Name: parallel measurement of circadian clock gene expression and hormone secretion in human primary cell cultures
Uploaded: 2016-11-11T15:00:00
Description: Watch this Scientific Journal Video about Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures at JoVE.com

We are grateful to our colleagues from the University of Geneva: Jacques Philippe for constructive comments on this work, Ueli Schibler for invaluable help with the development of the perifusion system and for scientific inspiration, Andr&#233; Liani for the having conceived design, manufacturing and commissioning of the perfusion system, Lesa-Technology LTD company for the assistance in the perifusion system and Drip-biolumicorder software development, George Severi for assistance with the perifusion experiments, Ursula Loizides-Mangold for critically reading the manuscript, and Anne-Marie Makhlouf for lentivirus preparations; to Etienne Lefai, St&#233;phanie Chanon and Hubert Vidal (INSERM, Lyon) for preparing human primary myoblasts; and to Domenico Bosco and Thierry Berney (Human Islet Transplantation Center, Geneva University Hospital) for providing human islets. This work was funded by the Swiss National Science Foundation Grant No. 31003A_146475/1, the Sinergia Swiss National Science Foundation Grant No. CRSII3-154405, Fondation Romande pour la Recherche sur Diab&#232;te, Bo Hjelt Foundation, Fondation Ernst et Lucie Schmidheiny, and Soci&#233;t&#233; Acad&#233;mique de Gen&#232;ve (CD).

Ethics statement: Manipulations included in this protocol were approved by the Ethics Committee of the Geneva University Hospital and by the Ethical Committee SUD EST IV (Agreement 12/111) 19. Human islets were isolated from pancreases of brain-dead multi-organ donors in the Islet Transplantation Centre at the University Hospital of Geneva (Switzerland) as described by us in references 16,18, or obtained from a commercial source.
1. Preparation of Primary Cell Culture
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The experimental settings described here are composed of lentiviral delivery of circadian bioluminescence reporters into cultured human primary cells, followed by subsequent in vitro synchronization and continuous recording of bioluminescence for several days, and parallel analysis of hormone secretion by the same cells. They represent an efficient approach for exploring molecular mechanisms and functional aspects of circadian clocks in human primary cells.
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The circadian timing system (from Latin &#34;Circa diem&#34;) has emerged in all light-sensitive organisms, as an adaptive mechanism to the rotation of the Earth. In mammals, it is organized in a hierarchical manner, encompassing the central clock, which is situated in the suprachiasmatic nucleus of the ventral hypothalamus, and peripheral (or slave) oscillators that are operative in different organs. Moreover, these cell autonomous self-sustained oscillators are functional in nearly every cell of the body 1. Photic signals represent a dominant synchronizing cue (Zeitgeber) for the SCN neurons, whereas neural and humoral signals emanating from the SCN reset the peripheral clocks. In addition rest-activity rhythms, that drive in turn feeding-fasting cycles, are further synchronizers for peripheral clocks 2. According to our current understanding, the molecular makeup of the core clock is based on transcriptional and translational feedback loops, which are conserved between organisms. This comprises the transcriptional activators BMAL1 and CLOCK, which together activate transcription of the negative core clock PER and CRY genes. High levels of PER and CRY proteins will inhibit their own transcription through inhibition of the BMAL1/CLOCK complex. An auxiliary loop consists of the nuclear receptors REV-ERBs and RORs, which also regulate the transcription of BMAL1 and CLOCK. Furthermore, posttranslational events including phosphorylation, sumoylation, acetylation, O-GlcNAcylation, degradation and nuclear entry of the core clock proteins represent an additional important regulatory layer in establishing the 24 hr oscillation cycle 3.
Accumulating evidence stems from studies in rodent models and highlights the critical role of the circadian system in the coordination of metabolic and endocrine functions 4-5. A number of large-scale transcriptome analysis suggest that feeding &#8211; fasting cycles play a central role in the synchronization of peripheral oscillators 6-8. In an agreement with these studies, metabolomic and lipidomic analysis in rodents and humans have revealed that a large number of metabolites oscillate in tissue, plasma, and saliva in a circadian manner 9-11. Importantly, most hormones exhibit circadian rhythms in blood 5,12-13. Moreover, circadian clocks of the corresponding hormone producing peripheral tissue might regulate hormone secretion locally. Cell-autonomous circadian oscillators have been described in rodent and human pancreatic islet cells 14-16. These oscillators play an essential role in regulating the pancreatic islet transcriptome and function 15,17-18. Furthermore, myokine secretion by human skeletal myotubes has been recently demonstrated to exhibit a circadian pattern, which is regulated by cell-autonomous oscillators operative in these cells 19.
Several approaches for studying circadian rhythms in humans in vivo have been widely used. For instance, plasma melatonin or cortisol levels as well as thoracic skin surface temperature (reviewed in references 3,20) have been studied to assess endogenous circadian clocks. Although these methods allow studying systemic circadian oscillations in vivo, they are far from providing a reliable assessment of free-running autonomous circadian rhythms in different organs and tissues. Nevertheless, such dissection from the systemic regulation would be an indispensable tool for understanding the specific effect of intracellular molecular clocks on the function of these cells. Therefore, a substantial effort has been undertaken to develop reliable approaches for studying human clocks in immortalized or primary cultured cells synchronized in vitro. Importantly, it has been demonstrated that clock characteristics measured in cultured primary skin fibroblast cells closely reflect the individual clock properties of the whole organism 21. The development of fluorescent and bioluminescent circadian reporters has greatly advanced this approach 22-27. Furthermore, studying primary cell clocks that are derived from different peripheral organs allows for the investigation of the molecular properties of human tissue-specific clocks 3,5,16,19-20,28. Thus, assessment of circadian clocks in in vitro synchronized primary explants or cells, by using bioluminescent reporters, represents a highly useful method to study the molecular makeup of human peripheral clocks and their impact on organ function.
In this article, we will present detailed protocols for assessing circadian gene expression in human primary islet and skeletal muscle cells synchronized in vitro as well as the impact of autonomous cellular clock disruption on the secretory function of these cells.

<table border="0" cellpadding="0" cellspacing="0" width="1006">
	<tbody>
		<tr height="19">
			<td height="19" style="height:20px;width:215px;">Trypsin-EDTA</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">25300-054</td>
			<td style="width:560px;">For muscle biopsy digestion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">DPBS no calcium no magnesium</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HAM F-10</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">41550-021</td>
			<td>For myoblasts culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FBS</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">10270</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicillin-Streptomycin</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">P0781-100</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Gentamycin</td>
			<td style="width:112px;">Axon&#160;</td>
			<td style="width:119px;">A1492.0001</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fungizone</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">15290-026</td>
			<td>Amphotericin B, supplement to culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">DMEM 1g/L glucose + Na pyruvate + glutamax&#160;</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">21885-025</td>
			<td>For myotubes culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">DMEM 1g/L glucose -Na Pyruvate - glutamax</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">11880-028</td>
			<td>Recording medium for LumiCycle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glutamax</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">35050-028</td>
			<td>L-alanyl-L-glutamine dipeptide, supplement to recording medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Accutase</td>
			<td style="width:112px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT-104</td>
			<td>Cell detachment solution, for islet cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CMRL</td>
			<td style="width:112px;">Gibco</td>
			<td style="width:119px;">21530-027</td>
			<td>Culture medium for islet cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium Pyruvate</td>
			<td style="width:112px;">Gibco</td>
			<td style="width:119px;">11360-039</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;width:215px;">15 ml High-Clarity Polipropylene Conical Tube</td>
			<td style="width:112px;">Falcon</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">F75 flask</td>
			<td style="width:112px;">BD Falcon</td>
			<td style="width:119px;">353136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">3.5 cm Petri dish&#160;</td>
			<td style="width:112px;">BD Falcon</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Foskolin</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">F6886</td>
			<td>Adenylyl cyclase activator, used for synchronization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Luciferin</td>
			<td style="width:112px;">Prolume LTD</td>
			<td style="width:119px;">260150</td>
			<td>Supplement to recording medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">OptiMEM&#160;</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">51985-026</td>
			<td>Serum-free Minimal Essential Medium (MEM) used for human islet cells transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Lipofectamine RNAiMAX reagent</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">13778-150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HiPerFect reagent</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">301705</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ON-TARGET plus siCLOCK smartpool&#160;</td>
			<td style="width:112px;">Dharmacon</td>
			<td style="width:119px;">L-008212-00</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ON-TARGET plus non targeting siRNA #1 (siControl)</td>
			<td style="width:112px;">Dharmacon</td>
			<td style="width:119px;">D-001810-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DNeasy Blood &#38; Tissue Kit&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">69504</td>
			<td>For myotubes DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">RNeasy Plus Mini kit&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td>For myotubes RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">QIAshredder&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">79654</td>
			<td>For myotubes RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">2 ml collecting tubes</td>
			<td style="width:112px;">Axygen</td>
			<td style="width:119px;">311-10-051</td>
			<td>To collect the medium with the perifusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tissue culture Plate, 6 Well</td>
			<td style="width:112px;">BD Falcon&#160;</td>
			<td style="width:119px;">353046</td>
			<td>To collect the medium with the perifusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">RNeasy Plus Micro kit&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">74034</td>
			<td>For islet RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Human IL-6 Instant ELISA kit&#160;</td>
			<td style="width:112px;">eBioscience</td>
			<td style="width:119px;">88-7066-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Human Insulin Kit Mercodia</td>
			<td style="width:112px;">Mercodia</td>
			<td style="width:119px;">10-1113-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hydrochloric acid, min,37%,p.a.</td>
			<td style="width:112px;">Acros organics</td>
			<td style="width:119px;">124630010</td>
			<td>Used for preparation of lysis buffer (375ml Ethanol+7.5%HCl+117.5%H2O)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol (&#62;99.8%)</td>
			<td style="width:112px;">Fluka Analytical</td>
			<td style="width:119px;">02860-1L</td>
			<td>Used for preparation of lysis buffer (375ml Ethanol+7.5%HCl+117.5%H2O)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Islets for Research</td>
			<td colspan="2">Prodo Laboratories</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name</td>
			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Equipment:</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Centrifuge</td>
			<td style="width:112px;">Heraeus</td>
			<td style="width:119px;">Megafuge 1.0R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water bath</td>
			<td style="width:112px;">VWR</td>
			<td style="width:119px;">1112A</td>
			<td>&#160;at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tissu culture hood</td>
			<td style="width:112px;">Faster&#160;</td>
			<td style="width:119px;">SafeFastElite</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">Tissu culture incubator</td>
			<td style="width:112px;">Heraeus</td>
			<td style="width:119px;">HeraCell 150</td>
			<td style="width:560px;">5% CO2 at 37 &#176;C, no water due to the LumiCycle installation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">Tissu culture incubator</td>
			<td style="width:112px;">Heraeus</td>
			<td style="width:119px;">HeraCell 150</td>
			<td style="width:560px;">5% CO2 at 37 &#176;C, no water due to the LumiCycle installation</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:215px;">Tissu culture incubator</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td style="width:119px;">Hera Cell 150i</td>
			<td style="width:560px;">5% CO2 at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Shaker</td>
			<td style="width:112px;">Heidolph Instruments</td>
			<td style="width:119px;">Unimax 1010</td>
			<td>For agitation of the siRNA mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">LumiCycle</td>
			<td style="width:112px;">Actimetrics</td>
			<td style="width:119px;"></td>
			<td style="width:560px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">LumiCycle software</td>
			<td style="width:112px;">Actimetrics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CosinorJ software</td>
			<td style="width:112px;">EPFL</td>
			<td style="width:119px;"></td>
			<td>Freely available at: http://bigwww.epfl.ch/algorithms/cosinorj/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheodyne titan MX&#160;</td>
			<td style="width:112px;">ERC GmbH</td>
			<td style="width:119px;"></td>
			<td>Control software that controls the timing of the automated switch</td>
		</tr>
	</tbody>
</table>

parallel measurement of circadian clock gene expression and hormone secretion in human primary cell cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Assessment of Islet Hormone Secretion with Parallel Circadian Bioluminescence Recording from Perifused Human Islet Cells
After providing a first molecular characterization of the circadian clock, operative in human islet cells 16, we aimed at studying the impact of clock disruption on islet function and transcription 18. We set up an efficient siClock transfection protocol in dispersed human islet ...

Watch this Scientific Journal Video about Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures at JoVE.com

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

The overall goal of this procedure is the parallel assessment of circadian bioluminescence and the secretory activity of human primary endocrine cells cultured and synchronized en vitro.This method can help to investigate the temporal pattern of cell secretion and how it can be regulated by cell-autonomous circadian clocks.The main advantage of this technique is that it allows us to monitor the circadian luciferins as reporter activity and, simultaneously collect outflow medium for measuring the levels of secreted hormones and myokines.To introduce circadian bioluminescence reporters into primary cells by lentiviral transduction, first, transfer the 3.5 cm petri dishes containing human myoblasts at 30%to 50%confluency to a laminar flow cabinet.Replace the human myoblast medium with 2 mL of growth medium.Then transduce the primary cell culture by pipetting lentivirus solution into the dish to obtain a multiplicity of infection equal to three.Incubate the cells overnight in a tissue-culture incubator.Change medium the next day.To achieve en vitro synchronization, add 10 micromolar adenylyl cyclase activator in 2 mL of medium per 3.5 cm petri dish containing the previously transfected primary cells.Incubate the cells for 60 minutes at 37

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Genetics

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