| Objective: To observe the effects of leucine intervention or calorie restriction on insulin resistance,lipid metabolism,serum bile acid levels,bile acid metabolism related genes and proteins expression in high fat diet-fed rats,and further explore the relationship between bile acid metabolism with insulin resistance and type 2 diabetes.To provide a new direction and theoretical basis for nutritional dietary intervention of type 2 diabetes.Methods: Thirty-two male adult SD rats were randomly divided into normal control group(ND),high fat diet control group(HFD),80% calories-restricted group(HFD+CR)and 1.5% leucine intervention group(HFD+Leu)according to their body weight.At the end of 32 weeks,oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)were performed to calculate the area under the curve(AUC)and to evaluate the insulin sensitivity in rats.Then the rats were sacrificed,the serum and tissues samples were collected.Serum insulin and glucose levels were determined using kits,and the homeostasis model assessment(HOMA-IR)was calculated.The levels of total cholesterol(TC)and triglycerides(TG)in serum and liver were detected by colorimetry method.The expressions of mRNA of bile acid and cholesterol metabolism related genes in rat liver were determined by real-time reverse transcription PCR(RT-PCR),including bile acid synthesis genes liver X receptor(LXRα),Farnesoid X receptor(FXR);cholesterol 7α-hydroxylase(CYP7A1),a key enzyme in the classic pathway for bile acid synthesis;cholesteral 27-hydroxylase(CYP27A1),a key enzyme in the alternative pathway for bile acid synthesis;cholesterol reverse transport regulation genes ATP-binding cassette transporters ABCA1,ABCG1,ABCG5,ABCG8.Western blot was used to analysize expression of hepatic bile acid synthesis-related proteins CYP7A1,CYP27A1,cholesterol synthesis key proteins sterol regulatory element binding protein-2(SREBP-2),3-hydroxy-3-methyl-glutaryl-CoA reductase(HMGCR).The levels of 16 bile acids in serum were determined by ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).Results:1.Weight and energy intake of rats(1)From the 5th week to the end of the experiment,the weight of the rats in the ND group was significantly different from HFD group,The weight of the HFD group was significantly higher than ND group(p<0.05).From the 13 th week to the end of the experiment,the body weight of the rats in the HFD group and the HFD+Leu group were significantly different,and the HFD+Leu group was significantly lower than HFD group(p<0.05).From the third week to the end of the experiment,the body weight of the rats in HFD+CR group was significantly lower than HFD group(p<0.05).(2)From the third week to the end of the experiment,the energy intake of the ND group and the HFD group were significantly different,and the intake of the HFD group was significantly increased(p<0.05).There was no significant difference in energy intake between the HFD+Leu group compared with the HFD group.The energy intake of HFD+CR group was 80% of HFD group.2.Fasting serum glucose,insulin level and insulin sensitivity of rats(1)After 32 weeks of high-fat diet,fasting serum glucose level,serum insulin level and insulin resistance index HOMA-IR were significantly higher in HFD group than ND group.(p<0.05),successful establishment of rat insulin resistance model.Compared with the HFD group,HOMA-IR was significantly lower in HFD+Leu group(p<0.05),and other indicators were not significantly changed.Fasting serum glucose,serum insulin level and HOMA-IR were significantly decreased in HFD+CR group(p<0.05),and abnormal glucose and lipid metabolism and insulin resistance were improved in rats.(2)Before the end of the experiment,the rats in each group underwent ITT after fasting.Compared with the ND group,the AUC of the HFD group was significantly increased(p<0.05),and the rats showed abnormal insulin tolerance.Compared with the HFD group,the AUC of the HFD+CR group was significantly lower(p<0.05),the insulin tolerance abnormality was improved,and there was no significant change in the HFD+Leu group.(3)Before the end of the experiment,the rats in each group were subjected to OGTT after fasting.Compared with the ND group,the AUC of the rats in HFD group was significantly increased(p<0.05),and the glucose tolerance was abnormal in rats;The AUC of both intervention groups was significantly lower(p<0.05),and the abnormal glucose tolerance in rats was improved.3.Lipid metabolism in serum and liver of ratsCompared with ND group,serum and liver lipid metabolism were significantly changed in HFD group: serum TC levels and liver TC levels were significantly increased in rats fed with high-fat diet(p<0.05).Compared with the rats in HFD group,serum TC levels,serum TG levels and liver TG levels were significantly decreased in the two intervention groups(p<0.05).Leucine or limited intervention significantly improved lipid metabolism in rats.4.Changes in serum bile acid levels in ratsCompared with ND group rats,the serum HDCA,UDCA,βMCA,THDCA,12 KLCA,free bile acid,tauroconjugate bile acid,and total bile acid were significantly lower in HFD group(p<0.05).Compared with HFD group,serum HDCA,TCDCA,free bile acid and total bile acid were significantly increased in HFD+Leu group(p<0.05);serum HDCA and free bile acid were significantly increased in HFD+CR group(p<0.05).The serum TCDCA level of HFD+Leu group was significantly higher than HFD+CR group(p<0.05).5.The mRNA expression of genes related to bile acid and cholesterol metabolism in ratsThe level of FXR mRNA in the liver of HFD group was significantly lower and the levels of ABCA1 and ABCG1 mRNA of HFD group were significantly higher than ND group(p<0.05).Compared with HFD group,the levels of LXR,FXR,CYP27A1,ABCA1,ABCG5 and ABCG8 mRNA in liver of HFD+Leu group were significantly increased(p<0.05).The levels of FXR,CYP7A1 and ABCG8 mRNA in the liver of HFD+CR group increased,and the levels of ABCA1 and ABCG1 mRNA decreased,but the differences were not significant.The LXR,CYP27A1,ABCA1,ABCG1 and ABCG5 mRNA levels in the liver of HFD+Leu group were significantly higher than those in HFD+CR group(p<0.05).6.The protein expression related to bile acid and cholesterol metabolism in ratsThe protein expression level of CYP27A1 in liver of HFD group was significantly lower than ND group(p<0.05).Compared with HFD group,the protein expression levels of CYP27A1,SREBP-2 and HMGCR in liver of HFD+Leu group were significantly increased(p<0.05);the expression of SREBP-2 in liver of HFD+CR group was significantly increased(p<0.05).There was no significant difference in expression of four proteins between HFD+Leu group and HFD+CR group.Conclusion: Leucine intervention significantly increased bile acid synthesis in insulin resistant rats,increased the reverse transport of cholesterol,reduced the accumulation of serum lipid,improved glucose and lipid metabolism and insulin sensitivity.Changes in bile acid synthesis and cholesterol transport related genes and proteins expression are possible mechanisms. |
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