton, J. D., King, V. M., Smith, A. G., Gant, T. W., Hastings, M. H.Kyriacou, C. P., 2002. 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