The Complex Interplay between AHNAKI and Calcium during Early Stages of Adipogenesis in 3T3-Ll Cells
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Authors
Sebastiani, Alejandra C.
Issue Date
2010
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
iv, 28 p.
Citation
Publisher
Kalamazoo College
License
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