Circadian Rhythms of PER2::LUC in Individual Primary Mouse Hepatocytes and Cultures

Casey J Guenthner,Lorna A Pyle,Penny C Molyneux,Alexander S Li,Tanya L Leise,Mary E Harrington,Jimmy K Yu,Martha E Luitje,Shin Yamazaki

doi:10.1371/journal.pone.0087573

Abstract

BackgroundHepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown.ResultsIn this study we isolated primary hepatocytes from transgenic Per2Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2−/− Per2Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes.ConclusionsOur results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.

Highlights

Circadian or daily rhythms are internally regulated by the hypothalamic suprachiasmatic nucleus (SCN) and are externally entrained by environmental factors such as light and food intake [1]
Yoo et al [6] showed that liver explants from Per2Luc transgenic mice remained rhythmic for more than 20 days in vitro, undisturbed, and rhythmicity was observed even when liver explants were prepared from SCN-ablated animals
In an initial set of experiments, collagen gel sandwich hepatocyte cultures were observed over 3 weeks from day in vitro 5 (DIV 5) to DIV 28 with medium changes on DIV 4, 5, 12, 13, 20, 21 and 28, 29

Summary

Introduction

Circadian or daily rhythms are internally regulated by the hypothalamic suprachiasmatic nucleus (SCN) and are externally entrained by environmental factors such as light and food intake [1]. Cells throughout the body can generate circadian oscillations using transcriptional-translational feedback loops involving several genes, including Period (Per2) and Cryptochrome 2 (Cry2) on the negative limb and Bmal on the positive limb of the main feedback loop [1]. Recent advances allow in vivo imaging of gene expression in the liver and demonstrate that the liver can support circadian cycles even in SCN-ablated mice [8,9]. These studies must be interpreted with caution, since it is possible that surgery may have synchronized the liver oscillators in [9], and in [8] only one circadian cycle was observed, with injections of luciferin and anesthesia at each measurement time point. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown

Methods

Results

Conclusion