Dataset Information

Phospholipase C beta 4 in mouse hepatocytes: rhythmic expression and cellular distribution.

ABSTRACT:

Background

Circadian regulated physiological processes have been well documented in the mammalian liver. Phospholipases are important mediators of both cytoplasmic and nuclear signaling mechanisms in hepatocytes, and despite a potentially critical role for these enzymes in regulating the temporal aspect of hepatic physiology, their involvement in the circadian liver clock has not been the subject of much investigation. The phospholipase C beta4 (PLCbeta4) enzyme is of particular interest as it has been linked to circadian clock function. In general, there is no knowledge of the role of the PLCbeta4 isozyme in mammalian hepatocytes as this is the first report of its expression in the mammalian liver.

Results

We found that in the liver of mice housed on a light:dark cycle, PLCbeta4 protein underwent a significant circadian rhythm with a peak occurring during the early night. In constant darkness, the protein rhythm was more robust and peaked around dusk. We also observed a significant oscillation in plcbeta4 gene expression in the livers of mice housed in both photoperiodic and constant dark conditions. The cellular distribution of the protein in hepatocytes varied over the course of the circadian day with PLCbeta4 primarily cytoplasmic around dusk and nuclear at dawn.

Conclusion

Our results indicate that PLCbeta4 gene and protein expression is regulated by a circadian clock in the mouse liver and is not dependent on the external photoperiod. A light-independent daily translocation of PLCbeta4 implies that it may play a key role in nuclear signaling in hepatocytes and serve as a daily temporal cue for physiological processes in the liver.

SUBMITTER: Klein BM

PROVIDER: S-EPMC2583973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo, hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo. Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia-inducible factors (HIF)-1/2? western blotting, and real-time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF-2? expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3-phosphate dehydrogenase [Gapdh], glucose transporter 1 [Glut1], pyruvate dehydrogenase kinase 1 [Pdk1], and lactate dehydrogenase A [Ldha]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (Cpt1), apolipoprotein B (Apob), and acetyl-coenzyme A carboxylase 1 (Acc1), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4? (Hnf4?) and farnesoid X receptor (Fxr) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF-2 signaling was involved in hypoxia-induced down-regulation of Fxr. Conclusion: Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. (Hepatology Communications 2018;2:299-312).