Two Alkaloids Of Piperlongum Attenuate Insulin Resistance Through Regulation Of Mitochondrial Biogenesis

Objective: In this study,the IR cell models of human hepatoma Hep G2 and C2C12 cells were established to observe the effects of PIP and GBN in improving IR.At the same time,investigating whether the two types of alkaloids in different IR cell models can come into play through adjusting the function of mitochondrion and mitochondrial biosynthetic signaling pathway,which is aimed at exploring the molecular mechanism of PIP and GBN in improving the glucose and lipid metabolism disorder.Methods: 一、IR model of Hep G2 cells was built to observe the effects of PIP and GBN on the improvement of glucose and lipid metabolism disorder and its effects on the regulation of relevant factors of mitochondrial function.1.IR model was established by using high glucose culture medium and fat emulsion to induce Hep G2 cells.2.Hep G2 cells were divided into the model group and drug groups.The drug intervention groups were split into PIP administration groups(2.5μM,5μM,10μM),GBN groups(0.5μM,2μM,8μM)and positive control group(MET).3.Culture supernatants were collected after the cells were respectively treated for 6h,12 h,24h,48 h.Glucose consumption was detected by glucose oxidase method.After the cells were treated for 24 h,we collected the cells to detect the contents of triglyceride and serum total cholesterol.Testing the mitochondrial DNA copy number with the method of fluorescent quantitative PCR and testing AMPK,p-AMPK,SIRT1 and PGC-1α protein expression by using western blotting analysis.二、IR model of C2C12 cells was made to observe the effects of PIP and GBN on the improvement of glucose metabolism disorder and its effects on the regulation of related factors of mitochondrial function.1.IR model was established with palmitic acid to induce C2C12 cells.2.The drug intervention groups were divided into three groups which were positive control group(MET),PIP administration groups(5μM,10μM,20μM)and GBN groups(0.5μM,2μM,8μM).3.Culture supernatants were collected after the cells were treated for 6h,12 h,24h,48 h respectively.Glucose consumption was detected by glucose oxidase method.After the cells were treated for 12 h,we collected the cells and detected the mitochondrial DNA copy number through fluorescent quantitative PCR method.Meanwhile,the cells of treated for 24 were gathered to test AMPK,p-AMPK,SIRT1 and PGC-1α protein expression by western blotting analysis.Results: 1.IR model was successfully established: After the Hep G2 cells were treated using the high glucose culture medium and the 10% fat emulsion for 12 h and the C2C12 cells were effected with the 0.5μM palmitateacid for 16 h,Glucose consumption of the models were significantly lower than control group,which showed the IR modesl of Hep G2 cells and C2C12 cells were successfully established.2.Hep G2 cells detection indicators:Compared with the model group,PIP,GBN and the positive drug metformin could increase the glucose consumption of IR cells,and the effect of PIP and GBN was more obvious with the prolongation of administration time,but the effect of different concentration of drugs had little difference on the glucose consumption of IR cells at the same time.The results mentioned above showed that the lower concentration of PIP and GBN could play a role in improving the glucose metabolism of hepatocytes.After the PIP and GBN acted on Hep G2 cells for 24 h,they could reduce the TG and TC content in a dose-dependent manner,indicating that the drugs improved the lipid metabolism of hepatocytes;PIP and GBN increased the mt DNA copy number of hepatocytes in a dose-dependent and time-dependent manner,and improved the function of mitochondria.To investigate the activity of AMPK,SIRT1 and PGC-1α in mitochondrial biogenesis-related genes,it was found that PIP and GBN could up-regulate the protein expression of AMPK and SIRT1 and down-regulate the protein expression of PGC-1α.3.C2C12 cells detection indicators: Compared with the model group,PIP,GBN and positive drug metformin could increase the glucose consumption of IR cells in a time-dependent manner.The effect of different concentration of drugs had little difference on the glucose consumption of IR cells at the same time,which showed that low concentration of PIP and GBN could improve the glucose metabolism of muscle cells.PIP and GBN could raise the mt DNA copy number in a dose-dependent and time-dependent manner,and adjust the function of mitochondria.PIP and GBN could up-regulate the expression of AMPK,SIRT1 and PGC-1α in mitochondrial biogenesis-related signaling pathways.Conclusion: 1.PIP、GBN and MET can improve the glucose and lipid metabolism disorder in the IR models of hepatic cells and muscle cells,they also reflect the characteristics of time-dependent and dose-dependent.Owing to the increase of the mitochondrial DNA copy number,the mitochondrial function is also improved.2.After detecting the activity of AMPK,SIRT1 and PGC-1α,the PIP、GBN and MET can up-regulate the expression of AMPK activity protein and SIRT1 in hepatocytes,so we can suggest that the amelioration effect of PIP and GBN on glucose and lipid metabolism and mitochondrial biogenesis owed to the way by adjusting the activity of AMPK and SIRT1.The PGC-1α protein expression in hepatocytes was down-regulated.We believe that PGC-1α is mainly involved in glucose metabolism,so its expression may be reduced by the effects of PIP and GBN.3.PIP、GBN and MET can up-regulate AMPK activity or protein expression of SIRT1 and PGC-1α in muscle cells.Thus,PIP and GBN may play an important role in improving the insulin resistance by regulating the mitochondrial biosynthetic pathway AMPK/SIRT1/PGC-1α in muscle cells.