Autonomous And Self-sustained Circadian Oscillators .
Diabetologia (2013) 56:497–507DOI 10.1007/s00125-012-2779-7ARTICLEAutonomous and self-sustained circadian oscillatorsdisplayed in human islet cellsP. Pulimeno & T. Mannic & D. Sage & L. Giovannoni &P. Salmon & S. Lemeille & M. Giry-Laterriere & M. Unser &D. Bosco & C. Bauer & J. Morf & P. Halban & J. Philippe &C. DibnerReceived: 25 July 2012 / Accepted: 18 October 2012 / Published online: 15 December 2012# The Author(s) 2012. This article is published with open access at Springerlink.comAbstractAims/hypothesis Following on from the emerging importance of the pancreas circadian clock on islet function andthe development of type 2 diabetes in rodent models, weaimed to examine circadian gene expression in human islets.The oscillator properties were assessed in intact islets aswell as in beta cells.Methods We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known asArntl)-luciferase reporter. Beta cells were stably labelled usinga rat insulin2 promoter fluorescent construct. Single-islet/celloscillation profiles were measured by combined bioluminescence–fluorescence time-lapse microscopy.Results Human islets synchronised in vitro exhibited selfsustained circadian oscillations of Bmal1-luciferase expressionat both the population and single-islet levels, with periodlengths of 23.6 and 23.9 h, respectively. Endogenous BMAL1and CRY1 transcript expression was circadian in synchronisedislets over 48 h, and antiphasic to REV-ERBα (also known asNR1D1), PER1, PER2, PER3 and DBP transcript circadianprofiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed isletcells were strongly oscillating as well, at population and singlecell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other.Conclusions/interpretation We provide for the first timecompelling evidence for high-amplitude cell-autonomouscircadian oscillators displayed in human pancreatic isletsand in dispersed human islet cells. Moreover, these clocksare synchronised between beta and non-beta cells in primaryhuman islet cell cultures.P. Pulimeno, T. Mannic and D. Sage contributed equally to this study.Electronic supplementary material The online version of this article(doi:10.1007/s00125-012-2779-7) contains peer-reviewed but uneditedsupplementary material, which is available to authorised users.P. Pulimeno : T. Mannic : L. Giovannoni : S. Lemeille :J. Philippe : C. Dibner (*)Division of Endocrinology, Diabetes and Nutrition,University Hospital of Geneva (HUG),Aile Jura 4-771.2, Rue Gabrielle-Perret-Gentil, 4,CH-1211 Geneva, Switzerlande-mail: Charna.Dibner@hcuge.chD. Sage : M. UnserBiomedical Imaging Group,Ecole Polytechnique Fédérale de Lausanne (EPFL),Lausanne, SwitzerlandP. Salmon : M. Giry-LaterriereDepartment of Neurosciences, Faculty of Medicine,University of Geneva,Geneva, SwitzerlandD. BoscoCell Isolation and Transplantation Centre, University Hospital,Geneva, SwitzerlandC. BauerNCCR Frontiers in Genetics, Bioimaging Platform, Sciences II,University of Geneva,Geneva, SwitzerlandJ. MorfDepartment of Molecular Biology, Sciences II,University of Geneva,Geneva, SwitzerlandP. HalbanDepartment of Genetic Medicine and Development,University Medical Centre, University of Geneva,Geneva, Switzerland
498Keywords Beta cells . Circadian clocks . Human pancreaticislets . Time-lapse microscopyAbbreviationsBMAL1 Brain and muscle ARNT-like protein 1CGECircadian gene expressCLOCK Circadian locomotor output cycles kaputCMRLConnaught Medical Research LaboratoriesmediumCRYCryptochromeGFPGreen fluorescent proteinGSISGlucose-stimulated insulin secretionMOIMultiplicity of infectionqPCRQuantitative PCRPERPeriodRIPRat insulin 2 promoterSCNSuprachiasmatic nucleusIntroductionCircadian oscillations of biological processes have beendescribed in virtually all light-sensitive organisms. Theyreflect the existence of underlying intrinsic clocks with near24 h oscillation periods. Circadian control of physiologyand behaviour is driven by a master pacemaker located inthe suprachiasmatic nucleus (SCN) of the hypothalamus,which orchestrates subsidiary oscillators in peripheralorgans. Rhythmicity in the SCN is entrained by externalZeitgeber (time-giver) cues, such as daily changes in lightintensity [1]. In mammals, the CLOCK (circadian locomotoroutput cycles kaput) and BMAL1 (brain and muscle ARNTlike protein 1) transcription factors activate the expressionof Per and Cry genes. Once the period (PER) and cryptochrome (CRY) proteins have reached a critical threshold,they attenuate the CLOCK/BMAL1-mediated activation oftheir own genes in a negative feedback loop [1, 2]. Thisrhythm-generating circuitry is functional in most cell types,including primary and immortalised cell lines [3, 4].There is growing evidence for connections between metabolic syndromes, including obesity and diabetes, and thecircadian clockwork [5]. Rhythmically expressed livergenes code for enzymes implicated in the metabolism offatty acids, cholesterol, bile acids and xenobiotics, and thedisruption of circadian oscillator function causes alterationsin metabolism [6]. Animals with a liver-specific Bmal1 (alsoknown as Arntl) disruption suffer from hypoglycaemia specifically during the resting phase [7]. Mice with disruptedcircadian clock function (homozygous mutants of Clock)develop hyperphagia, obesity and features of the metabolicsyndrome [8]. Further evidence of the interconnection between circadian oscillators and metabolism stems fromDiabetologia (2013) 56:497–507several recent studies on the core clock gene Rev-erbα (alsoknown as Nr1d1), which has been found to be indispensablefor proper lipid and carbohydrate metabolism [9–11]. Moreover, CRY1 and CRY2 modulate fasting glucose levelsthrough the inhibition of glucagon-induced gluconeogenesis[12]. Recently, evidence for self-sustained oscillators inmouse pancreatic islets has been provided by Bass andcolleagues [13]. This work indicated that Clock 19 mutantmice exhibited hyperglycaemia, elevated fasting glucoselevels and impaired glucose tolerance. A similar phenotypewas observed in Bmal1 knockout mice, suggesting thatthese metabolic alterations are not dependent on CLOCKfunction alone, but are general for core clock genes. Mostimportantly, this study revealed that ablation of the pancreatic clock might directly trigger onset of diabetes, representing an important milestone in our understanding of themolecular link between the clock, islet function and type 2diabetes aetiology [13]. In line with these findings,pancreas-specific Bmal1 knockout in a different mousemodel led to impaired insulin secretion [14].In humans, glucose homeostasis is tightly controlled bythe circadian system [15]. Genetic linkage analysis hasshown that CRY2 and PER2 might be associated with bloodglucose levels [16, 17]. Moreover, patients with circadianmisalignments show profound perturbations of plasma glucose and insulin levels [6]. Finally, core clock gene expression analysis in human islets demonstrated that mRNAlevels of PER2, PER3 and CRY2 might be downregulatedin isolated islets from individuals with type 2 diabetes incomparison with islets from healthy counterparts, suggesting the link between the human islet clock and type 2diabetes [18]. It is therefore of scientific and clinical importance to provide further insight into the emerging connectionbetween circadian oscillator function, metabolic regulationand type 2 diabetes in humans, with a specific focus on thehuman islet circadian oscillator molecular makeup.MethodsHuman islet preparation Human pancreases were obtainedfrom brain-dead multi-organ donors. Details of islet donorsare summarised in Table 1. Human islet isolations wereperformed at the Islet Transplantation Centre at the University Hospital of Geneva (Switzerland) as previously described [19, 20]. The use of human islets for researchwas approved by the local ethical committee. Islet puritywas estimated by dithizone staining; islet viability wasassessed by propidium iodide and fluorescein diacetatestaining. After purification islets were cultured in Connaught Medical Research Laboratories (CMRL) mediumfor 24–72 h before the experiments. Whole islets wereattached to dish surfaces covered with a laminin-5-rich
Diabetologia (2013) 56:497–507499Table 1 Islet donor ty(%)Viability(%)123456781–8MFFMMMMMM06, F02585656564852615655 4a29.323.819.729.120.323.127.828.625 4a909090909595858590 4a798587709496476077 17aM, male sex; F, female sexa1-min intervals. The Actimetrics LumiCycle Analysis program was used for analysis of rhythm variables (Actimetrics,Wilmette, IL, USA).Temperature entrainment Synchronisation with temperaturecycles was performed according to the protocol describedpreviously [27]. Briefly, simulated circadian body temperaturecycles (38–34 C) were generated in a custom-built incubatorequipped with photomultiplier tubes for simultaneous bioluminescence recording [27]. Luminescence traces are eithershown as raw or detrended data. For detrended time series,luminescence signals were smoothened by a moving averagewith a window of 60 data points and detrended by an additional moving average with a window of 24 h.Data are means SD, n08extracellular matrix derived from 804G cells [21]. Todissociate islets into single cells, islets were resuspended in Accutase (Innovative Cell Technologies,San Diego, CA, USA), incubated at 37 C until completelydissociated and then diluted in CMRL medium.Lentivectors and lentiviral production The Bmal1-luciferase (luc) lentivector was engineered by Liu and colleagues[22]. CMV-GFP lentivector (pLOX-CW-GFP) was previously described by us [23]. pRIP-GFP and pRIP-tomato wereengineered using the rat insulin 2 (RIP) promoter [23],controlling the production of green fluorescent protein(GFP) or the tdTomato red living colour [24], respectively.Maps and sequences of lentivectors are available at ourwebsite (http://medweb2.unige.ch/salmon/lentilab/). Lentiviral particles were produced by transient transfection in293T cells using the polyethylenimine method [25]. Lentiviral particles were harvested at 48 h post transfection, 100fold concentrated, titred and used for the transduction ofwhole islets or islet cells. Multiplicity of infection (MOI) ofthree was found optimal in this system.In vitro islet synchronisation and bioluminescence monitoring To synchronise islets/islet cells, dexamethasone wasadded to the culture medium at a final concentration of100 nmol/l. Following 30 min incubation at 37 C in a cellculture incubator, dexamethasone was replaced with normalculture medium, as previously described by us [26]. Bioluminescence patterns were monitored from human islets or isletcells 4 days following Bmal1-luc lentiviral transduction, asdescribed previously [3, 26]. Briefly, synchronised islets/isletcells were transferred to a light-tight incubator (37 C) in therecording medium (CMRL containing 100 μmol/l luciferin).Bioluminescence from each dish was continuously monitoredusing a Hamamatsu photomultiplier tube detector assembly(Hamamatsu City, Japan). Photon counts were integrated overBioluminescence time-lapse microscopy and data analysisWhole islets or dispersed islet cells were attached to glassbottom dishes and transduced with Bmal1-luc, or withBmal1-luc and RIP-tomato lentivectors, respectively. Fourdays after lentiviral transduction islets/islet cells weresynchronised with dexamethasone and subjected to combined bioluminescence–fluorescence imaging on an OlympusLV200 workstation (Bioimaging Platform, Geneva,Switzerland) equipped with a 63 UPLSAPO objective. Bioluminescence emission was detected for several consecutivedays using an EM CCD camera (Image EM C9100-13,Hamamatsu) cooled to 90 C using exposure times of30 min. The image series were analysed employing theImageJ 1.32 software (http://imagej.nih.gov/ij/). The imageanalysis has been done by an adapted version of the software Circadian Gene Express (CGE, http://bigwww.epfl.ch/sage/soft/circadian/), a Java ImageJ’s plugin that we previously developed for tracking cells in the context of circadianstudies [26, 28]. The measure of expression has beenadapted to the islets considering that the labelled cellsare mainly present at the periphery. The level of expression is obtained by subtracting the average of intensityinside a ring at the periphery of the islet and theaverage of intensity outside the islets, which representsthe local background level (Fig. 1e). CGE now includesa module to fit a model to a profile of expression. Themodel that we have chosen is a sinusoid:mðtÞ ¼ A sin!"2p tþθ þBTThe fitting is performed by a Levenberg–Marquardt optimisation procedure to recover the amplitude A and theperiod T. The bioluminescence image sequence has beende-noised using the PURE-LET method [29]. This methodis optimised for images corrupted by mixed Poisson–
500Diabetologia (2013) 56:497–507nitrogen and kept at 80 C. Total RNA was prepared usingRNeasy Plus Micro kit (Qiagen, Hombrechtikon, Switzerland). One μg of total RNA was reverse-transcribed usingSuperscript III reverse transcriptase (Invitrogen, Carlsbad,CA, USA) and random hexamers, PCR amplified and quantified as described previously [11]. Mean values for eachexperiment were calculated from technical triplicates ofPCR assays for each sample, and normalised to the meanof those obtained for GAPDH and 9S transcripts. GAPDHand 9S expression levels exhibited no significant variabilitythroughout each experiment and therefore served as internalcontrols. Primers used for this study are listed in electronicsupplementary material (ESM) Table 1.ResultsFig. 1 Cell-autonomous oscillators in human pancreatic islets.(a) Bmal1-luc and CMV-GFP co-expression in whole islets. Whole isletswere co-transduced with lentiviral particles expressing Bmal1-luc andCMV-GFP constructs. Four days after infection, GFP expression evaluation was performed in n020 islets from two donors (ten islets from eachdonor). At least half of the cells were GFP positive in all visualised islets(representative CMV-GFP transduced islet is shown). (b) Bmal1-lucoscillation profile in synchronised islets. Islets were synchronised withdexamethasone and Bmal1-luc bioluminescence was recorded using anActimetric Lumicycler for 200 h. Bmal1-luc oscillation profiles wererecorded for three parallel dishes for each donor, total of five donors. (c,d) Individual islet circadian bioluminescence recording (ESM Video 1,left). Human islets were cultured on 804G matrix-covered dishes andtransduced with Bmal1-luc viral particles. (c) Differential interferencecontrast (DIC) image. (d) Bioluminescence channel from the time-lapsemicroscopy images. (e) Trajectories of seven individual islets image byadapted CGE application (image corresponds to ESM Video 1, right). (f)Representative individual islet profile: the raw data (red curve) and thefitted sinusoid (black curve) are shownGaussian noise and it improves the signal-to-noise ratio ofthe bioluminescence signal.RNA analysis by real-time quantitative PCR Attached intactislets were synchronised by dexamethasone as describedabove, collected every 4 h for 48 h, deep-frozen in liquidRecording circadian bioluminescence from human pancreatic islets Bioluminescence profiles from cells producingluciferase driven from different circadian regulatory sequences can be recorded at the population level using photomultiplier tubes, or at the single-cell level using highlysensitive photon-counting microscopy [2]. We decided toemploy both approaches to decipher the human islet clockwork. First, we set up the experimental system for long-termrecordings of circadian reporter oscillations in cultured human islets. Whole islets were attached to dishes coveredwith a laminin-5-rich extracellular matrix derived from804G cells [21] and transduced with lentivectors expressingthe Bmal1-luc construct [30]. Transduction efficiency inwhole islets was evaluated using a CMV-GFP lentivector[23] co-transduced with Bmal1-luc. At least half of the cellswere GFP positive in all visualised islets, with intensiveGFP staining observed on the islet surface, and significantlyweaker penetration to the inner core (Fig. 1a). To access theimpact of lentiviral transduction on islet function, glucosestimulated insulin secretion (GSIS) was measured in theislet cells co-transduced with CMV-GFP and RIP-tomatolentivectors 5 days following the transduction, and compared with non-transduced counterparts (ESM Fig. 1, ESMMethods). No significant difference in GSIS was observedupon lentiviral transduction.Cell-autonomous and self-sustained circadian clocks displayed in isolated human islets To monitor circadian oscillation profiles in human islets, we first recorded circadianbioluminescence in the isolated human islets. Intact isletsexpressing Bmal1-luc lentivectors were synchronised by adexamethasone pulse 4 days after the viral transduction, andcircadian bioluminescence was continuously recorded for several days in an Actimetrics Lumicycler (Fig. 1b). We used theglucocorticoid hormone analogue dexamethasone for islet synchronisation since it was previously demonstrated to
Diabetologia (2013) 56:497–507synchronise circadian oscillators in cultured rat-1 and NIH3T3fibroblasts efficiently. In addition it transiently changes thephase of circadian gene expression in liver, kidney and heart[26, 31, 32], therefore representing a strong in vitro synchronisation stimulus. Our experiments revealed pronounced oscillations of Bmal1-luc expression with a period length of 23.6 0.4 h (n05; Fig. 1b), indicating the presence of functional isletautonomous circadian clocks in human islet population.We next sought to visualise single-islet oscillations employing a bioluminescent time-lapse microscopy approach. Bmal1luc transduced human islets were synchronised by a dexamethasone pulse and subjected to bioluminescence time-lapse microscopy recording for at least three consecutive days (ESMVideo 1 [left] and Fig. 1c, d). The Bmal1-luc bioluminescenceprofiles of individual human islets were analysed by anupdated version of the CGE software previously developedby us [28], specifically adapted to the islet analysis (ESMVideo 1 [right] and Fig. 1e). A representative profile of bioluminescence oscillations in a human islet is shown in Fig. 1f. Ofthe analysed single islets, 76% (13 out of 17) were oscillatingfor 3 days of recording, with an oscillation period of 23.99 1.20 h, closely matching the results obtained in the populationof human islets (Fig. 1b). Our single-islet recording experiments unambiguously demonstrated the presence of highamplitude cell-autonomous oscillators in human islets. Of note,this visualisation further ensures that Bmal1-luc oscillationswere recorded from the islets themselves and not from adjacentcells or tissue fragments, which represent 5–15% contamination of human islet preparations (Table 1).Multiple in vitro stimuli have previously been demonstrated to efficiently synchronise cultured cells [32]. Amongthem, forskolin has been used for in vitro synchronisation ofisolated mouse islets [13]. As demonstrated in ESM Fig. 2,short pulses of dexamethasone, horse serum or forskolinwere all able to strongly synchronise the islets isolated frommouse bearing the circadian Per2::luc reporter [33]. Furthermore, human islets expressing Bmal1-luc were subjected to simulated body temperature cycles oscillatingbetween 38 C and 34 C (Fig. 2a). Similar to dexamethasonesynchronisation, continuous temperature cycles resulted inhigh-amplitude circadian oscillations of Bmal1-luc bioluminescence in human islets (Fig. 2b, c).To validate our results obtained by circadian bioluminescence recording, we examined endogenous core clock geneexpression profiles in the s
Results Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9 h, respectively. Endogenous BMAL1 and
Background It is well established that affective disorders are associ-ated with sleep-wake and circadian disturbance [1-4]. Indeed, circadian dysfunction is postulated to play a role in the pathogenesis of mood disturbance onset, mainten-ance and recurrence [5-12]. In younger people with affective disorders, data on sleep-wake and circadian
higher) than that of a circadian rhythm. „Infradian rhythms – biological rhythms with a cycle of more than 24 hours (e.g. the human menstrual cycle). Circadian Rhythms „ Circadian is derived from a Latin phrase meaning "about a day“ [about (circa) and a day (dia)] „ Circadian rhyt
Circadian rhythms refer to self-sustained fluctuations with a period of approximately (circa) 1 day (diem) in various physiological processes. Circadian rhythmicity is ob-served for many hormones in circulation (i.e., corticosteroids) as well as for circulating immune cells and cytokines
Page 2 Autonomous Systems Working Group Charter n Autonomous systems are here today.How do we envision autonomous systems of the future? n Our purpose is to explore the 'what ifs' of future autonomous systems'. - 10 years from now what are the emerging applications / autonomous platform of interest? - What are common needs/requirements across different autonomous
a cell, these oscillations are self-sustained and cell autonomous, which constitute fundamental properties that characterize circadian rhythms. The activity of the cellular oscillators is orchestrated by the master clock, a pacemaker located in
4.19 Type 2 low frequency self-sustained oscillations, measured at E13. . . 56 4.20 Type 1 low frequency self-sustained oscillations, measured at E13. . . 57 4.21 Type one low frequency self-sustained oscillations, measured at E13. . 58 4.
used. The circadian preference of students was assessed using the reduced morningness-eveningness questionnaire (rMEQ). Several significant results were observed and there was a significant di erence in circadian preference among the three tested groups, with RN students scoring highest on the morningness scale and MLD students scoring the .
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