Molecular Mechanism And Role Of Metformin Regulating Autophagy In MIN6 Cells Via JNK/Beclin-1 Pathway

Objective: Type 2 diabetes is a disorder of glucose and lipid metabolism caused by insufficient insulin secretion or functional deficiency in the body.Defects in islet ? cells and insulin resistance are the key links in the pathogenesis of type 2 diabetes.Studies have shown that long-term hyperglycemia in the body causes islet ? cells and peripheral tissues to produce endoplasmic reticulum stress,oxidative stress,and inflammatory responses,resulting in islet ? cell function defects and insulin resistance.Autophagy is closely related to endoplasmic reticulum stress,oxidative stress,and inflammatory responses.Autophagy can clear damaged or dead organelles and intracellular biomolecules,and reduce endoplasmic reticulum stress,oxidative stress,and inflammatory responses to the islets.?-cell damage maintains the structure,number,and function of ?-cells.Existing evidence proves that metformin is involved in regulating the autophagy of pancreatic ? cells through autophagy-related signaling pathways,which provides a new direction for studying the mechanism of metformin's hypoglycemic mechanism.However,the specific molecular mechanism by which metformin regulates pancreatic ?-cell autophagy is unknown.Endoplasmic reticulum stress and oxidative stress interfere with insulin biosynthesis by activating c-JNK amino acid kinase(c-Jun NH2-terminal Kinase,JNK).Therefore,JNK signaling pathway plays an important role in pancreatic ?-cell autophagy and dysfunction.The specific mechanism by which the JNK signaling pathway regulates pancreatic ?-cell autophagy has not been clarified.In this thesis,we use MIN6 cell as a model and in vitro mimic high-concentration glucose environment to study the role of metformin in MIN6 cells and the regulation of autophagy via JNK/Beclin-1 pathway.Methods: MIN6 mouse cell lines were exposed to normal glucose(5.5mmol / L)and high glucose(33.3mmol / L)and the expression of autophagy-related proteins Beclin-1and LC3 were detected by Western blot analysis.Metformin,the autophagy-specific inhibitor 3-MA,and the specific JNK signal inhibitor SP600125 were then added to the cell culture media respectively or at the same time.Transmission electron microscopy was used to detect the submicroscopic structure of autophagosomes in cells;LC3fluorescence in the cytoplasm was observed by immunofluorescence.The levels of total protein JNK and phosphorylated p-JNK protein and the expression of autophagy-related proteins LC3 and Beclin-1 were detected by Western blot analysis.Results:1.Compared with the control group,transmission electron microscopy and Western Blot showed that the autophagy level of min6 islet cells was inhibited in mice after high glucose treatment.2.Metformin activates the level of autophagy in MIN6 cells.Western blot analysis showed that the expression of autophagy-related protein in metformin treatment group was higher than that in control group.LC3 immunofluorescence showed that the expression level of LC3 in metformin treatment group was higher than that in control group.3.The expression levels of p-JNK,Beclin-1 and LC3 protein are upregulated in the metformin-treated group;JNK-specific inhibitor SP600125 inhibited the expression activity of LC3 and Beclin-1.Conclusion: Metformin induces MIN6 cell autophagy through the JNK / Beclin-1signaling pathway,improves the inhibition of MIN6 cell autophagy by the high glucose environment,thereby slowing the development of diabetes.