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

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.

Human primary cells can tell body time: Dedicated to Steven A. Brown

“…A comparison between circadian transcriptomic and lipidomic analyses from serial skeletal muscle tissue biopsies collected in vivo and primary myotubes synchronized in vitro suggests a significant effect of the cell‐autonomous muscle clock on the temporal regulation of the transcriptional and metabolic landscape (Loizides‐Mangold et al, 2017; Perrin et al, 2018). Furthermore, siRNA‐mediated clock disruption in human primary cells has been developed for studying the effect of clock perturbation on gene transcription, lipid metabolism, hormone or cytokine secretion (Loizides‐Mangold et al, 2017; Perrin et al, 2015; Perrin et al, 2018; Petrenko et al, 2016; Saini et al, 2016).…”