The Function Of KLF15 Clock-Controlled Pathway On The Branched Chain Amino Acid Metabolism In The Muscle Of Siniperea Chuatsi During Starvation

Skeletal muscle is not only the main protein storage in the body,but also the tissue with active metabolism and high plasticity.Branched chain amino acids(BCAA)are the only amino acids metabolized at a high rate in skeletal muscle.Under starvation and other stress conditions,BCAA can enhance oxidation and provide energy to maintain normal physiological activities.The expression of Krüppel like factor 15(KLF15)in the muscle of adult mice showed a significant circadian rhythm and was involved in the regulation of BCAA metabolism in skeletal muscle.The Chinese perch(Siniperca chuatsi)has become one of the important species of freshwater aquaculture in China because of its delicious meat and high nutritional value.In this study,we studied the adaptive mechanism of core clock gene,KLF15 and key enzymes of BCAA metabolism in Siniperca chuatsi muscle under different starvation conditions.We screened the core clock gene and BCAA metabolism key enzyme gene which are closely related to KLF15 gene rhythm expression under different starvation time,and preliminarily verified KLF15-BCAA clock regulation pathway through dual luciferase reporter gene experiment and gene knockout technology.The main results were as following:1.The core biological clock genes with strong correlation with the rhythmic expression of KLF15 gene under different starvation duration were screened.In normal feeding,only Tim gene and KLF15 gene showed strong correlation.There was a strong correlation among Clock,Tim and KLF15 genes at 1 day of starvation.There was no correlation between KLF15 gene and core circadian clock gene at 3 days of starvation.Only Clock gene was strongly correlated with KLF15 gene at 5 days of starvation.There was a strong correlation among the genes of Clock,Arntl1,Per2 and KLF15.Among them,Clock gene had a strong correlation with KLF15 gene under multiple starvation durations,and it was positively correlated.The results showed that with the change of starvation duration,the core clock system will dynamically regulate KLF15 gene through the combination of different core clock genes so as to cope with the adverse effects of starvation.2.The key enzyme genes of BCAA metabolism were screened,which were strongly related to the rhythmic expression of KLF15 gene in different starvation time.The enzyme activity analysis showed that in five different starvation periods,the BCAT2 enzyme concentration showed significant circadian rhythms except for the destruction of circadian rhythm during 1 day of starvation.ALT activity had no circadian rhythm during normal feeding,but showed significant circadian rhythm after starvation.The results of gene expression showed that BCAT2 gene showed significant circadian rhythm expression during normal feeding,1 day of starvation,3 days of starvation and 5days of starvation,and its circadian rhythm disappeared after 7 days of starvation.ALT gene showed significant circadian rhythm expression in five different starvation duration.The correlation analysis between BCAT2 gene and KLF15 gene showed that there was a significant moderate positive correlation on the first day of starvation,and a significant strong positive correlation on the third and seventh day of starvation,but the circadian rhythm of BCAT2 gene disappeared on the seventh day of starvation.There was no significant correlation between other starvation duration.However,there was no significant correlation between ALT gene and KLF15 gene in the five treatment groups.In conclusion,KLF15 may mediate BCAA metabolism by dynamically and positively regulating BCAT2 m RNA expression and enzyme levels during different periods of starvation.3.Preliminary verification of KLF15-BCAA clock control pathway.Bioinformatics analysis demonstrated that there were nine potential Clock binding sites in the promoter region of KLF15 gene and one KLF15 binding site in the promoter region of BCAT2 gene.The dual luciferase reporter system preliminarily confirmed that Clock could significantly promote the transcription activity of KLF15(P<0.05),and KLF15 could also significantly promote the transcriptional activity of BCAT2(P<0.05).After intramuscular injection of vivo-morpholinos,the m RNA and protein levels of KLF15 were significantly decreased(P<0.05),and the m RNA level of BCAT2 was also significantly decreased(P<0.05).The m RNA level of ALT showed an decreasing trend,but there was no significant change(P>0.05).There were no significant changes in BCAT2 enzyme concentration,ALT activity,BCAA and Ala contents in muscle(P>0.05),but the BCAA and Ala contents in serum were notably decreased(P<0.05).In conclusion,KLF15 can positively regulate the transcriptional expression of BCAT2.