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

Summary

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.

Abstract

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.

Introduction

The circadian timing system (from Latin "Circa diem") 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 ....

Protocol

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

1. Human Pancreatic Islet Isolation, Dissociation and Culture
   NOTE: Coat every tube, plastic tip or pipette with Connaught Medical Resear....

Results

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 .......

Discussion

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.

The quality of the donor ma.......

Materials

List of materials used in this article

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