The Complex Interplay between AHNAKI and Calcium during Early Stages of Adipogenesis in 3T3-Ll Cells
Sebastiani, Alejandra C.
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Obesity, which is currently a major public health issue, results from excess adipose tissue. AHNAKI, may affect the onset of adipogenesis through its role in the phospholipase C gamma I (pLC𝛾l) signaling cascade at the plasma membrane of preadipocytes, in which intracellular calcium stores are released. High intracellular calcium has been shown to attenuate adipogenesis in 3T3-L I cells, a model cell line· for differentiation. As calcium homeostasis and AHNAKI function may be interdependent, this study sought to identify the effect of extracellular calcium on AHNAKI translocation from the nucleus to the plasma membrane, as well as the effect of AHNAKI on intracellular calcium mobilization in 3T3-LI cells. Subcellular localization of AHN'AKI in preadipocytes and early stage adipocytes cultured in normal and high extracellular calcium was determined. Subcellular fractionation assays, Western blotting, and densitometric analysis revealed a shift of AHNAKI from the nucleus to the plasma membrane in high extracellular calcium in preadipocytes. These results were corroborated by immunofluorescence studies. Immunocytochemical staining also showed. reduced AHNAKI in adipocytes. Calcium mobilization experiments employed the Fluo-3 AM system to measure relative fluorescence upon addition of phorbol 12-myristate 13- acetate (PMA), releasing calcium via an AHNAKI-dependent pathway. Levels of PM Ainduced calcium release were determined for AHNAKI transfected cells, AHNAKI knockdowns, adipocytes in the early stages of differentiation, as well as preadipocytes preincubated in high extracellular calcium. Calcium release seemed to be AHNAKI-dependent. Further investigation of the complex interaction of this protein and calcium during adipogenesis could reveal AHNAKI as a potential therapeutic target for obesity.