| Objective: Mitochondrial dynamics,the processes of mitochondrial fusion and fission maintain homeostasis,are precisely regulated by fusion/fission-related proteins,and play an important physiological role in mitochondrial metabolism,quality and function.The aberrant changes of these proteins can trigger mitochondrial dynamics imbalance,which cause mitochondrial dysfunctions and result various disease states.Recent studies have found that hepatic insulin resistance is closely related to mitochondrial dynamics imbalance.Extensive researches have focused on the role of outer mitochondrial membrane fusion proteines mitofusin 1/2(Mfn1/2)and mitochondrial fission protein Dynamin-like protein 1(Drp1)in hepatic insulin resistance.However,whether mitochondrial inner membrane fusion protein Optic Atrophy 1(Opa1)abnormality directly causes hepatic insulin resistance is still unknown.Sirtuin 1(SIRT1)has important effects on the regulation of diabetes and mitochondrial function,but whether it can improve mitochondrial function by regulating Opa1 is inconclusive.Berberine(BBR)is a traditional Chinese medicine in China,which has the effects of lowering blood glucose,improving mitochondrial function and hepatic insulin resistance.This article focuses on exploring whether berberine can prevent hepatic insulin resistance by activating SIRT1 to regulate mitochondrial fusion protein Opa1,and exploring its potential mechanism.Methods:In the in vivo study,8-weeks old wild type mice and db/db mice were purchased.Wild type mice defined as control group and db/db mice were divided into 2 group which established as model group and BBR treatment group.First,the mice were tested for fasting blood glucose,blood lipid,oral glucose tolerance,and insulin tolerance.At the same time,the liver was morphologically tested,and then liver proteins were extracted,and the expressions of SIRT1,Opa1,Mfn1 / 2,Drp1 and PEPCK/G6 pase mRNA expression were measured to investigate the effect of BBR on improving hepatic insulin resistance in type 2 diabetic mice and its relationship with SIRT1 regulating mitochondrial dynamics.In the in vitro study,firstly,we performed Opal silencing HepG2 cells,and observed the effects of silencing on key proteins of mitochondrial fusion/fission,mitochondrial complex I/V protein expression,ATP content,mitochondrial membrane potential,and key indicators of hepatic insulin resistance to investigate whether Opa1 silencing caused mitochondrial dysfunction and hepatic insulin resistance.Secondly,we established a palmitate(PA)-induced model of insulin resistance in HepG2 cells.Based on the model,we used SIRT1 overexpression and Opa1 silencing technology to observe SIRT1/Opa1 protein expression,ATP content,mitochondrial morphology and membrane potential,key indicators of hepatic insulin resistance;Finally,in order to explore whether BBR can treat hepatic insulin resistance by regulating SIRT1 to improve Opa1 expression,we gave BBR treatment on the basis of PA stimulation,while establishing SIRT1 silenced cells,and observed the expression of Opa1 protein,ATP content,mitochondrial membrane potential and key indicators of hepatic insulin resistance.Results:Compared with the normal group,the expression of fasting blood glucose,low-density lipoprotein,total cholesterol,and triglyceride in diabetic mice was significantly increased in in vivo studies.HE staining showed that hepatocytes were highly swollen,disorderly arranged,and appeared vacuoles;electron microscopy results showed that mitochondrial ridges were thinner and smaller in morphology;the expression of PEPCK/G6 pase mRNA was significantly increased.Western blotting results showed that the expression of mitochondrial fusion proteins Opa1/Mfn1 and SIRT1 in the model group was significantly down-regulated,while the expression of fission protein Drp1 was significantly increased in the model group.After 4 weeks of BBR treatment,fasting blood glucose,low-density lipoprotein,total cholesterol,and triglycerides were significantly reduced;HE staining showed that the drug could improve the morphological performance of the liver,and the sparse state of mitochondria was also significantly improved;PEPCK/G6 pase mRNA expression was significantly reduced.Western blotting results showed that the expression of mitochondrial fusion proteins Opa1/Mfn1 and SIRT1 in the liver of the model group was significantly down-regulated,while the expression of the mitotic protein Drp1 was significantly increased,indicating that SIRT1/Opa1 pathway is associated with BBR improving hepatic insulin resistance in diabetic mice.In in vitro studies,Opa1 silencing had no effect on mitochondrial fusion protein expression,but increased the expression of mitochondrial fission protein and the expression of mitochondrial complex I,ATP content,and mitochondrial membrane potential were significantly reduced.Opa1 silencing also caused the expression of PEPCK/G6 pase significantly up-regulated and impaired the sensitivity of pAKT to insulin,indicating that Opa1 silencing can impair mitochondrial function and cause hepatic insulin resistance.Using SIRT1 overexpression technology based on the PA model,it was found that the protein expression of SIRT1/Opa1,ATP content,and mitochondrial membrane potential were significantly up-regulated;mitochondrial morphology was greatly improved;the sensitivity of pAKT to insulin and glucose consumption were increased;and these effects were reversed when Opal was silenced.Finally,BBR treatment was given on the basis of PA stimulation.It was found that Opa1 protein expression,ATP content,and mitochondrial membrane potential were up-regulated.The sensitivity of pAKT to insulin and glucose consumption were also increased.But these effects were reversed after SIRT1 silencing.Conclusion: Opa1 silencing mediated mitochondrial fusion/fission imbalance could lead to hepatocyte insulin resistance,while SIRT1 improves hepatocyte insulin resistance by activating Opa1.As an effective hypoglycemic drug,Berberine may improve hepatic insulin resistance by regulating SIRT1/Opa1 pathway to treat type 2 diabetes. |
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