Effect Of ATP On Mitochondrial MECR Protein In The Mechanism Of Insulin Resistance

Background:MECR(mitochondrial 2-enoyl-acyl-carrier protein reductase)is an enzyme in the mitochondrial fatty acid synthase(mt FAS)pathway.The relationship between MECR and type 2 diabetes has not been reported.In this study,we examined the relationship between MECR and insulin resistance in high fat induced obese(DIO)mice.Under the condition of insulin resistance,MECR mRNA expression increased in the liver,but MECR protein level decreased.After berberine(BBR)was used to improve insulin resistance in DIO mice,MECR protein level returned to normal.It is suggested that insulin can induce MECR mRNA expression.In addition,under the condition of insulin resistance,the level of ATP in liver increased and the level of MECR protein decreased.BBR restored MECR protein level by reducing ATP level in the liver of DIO mice.These data suggest that in insulin resistant mice,insulin induces an increase in liver mRNA levels,but high levels of ATP in the liver cause a decrease in MECR protein.BBR reduced the ATP level in the liver of mice,restored the MECR protein level,and improved the insulin sensitivity of DIO mice.This study shows that the increase of MECR mRNA and the decrease of MECR protein are closely related to insulin resistance,and the decrease of MECR protein level by ATP may be involved in the occurrence of insulin resistance.Objective:To study the relationship between mitochondrial protein MECR and type 2 diabetes mellitus,and to explore the changes of MECR in insulin resistance.Methods:(1)The relationship between MECR and insulin resistance was explored by cell experiment: The change of MECR in insulin resistance was studied by qRT-PCR from mRNA level.(2)Distribution of MECR expression in mouse tissues: The expression of MECR protein was detected by Western blot.(3)To establish animal models to explore the relationship between insulin resistance and MECR: We establish high-fat mice,berberine treated high-fat mice model to determine the relationship between insulin resistance and MECR by HOMA-IR value detection,qRT-PCR technology and Western blot technology.(4)Detection of ATP level: Detecting the concentration of ATP in the liver tissue of animal model.ATP concentration and MECR protein expression were detected in the cell model to determine the relationship between ATP and MECR.(5)Related mechanism validation: The protein expression of lipoic acid,the end product of lipoic acid synthesis pathway,was detected in the cell model to verify whether MECR played a role through lipoic acid synthesis pathway.Then in the animal model to determine whether the ubiquitin proteasome inhibitor MG132 affects MECR degradation.(6)Supplementary experiment: The changes of MECR protein were observed in ATP deficient rats and the expression of MECR mRNA was determined.Results:(1)In the cell model,we found that after insulin treatment,MECR mRNA expression increased in a time-dependent manner.After forskolin treatment,the mRNA expression first decreased and then increased,but the overall expression decreased.After high level of glucose treatment,mRNA expression showed unstable changes.After hypoxia and TNF-? treatment,no significant changes were observed.(2)In the animal model,the expression of MECR protein in 9 kinds of mitochondria rich mouse tissues(brain,heart,liver,spleen,lung,kidney,skeletal muscle,colon and brown fat)was detected by Western blot.The protein content of skeletal muscle is the most abundant,about 7 times of that of brain,heart and liver.The expression of kidney,colon and brown fat decreased by half compared with brain,heart and liver.MECR expression in spleen and lung was the lowest,about half of that in kidney,colon and brown fat.(3)We found that there was insulin resistance in the liver of DIO mice,and the insulin resistance of DIO mice treated with BBR was improved.MECR protein expression in DIO mice was significantly reduced,and it was found that MECR protein expression was restored in BBR treated mice liver.It was also observed that MECR mRNA expression increased in DIO mice.(4)In cell experiments,we found that the expression of lipoylation increased when ATP was excess.In animal experiments,we found that the expression of lipoic acid increased in high-fat mice.When MG132(1 ? m)was added to the cell model,MECR expression decreased.(5)IF-KO mouse model was established.The result showed that MECR expression increased when ATP was deficient.We detected the mRNA expression of MECR by qRT-PCR and found no significant difference in mRNA.Conclusion:We found that the expression level of MECR gene is closely related to type 2 diabetes.It is suggested that the decrease of MECR protein level is related to the occurrence of insulin resistance,and the recovery of MECR protein level is related to the disappearance of insulin resistance.MECR protein level is regulated by ATP,which may mediate ATP signal to induce insulin resistance.