Hypoxic-ischemic injury in the nervous system is associated with the degradation of membrane phospholipids with subsequent release of free fatty acids (FFAs). It has been proposed that this increase in the release of FFA can cause fatty acid-induced apoptotic neuronal cell loss and would be at least in part responsible for the observed secondary injury seen following hypoxia/ischemia and nerve injury insult.
Purpose The present study uses NGF differentiated PC12 (NGFDPC12) cells to study neuronal apoptosis and to investigate the mechanism by which pathological concentrations of saturated FFA trigger neuronal cell death. We previously reported that NGFDPC12 cells exposed to palmitic acid (PA) show a dramatic loss of viability as early as 12 hours after treatment and exhibit an increase in the expression levels of several mRNAs of apoptosis-associated proteins such as BAK and βNIP-3 and hypoxic-inducing factor 1 alpha (HIF-1).
Methods To further study this observation, we performed a series of Western blots experiments using cell extract of NGFDPC12 cells exposed to PA bound to bovine serum albumin (BSA) (2:1 ratio) to determine the levels of βNIP-3 and its activator HIF-1. Cells were treated with the PA for 6, 9, 12, and 24 hours. Cells were then harvested and lysed in an extraction buffer with protease inhibitors. Next, 20 μg of total cellular protein were electrophoresed on 4-12% gradient SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane and blocked with 7.5% nonfat milk in TTBS for 1 hour. Blots were then probed with antibodies for HIF-1 or βNIP-3 followed by a secondary antibody conjugated to horseradish peroxidase. Antigen-antibody complexes were visualized with enhanced chemiluminescence.
Results Our Western blot results show an increase in both proteins HIF-1 and βNIP-3. HIF-1 and βNIP-3 have a maximum expression at 6 hours after PA treatment and gradually decreased thereafter.
Conclusion These findings suggest that these two apoptosis-associated proteins may play a role in the apoptotic cell damage mediated by saturated FFA in NGFDPC12 cells.
This work is supported in part by NIGMS award 2R25GM060507-05 to M.D.L.
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