(F-216) Stretch activation of yes-associated protein (YAP) in adipocytes

Introduction:: Adipose tissue is physiologically exposed to compound mechanical loading due to bodyweight and movement. The ability for adipocytes to sense and respond to physiological mechanical environments may behave as a link between obesity and the development of related metabolic actions. Yes-associated protein (YAP) is an on-off mechanosensitive transcriptional regulator that, when unphosphorylated, becomes activated and translocates to the nucleus to coordinate cell growth, differentiation, and potentially metabolism. We used a computer-regulated tension system to apply cyclic equiaxial stretch loading to differentiated 3T3-L1 adipocytes. We found that stretch applied to adipocytes induces YAP nuclear import for its activation.

Materials and Methods:: 3T3-L1 preadipocytes were seeded at a density of 3,000 cells/cm² on collagen-coated Bioflex stretch plates (Flexcell) and maintained in DMEM with 10% FBS and 1% P/S until 2-3 days post-confluence. After adipogenesis was induced, cells were differentiated until at least 90% of the cells displayed lipid accumulation. Differentiated adipocytes were exposed to 10% cyclic equiaxial strain at 1 Hz frequency for up to 2 h. For imaging, adipocytes were treated with anti-YAP followed by Alexa Fluor 488-conjugated conjugated antibody, and then stained with DAPI and Phalloidin. Expression of phosphorylated YAP (pYAP) and GAPDH were measured using western blotting, and the band intensities were quantified using ImageJ.

Results, Conclusions, and Discussions:: Mature adipocytes were exposed to a physiological level cyclic mechanical stretch and the activation of the transcriptional regulator YAP was determined. YAP was translocated to the nucleus as shown by immunofluorescent imaging (Fig. 1A) and quantification of subcellular localization intensity (Fig. 1B). Western blotting results show that YAP phosphorylation was reduced by stretch within 15 min (Fig. 1C), supporting the imaging results, as YAP dephosphorylation allows nuclear import. Our results indicate that cyclic stretch activates YAP and induces its translocation to the nucleus for differentiated adipocytes. Our findings support our hypothesis that stretch regulates YAP behavior in adipocytes, opening a new research field for mechanical load regulation of metabolic action in adipocytes potentially through YAP.

Acknowledgements (Optional): : NSF GRFP 1610400 (Bouzid), NIH/NIGMS COBRE NPOD Project Leader Grant (Lim; P20GM104320, PI: Zempleni), and University of Nebraska Collaboration Initiative (Lim).


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