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Cytokeratin 8 Inhibits Hepatic Glycogen Synthesis in Type 2 Diabetes Mellitus by Modulating Insulin-dependent IRS1/PI3K/Akt-GSK3β Pathway


 Purpose: Cytokeratin 8(CK8) is a cytoskeletal protein mainly expressed in the liver. Recent studies have found that CK8 was closely related to glycogen synthesis. However, the role and the underlying mechanisms of CK8 in hepatic glycogen synthesis in type 2 diabetes mellitus (T2DM) have remained to be fully elucidated. Therefore, this study aimed to investigate the effects and the underlying mechanisms of CK8 on hepatic glycogen synthesis in T2DM.Methods The T2D mouse model was constructed by high energy feed of 8-10 weeks old C57BL/6J male mice. The model was validated by OGTT test and ITT test. The liver samples of T2DM patients were collected and the expression levels of CK8, IRS1, PI3K, Akt, GSK3β, p-PI3K p-Akt and p-GSK3β were determined by Western blotting. Then, at the cellular level the murine NCTC 1469 cells were used, and to up-regulate and down-regulate the CK8 gene the overexpression plasmid was constructed and transfected and RNA interference technology was applied, and CK8, IRS1, PI3K, Akt, GSK3β, p-IRS1, p-PI3K p-Akt and p-GSK3β were detected by Western blotting. Immunohistochemistry was used to detect the level of glycogen synthase (GS) and glycogen staining experiments was performed by PAS. At the animal level, the T2D mouse model was used to up-regulate and down-regulate the CK8 gene using an adenovirus vector. The levels of CK8, PI3K, Akt, GSK3β, p-PI3K, p-Akt and p-GSK3β in rat liver were detected by Western blotting, immunohistochemistry was used to detect the level of GS and glycogen staining experiments was performed by PAS.Results The expression levels of IRS1, p-PI3K, p-Akt and p-GSK3β were significantly higher, while the expression levels of CK8 was significantly lower in control group than in the liver of T2D mice or T2DM patients. Upregulation of CK8 in murine NCTC 1469 cells treated with high-glucose medium, we found IRS1, p-IRS1, p-PI3K, p-Akt and p-GSK3β were significantly decreased, the level of GS was significantly decreased, and glycogen synthesis was inhibited compared with NCTC1469 cells transfected with empty vector. Downregulation of CK8 in murine NCTC 1469 cells murine treated with high-glucose medium, we found IRS1, p-IRS1, p-PI3K, p-Akt and p-GSK3β were significantly higher, the level of GS was significantly increased, and glycogen synthesis was promoted compared with NCTC1469 cells transfected with sh-NC. Upregulation of CK8 in the T2D mouse model, we found p-PI3K, p-Akt and p-GSK3β were significantly lower compared with T2D mouse model transfected with empty vector. The level of GS was significantly decreased, and glycogen synthesis was inhibited. Downregulation of CK8 in the T2D mouse model, we found p-PI3K, p-Akt and p-GSK3β were significantly increased, and the level of GS was significantly increased, and glycogen synthesis was promoted compared with T2D mouse model transfected with sh-NC.Conclusions Overall, this experiment provides a new molecular target for the treatment of T2DM by revealing the role of CK8 inhibiting hepatic glycogen synthesis in T2DM via regulating insulin-dependent IRS1/PI3K-Akt-GSK3β pathway. CK8 may play an important role in the pathogenesis of glycogen synthesis in T2DM.