Effect Of Tyr And Cys Combined With Formononetin On Hepatic Lipid Metabolism In Cold-Stimulated Mice

Livestock encountering cold stimulation are prone to slow growth,decreased production performance and reduced feeding efficiency.One study found that heart rate increased after 2hours of cold stimulation in meat sheep,respiratory rate decreased after 4 hours,and rectal temperature began to decrease after 12 hours.Lipids are important energy substances for livestock in low temperature environment,and the animal organism mainly consumes lipids to maintain normal metabolic functions under cold stimulation conditions.The liver is the central organ of the body to regulate lipid metabolism,providing energy for livestock and poultry to maintain the orderly occurrence of various physiological processes in a low temperature environment.Emergency energy mobilization in a hypothermic environment predisposes to mitochondrial dysfunction,and maintaining a healthy mitochondrial network is a prerequisite for safeguarding the body’s material metabolism.Mitophagy is a key component of the mitochondrial quality control system,which can remove damaged mitochondria to maintain mitochondrial health.Previous studies in our laboratory found that Tyrosine(Tyr)and Cysteine(Cys)as well-established feed additives can promote lipid metabolism in adipose and liver tissues and increase the level of energy metabolism in the body under cold stimulation conditions;Formononetin(FMN)is an isoflavonoid extracted from different plants.Previous laboratory studies also found that FMN acts synergistically with Tyr and Cys to promote lipid metabolism in the body.The biological role played by Tyr and Cys combined with FMN in hepatic lipid metabolism under cold stimulation conditions is not yet clear.Therefore,this study used mitophagy as a research target,aiming to investigate the effect of Tyr and Cys combined with FMN on hepatic lipid metabolism under cold stimulation conditions,and to provide a proven scheme of measures to improve the low-temperature adaptation of animal organisms.To initially explore the effect of Tyr and Cys combined with FMN on hepatic lipid metabolism,in this study,mice were treated with FMN by gavage on the basis of 3% Tyr and Cys in the diet of 4-week-old male C57BL/6 mice for 4 weeks,and liver tissues were collected at the end of gavage.The changes of TG and TC contents in mouse liver tissues were detected using triglyceride(TG)kit,total cholesterol(TC)kit,HE staining and oil red O staining.The results showed that the combination of Tyr and Cys with FMN led to a significant reduction in TG and TC contents and lipid droplet number in mouse liver tissues.The expression of lipid metabolism-related proteins in mouse liver tissues was detected by Western Blot technique,and it was found that the combination of Tyr and Cys FMN resulted in significantly higher expression levels of lipid synthesis-related proteins(FASN,ACC1,SREBP-1c)and very significantly higher expression levels of lipid catabolism-related proteins(PGC-1α,CPT1 A,ACOX1,PPAR-α)in mouse liver tissues compared with the control group(Control).The above results suggest that Tyr and Cys combined with FMN increased the utilization of TG and TC by mouse liver tissues.To confirm the effects of Tyr and Cys combined with FMN on liver lipid metabolism under cold stimulation conditions,4-week-old male C57BL/6 mice were randomly selected for 3 hours per day and placed in an artificial climate chamber at 4℃ for 4 weeks in this study;at the same time,FMN was given to mice by gavage on the basis of 3% Tyr and Cys in their diets,and liver tissues were collected at the end of gavage.Lipid metabolism,mitochondrial respiratory chain complexes and mitophagy related indexes were examined in liver tissue of each group of mice using TG and TC kits,HE staining,Oil Red O staining and Western Blot.The results showed that Tyr and Cys combined with FMN reduced the contents of TG and TC,the number of lipid droplets,the expression levels of lipid synthesis related proteins(FASN,ACC1,SREBP-1c),increased the expression levels of lipolysis-related proteins(PGC-1α,CPT1 A,ACOX1,PPAR-α)in the liver tissues of mice under cold stimulation condition compared with the cold stimulation group(CE).These results suggest that Tyr and Cys combined with FMN promote the utilization of TG and TC by liver tissues.Western Blot results showed that cold stimulation caused mitochondrial dysfunction in mouse liver tissue and induced PINK1-mediated mitochondrial autophagy to occur.In addition,Tyr and Cys combined with FMN led to adaptive enhancement of mitochondrial function in mouse liver tissue under cold stimulation,and the protein expression level of PINK1 was significantly increased and the occurrence of mitophagy was promoted.The above results indicate that Tyr and Cys combined with FMN can remove damaged mitochondria by promoting PINK1-mediated mitophagy pathway to maintain a healthy mitochondrial network under cold stimulation conditions.To verify the regulatory effect of FMN on PINK1-mediated mitophagy,this study used carbonyl cyanide m-chlorophenylhydrazone(CCCP)to construct a mitophagy model in AML12 cells,and FMN was treated with a time gradient.The optimal concentration and duration of action of CCCP and FMN were screened using CCK8,and changes in mitochondrial function and expression of mitophagy-related proteins were detected using Western blot.Mitochondrial membrane potential,Mito-ROS,autophagic flux and co-localization of LC3 with outer mitochondrial membrane localization protein(TOMM20)were detected using JC-1 fluorescent probe,Mito-SOX staining and immunofluorescent staining.Cell experiments showed that CCCP treatment decreased mitochondrial membrane potential,increased Mito-ROS production,impaired mitochondrial function,and induced PINK1-mediated mitophagy in AML12 cells compared with the control group.Compared with CCCP group,FMN treatment alleviated CCCP-induced mitochondrial dysfunction in AML12 cells,reduced the production of Mito-ROS,and maintained mitochondrial function by promoting PINK1-mediated mitophagy pathway to remove damaged mitochondria in AML12 cells.To verify the regulatory effect of Tyr and Cys combined with FMN on PINK1-mediated mitophagy,this study constructed a mitophagy model in AML12 cells using CCCP and stimulated AML12 cells with Tyr and Cys combined with FMN,and detected the changes of mitophagy-related protein expression using Western blot.The results showed that CCCP-induced Tyr and Cys combined with FMN treatment promoted PINK1-mediated mitophagy in AML12 cells.In conclusion,Tyr and Cys combined with FMN can maintain mitochondrial function by promoting PINK1-mediated mitophagy,enhance the expression of lipolysis related proteins and reduce the expression of lipid synthesis related proteins in mouse liver tissue under cold stimulation,thereby enhancing the utilization of TG and TC in liver tissue and improving the level of energy metabolism in the body.To compensate for the energy loss caused by cold stimulation and maintain the body’s metabolic homeostasis.