Effects Of Exercise Combined With Ketogenic Diet On Glucose Homeostasis And Hepatic Lipid Metabolism In STZ-induced T2DM Mice

Diabetes mellitus is one of the non-communicable diseases that threaten the health of the human body.It not only brings great burden to the patients,but also brings heavy economic burden to the country and the society.The International Diabetes Federation(IDF)dates show that 2016 of the world about 415 million adults with diabetes and it is estimated that the number will be 642 million in 2040.The pathogenesis of diabetes is very complex,so far,there is no therapy to completely cure diabetes.At present,the main treatment of type two diabetes mellitus(T2DM)is drug therapy,diet therapy and reasonable physical activity.Because of the safety and limited efficacy of the drug,more and more attention has been paid to non-drug therapy.The clinical study found that the ketogenic diet as a diabetic intervention can produce positive effect on glucose homeostasis and widely used by T2DM patients.However,in recent years,studies show that the long-term ketogenic diet may lead to diabetic mice fat metabolism in liver tissue,and even induce fatty liver.Clinical and animal research have shown that different forms of exercise can have a positive effect on the prevention and treatment of fatty liver.But,if exercise can improve the liver lipid metabolism combined with ketogenic diet is to be further understood.Objective:(1)Effects of ketogenic diet on glucose and lipid metabolism in diabetic mice.(2)To explore the impact of the movement of ketogenic diet combined with different ways of lipid metabolism in the liver of diabetic mice and its mechanism.Methods:85 3-5 week old male C57BL/6J mice were purchased from East China Normal University animal research center,after 3 days of feeding mice,10 mice as normal control group(NC)randomly selected,with chow diet.The other 75 mice were fed with high fat diet for 4 weeks,followed by intraperitoneal injection of STZ at dose of 100 mg/kg BW.The fasting blood glucose level was higher thanll.1 mmol/1 for three times as type Ⅱ diabetes mellitus mice.In this experiment a total of 58 mice were successfully modeled,randomly selected 32 from them,according to the body weight and blood glucose level,were randomly divided into diabetic control group(HFD,n=8),the ketogenic diet group(KD,n=8),the ketogenic diet high intensity intermittent exercise group(KH,n=8),the ketogenic diet aerobic exercise group(KA,n=8).Every time before and after exercise were 5 min,8 m/min preparation and finishing activities,KH took 8 weeks and no grade treadmill training,high intensity exercise for 4 min,2 min active rest,speed from 17 m/min to 24 m/min,60 minutes each time,3 times one week;KA took 8 weeks no grade treadmill training,12 m/min speed 60 min,60 min each time,5 times one week.Results:(1)Compared with Group NC,Group HFD,FBG(P<0.01)HbAlc(p<0.05),serum Glu(p<0.05),Insulin(p<0.05),HOMA-IR(p<0.05)and daily water intake(p<0.05)increased significantly of mice,HOMA-ISI decreased significantly(p<0.05),group KD HOMA-ISI(p<0.05)increased significantly,serum Insulin(p<0.05),HOMA-IR(p<0.05)weight(p<0.05)decreased significantly,FBG,HbAlc,Glu and serum on water intake had no significant difference.Compared with Group NC,group KH HOMA-ISI(p<0.05)increased significantly,HOMA-IR(p<0.05),serum Insulin(p<0.05)and body weight(p<0.05)decreased significantly,there is no significant difference of FBG,HbAlc,Glu,and the amount of water.Group KA serum Insulin(p<0.05),weight(p<0.05)decreased significantly,FBG,HbAlc,Glu,HOMA-IR,HOMA-ISI and the amount of water have no significant difference.Compared with Group HFD,Group KD,Group KH and Group KA HOMA-ISI(p<0.05)were significantly increased,while FBG(p<0.01),HbAlc(p<0.01),serum Insulin(p<0.05),Glu(p<0.05),HOMA-IR(p<0.05),weight(p<0.05)and the day drinking water(p<0.05)decreased significantly.Compared with Group NC,Group KD,Group KH and Group KA of mice PCK1 expression of mRNA G6PC decreased significantly(p<0.01).Compared with Group HFD,the relative mRNA expression of PCK1 and G6PC in Group KD,Group KH,Group KA were significantly decreased(p<0.01).(2)compared with Group NC,Group HFD of serum ALT(p<0.01),TC(p<0.05),LDL-C(p<0.05),liver index(p<0.01),liver TG and NEFA level increased significantly,epididymal fat%(p<0.05)significantly decreased,serum TG,HDL-C and AST level had no significant change.Group KD serum TC(p<0.05),TG(p<0.05),HDL-C(p<0.05),ALT(p<0.01),AST(p<0.05),epididymal fat%(p<0.01),liver index(p<0.01),liver TG(p<0.01),NEFA(p<0.05)was significantly increased.Compared with Group HFD,Group KD of serum HDL-C(p<0.05)and epididymal fat%(p<0.01)significantly increased,LDL-C,liver index reduced significantly,serum TC、TG、ALT、AST,liver NEFA level had no significant difference.Liver HE and Oil Red O staining showed that the structure of hepatic lobule is clear in NC.But,there were amount of fat vacuoles in the liver of HFD mice and there were more obvious fatty vacuoles in mice liver of group KD and liver MASSON staining showed.Compared with the Group NC,the liver of the Group KD showed obvious fibrosis yet.(3)Compared with group NC,Group HFD lipid metabolism related gene CIDEA(p<0.01),SCD1(p<0.05),SREBP1(p<0.05),ACC1(p<0.01),ACC2(p<0.01),CPT1A(p<0.01)mRNA relative expression level increased significantly and FAS,AMPKal had no significance,AMPKa2 level is significantly decreased.The mRNA relative expression level of CIDEA,FAS,SCD1,ACC1,ACC2,SREBP1,CPT1A are significantly increased(p<0.01),in group KD,and down regulate the AMPKal(p<0.05)and AMPKa2(p<0.01)level.Compared with Group HFD,the relative mRNA expression level of SCD1(p<0.05),AMPKa2(p<0.05),CPT1A(p<0.01)in Group KD increased significantly in group KD and CIDEA,FAS,SREBP1,ACC1,ACC2,AMPKa1 have no significant change.(4)Compared with group NC,the protein levels of FAS increased significantly,ATGL、P-ACC/ACC ratio(p<0.05)decrease significantly,HSL,MGL,PPARa,PPARy,CPT1A protein and P-ACC/ACC protein ratio did not change significantly,in mice liver of group HFD.The ratio of P-AMPK/AMPK protein decreased significantly(p<0.05),FAS(p<0.01),PPARy(p<0.05),CPT1A(p<0.05)protein level increased significantly,in group KD,But,protein level of HSL,ATGL,MPGL,PPARa and ratio of P-ACC/ACC had no significant change.Compared with Group HFD,FAS,P-AMPK/AMPK ratio decreased significantly(p<0.05),the protein level of ATGL,CPT1A,PPARa are significantly increased(p<0.05),and the protein level of HLS,MGL,PPARy and P-ACC/ACC protein ratio did not change significantly of mice liver in group KD.(5)Compared with Group KD,the level of serum TG(p<0.05),serum ALT(p<0.01),serum AST(p<0.05),serum HDL-C(p<0.01),liver TG(p<0.01),NEFA(p<0.01)level are decreased significantly,serum LDL-C levels are significantly higher(p<0.01)in group KA,but,liver index and epididymal fat%have not change significantly.Level of serum LDL-C(p<0.01)increased significantly,also the level of serum ALT(p<0.05),TC(p<0.05),liver TG(p<0.05)in the liver was significantly decreased in the group KH.But,the levels of serum TG,AST,liver NEFA,liver index and epididymal fat%do not change significantly.Compared with Group KD,liver HE and oil red staining showed that the morphology of hepatocytes in Group KA tended to be orthergasia,and the content of lipid droplets decreased significantly.The cell structure is clearer,less lipid content of mice liver in group KH.Liver MASSON staining showed that compared with the Group KD,the degree of fibrosis of KH decreased slightly but liver fibrosis is significantly decreased in Group KA.(6)Compared with group KD,the relative mRNA expression of G6PC decreased significantly(p<0.05)in liver tissue of KH group and KA group,and the activity of G6PC in liver tissue of KA group decreased significantly(p<0.05),but there is no significant difference in group KH.Compared with the KD group,the ketogenic diet combined with high intensity intermittent exercise or aerobic exercise intervention can make the liver of diabetic mice PCK1 mRNA relative expression(p<0.01)andactivity(p<0.05)significantly decreased。Compared with the group KD,CIDEA(p<0.01),FAS(p<0.01),SCD1(p<0.05),SREBP1(p<0.01)ACC1(p<0.01)mRNA expression are decreased significantly,when ACC2(p<0.05),AMPKa2(p<0.01),CPT1A(p<0.05)are increased significantly,but no obvious change of AMPKal in the liver of mice in group KH.Compared with the group KD,The FAS,SCD1,SREBP1,ACC1,ACC2 and CIDEA relative expression of mRNA are decreased significantly(p<0.01)while AMPKal,AMPKa2 and CPT1A(p<0.01)are increased significantly in liver tissue of group KA.(7)Compared with Group KD,the protein level of FAS,HSL and ATGL of mice liver had no significant change in Group KA.But,the protein level of MGL,CPT1A and P-AMPK/AMPK,P-ACC/ACC were significantly higher(p<0.05).Compared with Group KD,the protein level of HSL ATGL,MGL,CPT1A and P-ACC/ACC ratio do not change significantly and the protein level of FAS was significantly decreased(p<0.05)with the ratio of P-AMPK/AMPK increased significantly(p<0.05)in mice liver of group KH.Conclusion:(1)the ketogenic diet,as well as ketogenic diet combined with exercise intervention can significantly improve glycemic control in diabetic mice.(2)Long term ketogenic diet may lead to liver fat accumulation in diabetic mice.(3)The ketogenic diet combined with either aerobic exercise or high intensity intermittent exercise can effectively alleviate the liver fat accumulation,and aerobic exercise effect is more obvious.(4)Aerobic exercise may activate the AMPK/ACC/CPT1A signaling pathway to accelerate the oxidation of fatty acids and slow the progression of fatty liver.