The Molecular Mechanism Of The Circadian Clock Gene ROR? Regulating Betatrophin

Many mammals have physiological circadian rhythms,such as sleep-awakeness,blood pressure,serum hormone levels and energy metabolism all show a fluctuation for the period of 24-hour,which adapts to the diurnal cycle of Earth's rotation.In mammals,the clock system is divided into two categories:central and peripheral biological clocks.The central clock is located in the hypothalamus suprachiasmatic nucleus(SCN),which accepts and integrates external optical signals through the nervous control and endocrine regulation,affecting physiological processes in tissues and organs.The peripheral clock is synchronized by the central clock operation,maintaining the body's internal optimization to use energy and to adapt to the external environmental factors,such as changes in light and feeding cycles.Biological clock controls the body's neuroendocrine system and metabolic system precisely.Studies have confirmed that approximately 43%genes in the entire mammalian genome exhibit circadian oscillations,many of which encode metabolic enzymes that play key roles or serve as transcription factors in various metabolic processes such as hepatic gluconeogenesis,fat metabolism and bile acid metabolism.Light cues synchronize the master clock that conveys temporal cues to other peripheral clocks via neuronal and hormonal signals.On the other hand,feeding can reset the phase of most peripheral clocks.In addition to the neuroendocrine regulation of circadian clock,hormones also orchestrate clock operation as internal time-givers.For example,adrenal glucocorticoids,pineal melatonin and adipocyte-derived leptin participate in the internal synchronization(coupling)within the multi-oscillatory network.Furthermore,pancreatic insulin is involved in food synchronization of peripheral clocks,while stomach ghrelin provides temporal signals modulating behavioral anticipation of mealtime.Circadian desynchronization induced by shift work or chronic jet lag has harmful effects on metabolic regulation,thus favoring diabetes and obesity.Conversely,adequate timing of endocrine therapies can promote phase-adjustment of the master clock(e.g.via melatonin agonists)and peripheral clocks(e.g.via glucocorticoid agonists).Recent studies have reported that betatrophin is a novel hormone-like protein which is dominantly secreted by the liver and fat in mice,and it plays important roles in triglyceride and glucose metabolism.However,the relationship between betatrophin and the clock remains unknown.In the current study,we first placed mice in a completely dark environment,and found the expression of betatrophin showed a rhythmic oscillation in various metabolic organs such as liver,white and brown adipose tissues.By using restriction feeding experimentss,we found that feeding time switch reversed the oscillation phase of hepatic betatrophin expression,indicating that betatrophin expression is regulated by the periphery clock.In addition,the expression levels of betatrophin in mouse liver were decreased by fasting,while recovered after re-feeding.Interestingly,betatrophin expression was significantly increased in liver of ob/ob and high-fat diet-induced obese mice,showing that nutritional signals also regulate betatrophin expression.At the molecular level,RORa(retinoic acid receptor a,RORa)is a nuclear receptor and activates the transcriptional activity of Bmall/Clock.It also plays a key role in the reglation of lipid metabolism.Our study found that betatrophin proximal promoter contained two typical RORa binding sequences(RORE).Indeed,luciferase reporter gene assays proved that RORa enhanced the transcriptional activity of betatrophin promoter,and this activation effect disappeared when RORE motifs were mutated.ChIP assays confirmed that RORa binds to RORE in the betatrophin promoter and changed the chromatin structure into an activation state.In addition,adenovirus-mediated overexpression of ROR? in HepG2 cells upregulated the expression of betatrophin.Functionally,serum shock experiments indicated that knockdown of betatrophin by siRNA interference in HepG2 cells altered the oscillation patterns of lipid metabolism-related genes.In summary,our study suggests that betatrophin is a new clock-control gene(CCG),and is positively regulated by clock gene RORa.In addition,betatrophin may be involved in the regulation of lipid metabolic gene oscillations.