Bax signaling regulates palmitate-mediated apoptosis in C(2)C(12) myotubes.
Insulin resistance is a primary characteristic of type 2 diabetes. Several lines of evidence suggest that accumulation of free fatty acids in skeletal muscle may at least in part contribute to insulin resistance and may be linked to mitochondrial dysfunction, leading to apoptosis. Palmitate treatment of several cell lines in vitro results in apoptosis and inhibits protein kinase B (Akt) activity in response to insulin. However, the role of Bax and Bcl-2 in regulating palmitate-induced apoptosis has not been well studied. Therefore, the purpose of this study was to determine whether palmitate-induced apoptosis in C(2)C(12) myotubes is dependent on Bax to Bcl-2 binding. An additional purpose of this study was to determine whether the changes in Bax to Bcl-2 binding corresponded to decreases in Akt signaling in palmitate-treated myoblasts. Apoptotic signaling proteins were examined in C(2)C(12) myotubes treated overnight with palmitate. Bax to Bcl-2 binding was determined through a coimmunoprecipitation assay that was performed in myotubes after 2 h of serum starvation, followed by 10 min of serum reintroduction. This experiment evaluated whether temporal Akt activity coincided with Bax to Bcl-2 binding. Last, the contribution of Bax to palmitate-induced apoptosis was determined by treatment with Bax siRNA. Palmitate treatment increased apoptosis in C(2)C(12) myotubes as shown by a twofold increase in DNA fragmentation, an approximately fivefold increase in caspase-3 activity, and a 2.5-fold increase in caspase-9 activity. Palmitate treatment significantly reduced Akt protein expression and Akt activity. In addition, there was a fourfold reduction in Bax to Bcl-2 binding with palmitate treatment, which mirrored the reduction in Akt(Ser473) phosphorylation. Furthermore, treatment of the C(2)C(12) myotubes with Bax siRNA attenuated the apoptotic effects of palmitate treatment. These data show that palmitate induces Bax-mediated apoptosis in C(2)C(12) myotubes and that this effect corresponds to reductions in Akt(Ser473) phosphorylation.
Peterson
Jonathan M
JM
West Virginia University School of Medicine, Morgantown, WV 26506, USA.
Wang
Yan
Y
Bryner
Randall W
RW
Williamson
David L
DL
Alway
Stephen E
SE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002460:Cell Line; D053938:DNA Fragmentation / Q000187:drug effects; D051379:Mice; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000378:metabolism; D019308:Palmitic Acid / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*
D051017:Apoptosis Regulatory Proteins; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D019308:Palmitic Acid; D051058:Oncogene Protein v-akt