Klotho induces insulin resistance possibly through interference with GLUT4 translocation and activation of Akt, GSK3β, and PFKfβ3 in 3T3-L1 adipocyte cells

Mohamad Hasannejad , Seyed Ziaaldin Samsamshariat , Armita Esmaili, Ali Jahanian-Najafabadi


Klotho is considered as an anti-aging factor inducing insulin resistance and involved in type 2 diabetes. However, mechanisms by which klotho induces insulin resistance remain to be understood.  Thus, in this study, we aimed to evaluate possible interference points of klotho with insulin signaling pathways in 3T3-L1 adipocyte cells by focusing on phosphorylation levels of Akt, GSK3β, PFK-fβ3, and GLUT4 translocation. Differentiation of 3T3-L1 cells to the adipocyte-like cells were performed using specific differentiation kit and confirmed by mRNA expression assay of PPARγ using qRT-PCR, and Sudan black staining of lipid droplets. Then cells were co-treated with klotho and insulin. Expression and translocation of GLUT4 mRNA were evaluated using qRT-PCR and Alexa flour 488 conjugated GLUT4 antibody, respectively. P-Akt/Akt, p-GSK3β/GSK3β, and p-PFKfβ3/PFKfβ3 ratios were determined in insulin and klotho/insulin treated cells using western blot. Our result indicated that GLUT4 expression were decreased to 0.72 ± 0.16 fold in insulin treated cells, however it was calculated 1.12 ± 0.25 fold in klotho/insulin treated cells. In addition, klotho prevented GLUT4 membrane translocation by 27.2% in comparison with insulin-treated cells (P < 0.05). Interestingly, in insulin/klotho co-treated cells,  phospho-levels of Akt, GSK3β, and PFKfβ3 proteins was decreased to 2.34 ± 0.14, 2.29 ± 0.63,  and  1.95 ± 0.37 fold in comparison with the insulin cells, (P < 0.05). In conclusion, our study indicated that klotho induces insulin resistance in adipocytes possibly through prevention of GLUT4 translocation, and interfere with phosphorylation of Akt, GSK3β, and PFKf3β intracellular signaling mediators by insulin.


Adipocyte; Insulin resistance; Klotho; 3T3-L1 cell.

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