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

Abstract

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.

Upregulation of Klotho Aggravates Insulin Resistance in Gestational Diabetes Mellitus Trophoblast Cells

“…Still, these results were reversed after klotho overexpression. This was consistent with the fact that klotho could reduce the phosphorylation of Akt to weaken insulin signal transduction [ 22 ]. These results suggested that klotho gene knockdown could activate the IGF-1/PI3K/Akt/mTOR signaling pathway in trophoblast cells.…”

“…Still, these results were reversed after klotho overexpression. is was consistent with the fact that klotho could reduce the phosphorylation of Akt to weaken insulin signal transduction [22].…”

“…It has been proven that klotho is closely related to the insulin signaling pathway of cells [ 22 ]. This study further verified whether klotho damages the insulin signaling pathway of trophoblast cells caused by HG.…”

Serum Klotho and insulin resistance: Insights from a cross-sectional analysis

“…This weakens intracellular signal transduction, prevents the translocation of Glucose Transporter Type 4, and triggers insulin resistance in adipocytes. [25,26] Furthermore, Klotho plays a significant role in the internal regulation of insulin activity as a negative feedback mechanism: insulin promotes the shedding of Klotho, elevating serum Klotho protein levels, thereby hindering peripheral insulin signaling and blocking extended insulin activity. [27] On the other hand, some studies have found that in T2DM mice models, Klotho could improve insulin sensitivity and hepatic glucose homeostasis.…”

A Survey of Genetic Risk Factors for Alzheimer’s Disease

“…The Klotho gene is located on Chromosome 13 and has a length of 1012 amino acids (25). The Klotho gene codes for the protein Klotho (KL), which is an anti-aging protein that protects oxidative stress (25), provides insulin resistance (26), and aids calcium homeostasis (27). The KL-VS variant of the Klotho gene plays a role in AD pathology.…”