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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 Front+Endocrinol+(Lausanne)
2017 ; 8
(ä): 68
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High-Resolution Recording of the Circadian Oscillator in Primary Mouse ?- and
?-Cell Culture
#MMPMID28439257
Petrenko V
; Gosmain Y
; Dibner C
Front Endocrinol (Lausanne)
2017[]; 8
(ä): 68
PMID28439257
show ga
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 ?-cell clocks have been
extensively studied during the last years, ?-cell clocks and their role in islet
function and orchestration of glucose metabolism stayed unexplored, largely due
to the difficulty to isolate ?-cells, which represents a considerable technical
challenge. Here, we provide a detailed description of an experimental approach
for the isolation of separate mouse ?- and ?-cell population, culture of isolated
primary ?- and ?-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 ?- and ?-cells, and luciferase reporter for
monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell
sorting allowed separating pure ?- and ?-cell populations from isolated islets.
Experimental conditions, developed by us for the culture of functional primary
mouse ?- and ?-cells for at least 10?days, will be highlighted. Importantly,
temporal analysis of freshly isolated ?- and ?-cells around-the-clock revealed
preserved rhythmicity of core clock genes expression. Finally, we describe the
setting to assess circadian rhythm in cultured ?- and ?-cells synchronized in
vitro. The here-described methodology allows to analyze the functional properties
of primary ?- and ?-cells under physiological or pathophysiological conditions
and to assess the islet cellular clock properties.
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