The Effects Of Dietary Restriction On Clock Gene Methylation And Liver Aging-related Markers

Objective : To investigate the effects of caloric restriction and alternate day fasting on clock gene methylation and liver aging markers in 12-month-old mice.Methods:Thirty male 12-month-old mice were randomly divided into three groups:control group(CON group),caloric restriction group(CR group)and alternate day fasting group(ADF group).Mice were given free diet,caloric restriction(60% of control group daily intake)and alternate day fasting(fasting and feeding each 24h)for 16 weeks,the general condition of the mice was observed and the body weight of the mice was measured every week;the fasting blood glucose of the mice was measured at 0,8 and 16 weeks respectively.After 16 weeks of feeding,mice were killed by anesthesia.Detection of glucose and lipid metabolism and inflammatory markers:serum insulin,IL-6,TC,TG levels;DNA methylation:Npas2,Rev-Erbα;liver oxidative stress related indicators: SOD activity,GSH-Px activity,MDA content;liver tissue β-galactosidase staining.Results:After 16 weeks of feeding,the changes of mice in each group were as follows:(1)Body weight change:there was no significant change in body weight in control group;he body weight of CR group kept decreasing and tend to stabilize nine weeks later;the body weight of ADF group increased and then decreased slightly ten weeks later.(2)The indexes of glucose and lipid metabolism:the fasting blood glucose dropped down lower(P<0.05),while insulin and TC in CR group dropped down much lower than those in control group(P<0.01);the fasting blood glucose in ADF group dropped down lower at the end of the eighth week(P<0.05),while it increased much higher at the end of the sixteenth week(P<0.01),insulin dropped down lower(P<0.05);the fasting blood glucose in CR group increased slightly higher at the end of the eighth week(P>0.05),while it dropped down much lower at the end of the sixteenth week(P<0.01),insulin dropped down slightly lower(P>0.05),but not statistically significant,TC dropped down much lower(P<0.01).(3)IL-6:IL-6 in CR group dropped down much lower than that in control group(P<0.01);IL-6 in ADF group dropped down lower than that in control group(P<0.05);IL-6 in CR group dropped down slightly lower than that in ADF group(P>0.05),but not statistically significant.(4)DNA methylation level:the methylation percentage of Npas2 promoter region in ADF group increased higher than that in control group(P<0.05);the methylation percentage of Npas2 promoter region in CR group dropped down much lower than that in ADF group(P <0.01).(5)Liver β-gal staining results:the blue granules(representing senescent cells)in CR group dropped down much lower than those in control group(P<0.01);the blue granules in ADF group dropped down lower than those in control group(P<0.05);the blue granules in CR group dropped down slightly lower than in ADF group(P>0.05),but not statistically significant.(6)The changes of oxidative stress index:the activity of SOD in CR group increased much higher(P<0.01),the activity of GSH increased higher,while MDA content dropped down lower than those in control group(P<0.05);the activitives of SOD and GSH in ADF group increased higher(P<0.05),while MDA content dropped down slightly lower than those in control group(P>0.05),but not statistically significant;the activitives of SOD and GSH in ADF group increased slowly higher,while MDA content dropped down slightly lower than those in ADF group(P>0.05),but not statistically significant.Conclusion:This study found that dietary restriction can improve aging-related markers,and have little effect on the methylation of clock genes,but alternate day fasting increases the level of Rev-Erbα methylation.