Molecular Mechanism On Circadian Clock Protein BMAL1 Regulating Rhythmic Accumulation Of Liver Glycogen Through Glycogen Synthase 2

Hepatic glycogen synthesis is a key link for the body to maintain the glucose homeostasis,and the disorder of the body’s glucose metabolism is closely related to the incidence of several metabolic disease,including diabetes and fatty liver.Glycogen synthase 2（GYS2）is the rate-limiting enzyme of liver glycogen synthesis,and its m RNA and protein expression levels show obvious circadian rhythmic changes in mice livers.The expression of GYS2 m RNA and protein in the livers of mice lacked Clock or Per2 gene function was significantly lower than that of wild-type（WT）mice,and their circadian rhythmic changes were lost.The mammalian central circadian clock（suprachiasmatic nucleus SCN）and the peripheral circadian clock（liver,muscle and pancreas）coordinately regulate the homeostasis of glucose metabolism from multiple levels.As the core transcription factor of the circadian clock system,BMAL1deficiency leads to a significant decrease and loss of rhythm in the expression of Gys2 m RNA in the liver of mice.However,it is still unclear how BMAL1 regulates the rhythmic expression of Gys2 in liver,and how it affects the rhythmic accumulation of liver glycogen,and further research is urgently needed.In this study,WT mice,Bmal1 systemic knockout mice(Bmal1^-/-)and Bma1 liver-specific knockout mice(L-Bmal1^-/-)were used as models,q PCR,Western Blot,PAS staining,hepatocyte primary culture,the dual luciferase report assay,biofluorescence measurement system,and the electrophoretic mobility shift assay（EMSA）techniques were used to investigated the molecular mechanism of the BMAL1 regulating the rhythmic accumulation of liver glycogen through GYS2.The main research results are as follows:1.Under both ZT and CT conditions,the changes of circadian clock genes and Gys2 gene expression,liver glycogen content and serum glucose concentration in liver of WT mice were explored under ad libitum feeding state.The q PCR results showed that the m RNA expression levels of circadian clock genes（Per1,Per2,Per3,Bmal1,Clock,Nr1d1,Dbp）and Gys2 gene in WT mice liver had rhythmic changes under both ZT and CT conditions.The results of PAS staining and quantitative detection of liver glycogen showed that there were obvious circadian rhythmic changes in mouse liver glycogen content under ZT and CT conditions.In addition,there are rhythmic changes in serum glucose concentration under both ZT and CT conditions.The above results suggest that the circadian clock may play an important role in regulating the rhythmic expression of Gys2 and the rhythmic accumulation of liver glycogen,while light has no significant effect on the rhythmic expression of liver Gys2 gene m RNA and the rhythmic accumulation of liver glycogen in mice.Note:Zeitgeber Time（ZT）,ZT0 is the start of light,ZT12 is the end of light;Circadian Time（CT）,the mouse is placed in a continuous dark environment,CT0 is the beginning of the main sightseeing photo,and CT12 is the end of the main sightseeing photo.2.Under CT conditions,the diurnal changes of Gys2 gene m RNA expression,liver glycogen content,and serum glucose concentration in the liver of WT mice after fasting were explored.The q PCR results showed that the m RNA expression levels of circadian clock genes（Per2,Per3,Bmal1,Clock,Nr1d1,Dbp）and Gys2 gene in WT mice livers were decreased significantly under continuous starvation,but still exhibiting circadian rhythmic changes.At the same time,the results of PAS staining and quantitative detection of liver glycogen showed that the liver glycogen content of mice after fasting decreased sharply with the prolongation of fasting time,but still maintaining circadian rhythmic accumulation.In addition,the serum glucose concentration of mice under continuous starvation displayed a downward trend,but there were still significant circadian rhythmic changes.The above results indicate that the circadian clock may play an important role in regulating the rhythmic expression of Gys2 and the rhythmic accumulation of liver glycogen,while and feeding is not a necessary condition for determining the rhythmic expression of Gys2 gene m RNA and the rhythmic accumulation of liver glycogen in mice.3.Under CT conditions,the effects of systemic knockout of Bmal1 gene on the m RNA and protein expression of liver GYS2,liver glycogen content and serum glucose homeostasis were explored in mice.The results showed that systemic knockout of Bmal1 gene resulted in a significant decrease in the m RNA expression of Clock,Per2,Nr1d1 and Dbp genes in the liver of mice,and the m RNA expression of Per1 and Cry1 genes was up-regulated.Compared with the control mice,the m RNA expression of Gys2 gene in the liver of Bmal1^-/-mice also decreased significantly and lost rhythm changes.At the same time,the expression of GYS2protein in the liver of Bmal1^-/-mice decreased.In addition,the results of PAS staining and quantitative detection of liver glycogen content of Bmal1^-/-mice was lower than that of the control mice,and the rhythmic changes were lost.The results of mouse primary hepatocyte detection showed that GYS2 m RNA and protein expression of Bmal1^-/-mice were significantly lower than those of control mice.Glucose tolerance test and insulin tolerance test data show that Bmal1^-/-mice have abnormal glucose metabolism,which manifested by impaired glucose tolerance and decreased insulin sensitivity.4.Under CT conditions,the effects of liver-specific knockout of Bmal1 gene on the m RNA and protein expression of GYS2,liver glycogen content and serum glucose homeostasis were further explored in mice.The q PCR results showed that the liver-specific knockout of Bmal1 gene significantly decreased the m RNA expression of circadian clock genes（Nr1d1,Dbp）in the liver of mice,while the m RNA expression of Clock and Cry1 genes increased significantly,and other circadian clock genes（Per1,Per2）were no significant change in m RNA expression.Compared with the control mice,the expression of Gys2 gene m RNA in the liver of L-Bmal1^-/-mice decreased significantly,and the rhythmic changes were lost.Western Blot results showed that the expression of GYS2 protein in the liver of L-Bmal1^-/-mice decreased compared with the control group.In addition,the results of PAS staining and quantitative detection of liver glycogen content of L-Bmal1^-/-mice was lower than that of the control mice,and the rhythmic changes were weakened.The results of the mouse primary hepatocyte detection showed that the GYS2 m RNA and protein expression of L-Bmal1^-/-mice were significantly lower than those of the control mice.Glucose tolerance test and insulin tolerance test showed that,L-Bmal1^-/-mice had abnormal glucose metabolism ability,which manifested impaired glucose tolerance and decreased insulin sensitivity.5.In order to further analyze the molecular mechanism of the circadian clock protein BMAL1 regulating the rhythmic transcription of Gys2 gene,this study constructed the luciferase reporter gene plasmids which containing two E-box or E-box mutant DNA sequences at the end of the 7th intron of the Gys2 gene.The results of the dual luciferase report assay showed that the relative luciferase activity of the single E-box element mutation group and the double E-box element mutation group both showed different degrees of decline.Among them,the luciferase expression activity of the double E-box element mutation group decreased the most,reaching more than 95%.Using the Kronos AB-2550 real-time biofluorescence measurement system,we further found that the luciferase activity of the double E-box element mutation group was significantly lower than that of the WT control group,and the rhythmic changes were lost.EMSA results showed that BMAL1 protein specifically binds to the two E-box elements at the seventh intron of Gys2.In summary,this study found that the liver-specific knockout of the Bmal1 gene resulted in impaired liver glycogen synthesis in mice and weakened rhythmic changes,confirming that the rhythmic accumulation of liver glycogen in mice was directly regulated by the liver circadian clock system.In addition,the molecular mechanism on circadian clock protein BMAL1 regulation of Gys2 rhythmic expression through binding the two E-box elements within the 7th intron of Gys2,which in thus affects rhythmic accumulation of liver glycogen,was further dissected.This study will provide a more comprehensive understanding of the regulation network of liver glucose metabolism from the perspective of the circadian clock,and provide a basic and theoretical proof for further analysis of the molecular mechanism of the liver circadian clock on regulating the homeostasis of glucose metabolism,and also provide novel ideas for the prevention and treatment of diseases related to the body’s glucose metabolism.