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

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.


Keywords


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

Full Text:

PDF

References


Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390(6655):45-51.

Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, et al. Suppression of aging in mice by the hormone Klotho. Science. 2005;309(5742):1829-1833.

Watson K, Nasca C, Aasly L, McEwen B, Rasgon N. Insulin resistance, an unmasked culprit in depressive disorders: Promises for interventions. Neuropharmacology. 2018;136(Pt B):327-334.

Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, et al. Secreted Klotho protein in sera and CSF: implication for post‐translational cleavage in release of Klotho protein from cell membrane. FEBS Lett. 2004;565(1-3):143-147.

Le Tilly E, Ong T, Abadie J, Guicheux J, Beck L, Vinatier C. Involvement of the anti-aging protein Klotho in chondrocyte autophagy and apoptosis during osteoarthritis. Osteoarthr Cartil. 2018;26:S20-S21.

Chen CD, Podvin S, Gillespie E, Leeman SE, Abraham CR. Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17. Proc Natl Acad Sci U S A. 2007;104(50):19796-19801.

Razzaque MS. The role of Klotho in energy metabolism. Nat Rev Endocrinol. 2012;8(10): 579-587.

Utsugi T, Ohno T, Ohyama Y, Uchiyama T, Saito Y, Matsumura Y, et al. Decreased insulin production and increased insulin sensitivity in the klotho mutant mouse, a novel animal model for human aging. Metabolism. 2000;49(9):1118-1123.

Quarles LD. Fibroblast growth factor 23 and α-Klotho co-dependent and independent functions. Curr Opin Nephrol Hypertens. 2019;28(1):16-25.

Inci A, Olmaz R, Sarı F, Coban M, Ellidag HY, Sarıkaya M. Increased oxidative stress in diabetic nephropathy and its relationship with soluble Klotho levels. Hippokratia. 2016;20(3):198-203.

Kuro-o M. Klotho and aging. Biochim Biophys Acta. 2009;1790(10):1049-1058.

Unger RH. Klotho-induced insulin resistance: a blessing in disguise? Nat Med. 2006;12(1):56-57.

Rhodes CJ, White MF, Leahy JL, Kahn SE. Direct autocrine action of insulin on β-cells: does it make physiological sense? Diabetes. 2013;62(7): 2157-2163.

Kido Y, Burks DJ, Withers D, Bruning JC, Kahn CR, White MF, et al. Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2. J Clin Invest. 2000;105(2):199-205.

Pessin JE, Saltiel AR. Signaling pathways in insulin action: molecular targets of insulin resistance. J Clin Invest. 2000;106(2):165-169.

Ost A, Danielsson A, Lidén M, Eriksson U, Nystrom FH, Stralfors P. Retinol-binding protein-4 attenuates insulin-induced phosphorylation of IRS1 and ERK1/2 in primary human adipocytes. FASEB J. 2007;21(13):3696-3704.

Norseen J, Hosooka T, Hammarstedt A, Yore MM, Kant S, Aryal P, et al. Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing pro-inflammatory cytokines in macrophages through a JNK-and TLR4-dependent and retinol-independent mechanism. Mol Cell Biol. 2012:32(10):2010-2019.

Li D, Dobrowolska G, Aicher LD, Chen M, Wright JH, Drueckes P, et al. Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle. J Biol Chem. 1999;274(46):32988-32996.

Eckel R, Fujimoto W, Brunzell J. Effect of in-vitro lifespan of 3T3-L1 cells on hormonal responsiveness of lipoprotein lipase activity. Int J Obes.1981;5(6):571-577.

Tafuri SR. Troglitazone enhances differentiation, basal glucose uptake, and Glut1 protein levels in 3T3-L1 adipocytes. Endocrinology. 1996;137(11):4706-4712.

Shahrestanaki MK, Aghaei M. A3 receptor agonist, Cl-IBMECA, potentiate glucose-induced insulin secretion from MIN6 insulinoma cells possibly through transient Ca2+ entry. Res Pharm Sci. 2019;14(2):107-114.

Koshy S, Alizadeh P, Timchenko LT, Beeton C. Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry. J Vis Exp. 2010 (45). pii: 2429. DOI: 10.3791/2429.

Ataei N, Aghaei M, Panjehpour M. Cadmium induces progesterone receptor gene expression via activation of estrogen receptor in human ovarian cancer cells. Res Pharm Sci. 2018;13(6):493-499.

Shahrestanaki MK, Arasi FP, Aghaei M. Adenosine protects pancreatic beta cells against apoptosis induced by endoplasmic reticulum stress. J Cell Biochem. 2018;120(5):7759-7770.

Shahrestanaki MK, Arasi FP, Aghaei M. IPP-1 controls Akt/CREB phosphorylation extension in A2a adenosine receptor signaling cascade in MIN6 pancreatic β-cell line. Eur J Pharmacol. 2019;850:88-96.

Jaldin-Fincati JR, Pavarotti M, Frendo-Cumbo S, Bilan PJ, Klip A. Update on GLUT4 vesicle traffic: a cornerstone of insulin action. Trends Endocrinol Metab. 2017;28(8):597-611.

Ma J, Nakagawa Y, Kojima I, Shibata H. Prolonged insulin stimulation down-regulates GLUT4 through oxidative stress-mediated retromer inhibition by a protein kinase CK2-dependent mechanism in 3T3-L1 adipocytes. J Biol Chem. 2014;289(1):133-142.

Trefely S, Khoo PS, Krycer JR, Chaudhuri R, Fazakerley DJ, Parker BL, et al. Kinome screen identifies PFKFB3 and glucose metabolism as important regulators of the insulin/IGF-1 signalling pathway. J Biol Chem. 2015;290(43):25834-25846.

Minokoshi Y, Kahn CR, Kahn BB. Tissue-specific ablation of the GLUT4 glucose transporter and the insulin receptor challenge assumptions about insulin action and glucose homeostasis. J Biol Chem. 2003; 278(36):33609-33612.

Lorenzi O, Veyrat-Durebex C, Wollheim CB, Villemin P, Rohner-Jeanrenaud F, Zanchi A, et al. Evidence against a direct role of klotho in insulin resistance. Pflugers Arch. 2010;459(3):465-473.

Chihara Y, Rakugi H, Ishikawa K, Ikushima M, Maekawa Y, Ohta J, et al. Klotho protein promotes adipocyte differentiation. Endocrinology. 2006;147(8):3835-3842.

Fan J, Sun Z. The antiaging gene klotho regulates proliferation and differentiation of adipose‐derived stem cells. Stem Cells. 2016;34(6):1615-1625.

Samms RJ, Cheng CC, Kharitonenkov A, Gimeno RE, Adams AC. Overexpression of β-klotho in adipose tissue sensitizes male mice to endogenous FGF21 and provides protection from diet-induced obesity. Endocrinology. 2016;157(4):1467-1480.

Wang Q, Somwar R, Bilan PJ, Liu Z, Jin J, Woodgett JR, et al. Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol Cell Biol. 1999;19(6):4008-4018.


Refbacks

  • There are currently no refbacks.


Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.