Bax signaling regulates palmitate-mediated apoptosis in C₂C₁₂myotubes

Abstract

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.

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone, a radiolytic product of palmitic acid, in human lymphoma U937 cells

“…An oxidative endpoint marker of DNA, 8-oxo-guanine has attracted particular attention since it is mutagenic it causes G-to-T transversions upon replication and has been suggested to play an important role in carcinogenesis [38]. In the current study, 2-DCB enhanced intracellular ROS generation in a time-dependent manner and the pretreatment of cells with NAC significantly prevented 2-DCB-induced apoptosis, indicating that Two major pathways, the extrinsic and intrinsic pathways, leading to caspase activation have been found [10,15]. Our data showed the activation of Fas and caspase-8 after the treatment with 2-DCB and PA.…”

“…Moreover, Bid was cleaved by 2-DCB and PA. Bid can be cleaved by caspase-8, and the cleaved Bid as the carboxyl-terminal fragment translocates to the mitochondria to induce the release of cytochrome c [4]. Cytochrome c functions with Apaf-1 to induce caspase-9 activation, thereby initiating the apoptotic caspase cascade [15]. These two apoptotic pathways may be interconnected by the caspase-8-mediated cleavage of Bid, which triggers the activation of the mitochondrial pathway.…”

“…Recent studies showed that PA could induce apoptosis in human lung carcinoma cells, hepatocytes, renal cells, smooth muscle cells and endothelial progenitor cells [8][9][10][11][12]. The mechanisms of apoptosis induced by PA are involved in endoplasmic reticulum stress, mitochondrial dysfunction, PKC activation and increased oxidative stress [10,[13][14][15][16]. However, the effects of 2-DCB, a radiolytic product of PA, on apoptosis induction have never been shown.…”

Brain and Muscle Arnt-like 1 is a Key Regulator of Myogenesis

“…The supernatant was collected as the cytosolic fraction and total cellular protein was obtained separately. Nuclear protein was extracted as described (Peterson et al, 2008). Briefly, cells were harvested in hypotonic buffer containing 50 mM NaCl and the nuclear pellet was collected, washed and subsequently lysed in buffer containing 500 mM NaCl.…”

High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle

“…5 In vitro studies have demonstrated that exposure of myotubes to a high-fat (HF) environment results in increased caspase-3 activity 6 and DNA fragmentation. [7][8][9] Similarly, MCK(m)-hLPL mice (which accumulate muscle lipids) have a higher percentage of apoptotic nuclei in muscle, 5 while triacylglycerol infusion into lean mice increases muscle caspase-3 activation. 10 Additionally, 16 weeks of HF feeding has been shown to increase caspase-3 activity in mouse muscle.…”