Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA209

Poster Communications

Novel method to study circadian regulation of insulin secretion in vitro

Z. Novosadova1,2, A. Sumova1

1. Department of neurohumoral regulation, Czech Academy of Sciences, Prague, Czech Republic. 2. Department of Genetics and Microbiology, Charles University, Prague, Czech Republic.

Insulin secretion from pancreatic islets is tightly controlled by a complex mechanism. There is an emerging evidence for the role of circadian system in these regulations. Circadian system in mammals consists of central light-entrainable oscillator located in the brain in suprachiasmatic nuclei (SCN) and peripheral oscillators in other cells in the body. Peripheral oscillators are entrained by the SCN and also by other cues (e.g. timing of food intake). The clock mechanism is based on molecular circuits involving so called clock genes, namely Bmal1, Clock, Per1, Per2 and Cry. Using ex vivo approach, it is possible to study intrinsic properties of peripheral oscillators in conditions when they are not under influence of the entraining signals. This method allows us to study mechanism of how circadian clock in pancreas contributes to regulation of insulin secretion in vitro. Long-term measurement of biological rhythmicity is essential for analysis of circadian rhythms which cannot be assessed based only on data from one cycle. Therefore, we propose a novel method to study islets in organotypic explants from pancreas, because the currently available systems using isolated islets of Langerhans are not well suited for this prolonged incubation. We prepared organotypic explants from pancreas of transgenic mice expressing clock gene Per2 fused with gene for luciferase enabling us to detect bioluminiscence rhythms of the pancreatic clock. Pancreatic explants exhibited robust rhythms in Per2 production for more than 10 days. Islets within the organotypic explants were subjected to repeated stimulation by higher glucose concentrations in media to test their viability. Insulin content was then measured by ELISA. After 3 days of incubation in low glucose (5mM), the insulin production was steady (up to 0,4ng/ml per day). Upon the stimulation with higher glucose (20mM), the islets within pancreatic explants exhibited higher insulin excretion rate (up to 1ng/ml per day). We can estimate the number of viable islets from the amount of excreted insulin into media. We were able to detect roughly 2 medium sized islets. The stimulations by higher glucose content in media were repeated at least twice, interrupted with low glucose (3mM), and insulin content changed accordingly. The results provided evidence for viability of the islets within the explants. The results allow us to propose a novel experimental approach for studying of long-term insulin secretion in pancreatic organotypic explants in vitro and evaluation of internal clock functions. Additionally, this novel method could be used in future for testing of drugs influencing the insulin excretion.

Where applicable, experiments conform with Society ethical requirements