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.
Highlights
The circadian system represents a complex anticipatory mechanism developed during evolution in most organisms, allowing to coordinate a plethora of physiological functions to the daily changes of geophysical time
The major obstacle for studying α- and β-cells is that they are organized in the tight three-dimensional structure within the of the sorted cells
Note that Cherry-positive β-cells have greater granularity when compared to Venus-positive α-cells. (C) Dot plots for DRAQ7-based assessment of sorted cell viability, indicating approximately 90% alive cells in the preparation. (D) Representative histograms showing number of dead α- and β-cells among analyzed events and (E) corresponding quantitative data from 12 independent experiments, suggesting higher cell death for β-cell population, as compared to α-cells. (F) Histogram indicating average numbers of obtained α- and β-cells per mouse (N = 12 independent experiments with three to six mice per experiment), statistical difference illustrate more than three times greater content of β-cells within sorted population, as compared to α-cells
Summary
The circadian system represents a complex anticipatory mechanism developed during evolution in most organisms, allowing to coordinate a plethora of physiological functions to the daily changes of geophysical time. Within this system, a master pacemaker in the hypothalamus orchestrates. The molecular composition of central and peripheral oscillators is identical, and it relies on primary and secondary feedback loops of transcription and translation of key core clock components [4]. Studies in clock-deficient genetic rodent models suggest that a number of metabolic defects develop in mice that are deficient for one or two core clock components [11, 12]. Clock mutant mice develop hyperphagia, obesity, hyperglycemia, and hypoinsulinemia [12]
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