High-Resolution Recording of the Circadian Oscillator in Primary Mouse α- and β-Cell Culture

Abstract

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

The importance of being rhythmic: Living in harmony with your body clocks

“…31,54,61,[65][66][67][68][69][70][71][72][73][74][75][76][77] Interestingly, circadian phases of the core clock components, as well as of a high number of key functional genes, were not aligned between α-and β-cells within the pancreatic islet, which may play a role in fine-tuning of temporal profiles of insulin and glucagon secretion. [61][62][63][64] In agreement with rodent studies, emerging work in humans conducted through serial tissue biopsies collected across 24 hours, and in primary cells synchronized in vitro, suggest that a considerable portion of transcripts in these organs exhibit rhythmic expression. 31,41,[78][79][80] Circadian transcription reconstruction analysis through cyclic ordering by periodic structure (CYCLOPS) conducted by John Hogenesch and colleagues across 13 tissues derived from a cohort of over 600 human donors provides a valuable database for future studies of clock outputs in humans.…”

“…Detoxification and metabolic function of the liver, response to insulin by skeletal muscle or white adipose tissue, as well as hormone and cytokine secretion by pancreatic islet and other endocrine organs are all subject to circadian variations ( Figure 1B). 48,[57][58][59][60][61][62][63][64] Around-the-clock transcriptome profiling conducted in different organs strongly suggests that the temporal orchestration of metabolism represents a major purpose of circadian clocks ( Figure 1B; Figure 2, left panel). 31,54,61,[65][66][67][68][69][70][71][72][73][74][75][76][77] Interestingly, circadian phases of the core clock components, as well as of a high number of key functional genes, were not aligned between α-and β-cells within the pancreatic islet, which may play a role in fine-tuning of temporal profiles of insulin and glucagon secretion.…”

Time zones of pancreatic islet metabolism

“…Our own recent study focuses on the molecular characterization of the α‐ and β‐cellular clocks, their interactions, impact on gene transcription and islet hormone secretion . In order to assess in parallel α‐ and β‐cell circadian properties, experimental settings that allow the simultaneous monitoring of the circadian clock in separated α‐ and β‐cells have been developed . This was achieved by combining the proglucagon (Gcg)‐Venus mouse strain for α‐cell‐specific labelling, the β‐cell‐specific rat insulin2 promoter (RIP)‐Cherry reporter mouse, and the Period2::Luciferase (Per2::Luc) knock‐in mouse, allowing for continuous assessment of circadian bioluminescence at population and single‐cell levels.…”

“…Moreover, temporal islet‐ and islet‐regulating hormone misalignment impacts on the development of metabolic diseases. Indeed, studies of genetic mouse models with whole‐body and islet‐specific clock disruption have shown perturbed insulin and glucagon secretion (Figure ) leading to the development of overt T2D . Similarly, clock disruption in the human islet results in decreased overall levels and perturbed temporal patterns of insulin and glucagon secretion (Figure and Petrenko and Dibner, unpublished data).…”

“…47 In order to assess in parallel αand β-cell circadian properties, experimental settings that allow the simultaneous monitoring of the circadian clock in separated αand β-cells have been developed. 89 This was achieved by combining the proglucagon (Gcg)-Venus mouse strain for α-cellspecific labelling, 90 the β-cell-specific rat insulin2 promoter (RIP)-Cherry reporter mouse, 91 and the Period2::Luciferase (Per2::Luc) knock-in mouse, 92 allowing for continuous assessment of circadian bioluminescence at population and single-cell levels. Strikingly, the group of genes that is rhythmically expressed in αand β-cells with distinct circadian properties comprises mostly the core-clock components.…”