| Objective:Liraglutide is a newly antidiabetic drug in the treatment of type 2diabetes,it can not only promote insulin secretion,but also can increase the sensitivity of pancreatic tissue to insulin.Insulin resistance(IR)is one of the pathogenesis of type 2 diabetes mellitus and metabolic syndrome.Former studies have found that IR can be improved by liraglutide,however,the mechanism is not yet clear.In our study,the high fat diet rats were injected in liraglutide of different concentrations,then its effect on the rat liver lipid accumulation and insulin sensitivity were detected;we observe the detection of hepatic endoplasmic reticulum chaperone GRP78,endoplasmic reticulum stress related molecule JNK,the expression of insulin signaling pathway related molecules GLUT-2,AKT.To investigate whether the liraglutide can improve IR,reduce liver fat deposition mechanism,providing a new theoretical basis for the clinical.Method:Wistar rats(n = 54,approximately 250 g)were randomly distributed into two initial groups: normal chow(NC)group and high-fat diet(HFD)group after a one-week acclimatization period.After 8 weeks of feeding,we randomly chose 5 rats in each group and assessed insulin sensitivity in order to determine whether the insulin-resistant rat model was successfully established by hyperinsulinemic-euglycemic clamp technique.Next,the insulin-resistant rats in the HFD group were randomly subdivided into three groups:HFD,LL,and HL(n = 11 in each group)groups,were fed a high-fat diet but treated withdifferent doses(100 and 200 ug/kg)of liraglutide.The NC group(n = 11 continued to be fed with normal chow and was given 0.5 ml/kg of normal saline.All reagents including saline and liraglutide were injected subcutaneously twice daily for 2 weeks.Then 5 rats were randomly selected from each groups for calculation of GIR clamp test and oral glucose tolerance(OGTT)the calculation of the area under the curve of glucose in rats(AUC).The remaining 6 rats in each group weighted the body mass.Rats were killed and serum was obtained from abdominal aorta,which was used to detect FFA,TCH,TG,HDL-C,LDL-C,ALT and AST.The rat liver specimen was used to detect the TG in liver,observe the ultrastructure by electron microscopy.Western blot technique was used to detect the expression of GRP78,GLUT-2,p-JNK and p-AKT protein in the liver,and the expression of GRP78 and GLUT-2 mRNA were detected by Realtime-PCR.Results:1 Comparison of the general situation of the four groups of ratsCompared with NC group,the body weight(BW),FFA,TG,LTG were significantly increased in the HFD group(P<0.05);compared with HFD group,TG,BW,FFA,LTG,AST and ALT were significantly decreased in the HL group(P<0.05).Compared with the HFD group,there were only FFA,AST,ALT decreased in the LL group(P<0.05).Compared with the LL group,the serum indicators of HL group were significantly improved,and the FFA,LTG and TG were also significantly decreased(P<0.05).2 Comparison of glucose infusion rate in the four groups of ratsAfter eight weeks of feeding:Compared with contol group,the GIR was decreased in the HFD group(30.21±2.51vs20.41±3.64mg/kg.min)(P<0.05).After 2 weeks of liraglutide injection,compared with HFD group,the GIR in the HL group increased(18.83±1.95)vs(25.80±1.40)mg/kg.min(P<0.05),the GIR was also increased in LL group(18.83±1.95)vs(21.55±2.46)mg/kg.min(P<0.05).Compared with LL group,the GIR was increased in HL group(21.55±2.46)vs(25.80±1.40)mg/kg.min(P<0.05).3 Comparison of the results of oral glucose tolerance test between the four groupsCompared with NC group,the blood glucose and AUC of HFD group in 0min,30 min,60min,120 min four points were significantly increased(P<0.05).Both of the blood glucose and AUC in HL group rats were decreased at four points compared with HFD group(P<0.05).The blood glucose and the AUC this four points of LL group were decreas ed.However,there is significant of blood glucose and AUC only at 120 min.(P<0.05).The blood glucose and AUC of HL group in 0min,120 min two points were significantly decreased when compared with LL group(P<0.05)4 Morphological of liver observation by a light microscopeThe structure of the liver cell wall and internal organelle are complete.The hepatic lobule and central vein are normal,and the hepatic cord with radial arrangement is observed clearly around the central vein in the NC group.The HFD group rat liver tissue visible severe diff use fattydegeneration of the liver tissue damage and fat deposition.The l ipid droplet vacuoles in cytoplasm decreased obviously in the LL group.The structure of hepatic lobe and hepatic sinus of HL group was clear,and the cells arranged neatly.5 Ultrastructure of hepatocytes observed by an electron microscopeElectron microscopy revealed normal liver ultrastructure in control rat livers: In the NC group,the cytoplasm consisted of rich organelles,especially the clear vision of the rough endoplasmic reticulum(RER).The most palpable in the HFD group was the increased number of lipid droplets in the cytoplasm,obvious RER dilatations and degranulation significantly.Notably,after liraglutide treatment for 2 weeks,the dilatations of the RER got some relief,although they did not fully recover;while there is still have mild damage in liver cells.Compared with the LL group,the RER in the HL group recovered to normal state.6 The expression of mRNA in GRP78,GLUT-2 in liver of four groupsCompared with NC group,in the HFD group,the expression mRNA in GRP78 was increased,and the expression of mRNA in GLUT-2 was decreased(P<0.05);Compared with HFD group,in the LL and HL groups,the expression of mRNA in GRP78 were both decreased(P<0.05),and the change in the HL group was more obvious(P<0.05).Compared with HFD group,in the LL and HL groups,the expression of mRNA in GLUT-2 were both increased(P<0.05),and the change in the HL group was more obvious(P<0.05).7 The expression of protein in GRP78,GLUT-2,p-AKT and p-JNK in liver of the four groupsCompared with NC group,the expression of the protein of GRP78 and p-JNK upregulated in HFD group(P<0.05),the expression of GLUT-2 and p-AKT down regulate(P<0.05).Compared with HFD group,the expression of the protein of GRP78 and p-JNK down regulate(P<0.05),the expression of GLUT-2 and p-AKT upregulated in the LL and HL groups(P<0.05).Compared with LL group,the expression of the protein of GRP78 and p-JNK down regulate(P<0.05),the expression of GLUT-2 and p-AKT upregulated in the LL and HL groups in the HL group(P<0.05)8 The results of single correlation analysis of four groupsGIR was negatively correlated with body weight,FFA,LTG,and TG(P<0.01).The expression protein of GRP78 was positively correlated with FFA,LTG and TG(P<0.01),and negatively correlated with GIR(P<0.01).The expression protein of GLUT-2 protein expression was negatively correlated with FFA,LTG,TG(P<0.01),and GIR was positively correlated(P<0.01).Conclusion:1 High-fat diet can cause glucose and lipid metabolism in rats,and increase the liver fat deposition,decreased glucose tolerance and IR.2 Liraglutide improved the glycolipid metabolism,IR and glucose tolerance,reduced lipid accumulation in the liver in a dose-dependent manner.3 Liraglutide could be a protective effect on endoplasmic reticulum stress in the liver,reduce liver fat deposition,promote the uptake of glucose in liver cells,improve IR in a dose-dependent manner,and these may related by the GRP78/JNK/AKT/GLUT-2 pathway.. |
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