Research ArticleSynergistic effect of cAMP and palmitate in promoting altered mitochondrial function and cell death in HepG2 cells
Introduction
Exposure of non-adipose cells, including β-cells, cardiomyocytes and hepatocytes, to excess free fatty acids (FFAs) has been shown to induce lipotoxicity and cell death [1], [2], [3], [4], [5], [6], [7], [8], [9]. While elevated unsaturated FFAs, such as oleate and linoleate, are better tolerated, elevated saturated FFAs, such as palmitate, can cause cellular damage and even cell death [1], [2]. cAMP (cyclic adenosine monophosphate), an important second messenger, has been shown to protect pancreatic β-cells from palmitate-induced apoptosis [10], [11]. In addition, cAMP was also reported to protect hepatocytes from bile acid [12], [13], Fas ligand [13], [14] and TNF-α [13], [15] induced apoptosis. Previous study in our group indicated that intracellular cAMP level in HepG2 cells was reduced significantly by palmitate, but not oleate or linoleate [16]. Therefore, we initially hypothesize that the down-regulation of cAMP by palmitate may play a role in the induction of cell death. However, restoring cAMP levels in HepG2 cells did not rescue the cells from palmitate-induced toxicity. Moreover, when cAMP level was increased to a high concentration, it synergized with palmitate to promote cell death. cAMP has been proposed as a potential drug target for type 2 diabetes [17], since it appears to enhance insulin secretion. Given that FFAs, which are elevated in obesity and diabetes [18], [19], [20], can induce hepatotoxicity, it warrants evaluating the effect of cAMP in the presence of elevated FFAs. In this study, we assess the effect of cAMP on hepatotoxicity and cell death under elevated levels of FFAs.
Mitochondria serve as an integrator for cell death and survival signals [21], [22], regulating both apoptosis and necrosis. The onset of mitochondrial membrane permeabilization (MMP) releases a number of cytotoxic proteins, such as cytochrome C and Smac (second mitochondria-derived activator of caspase), from the intermembrane space of mitochondria [23]. Release of these cytotoxic proteins activates both caspase-dependent and independent cell death [23] and such release can be controlled by mitochondrial morphology. In healthy cells, the mitochondria display an elongated and interconnected structure. When a cell becomes apoptotic, its mitochondrial network is disrupted into short and disconnected structure [24], [25]. Mitochondrial function is also altered in necrosis, manifesting in diminished mitochondrial ATP production [26]. Apoptosis and necrosis can occur simultaneously in the same cell. Cells in late stage apoptosis may contain necrotic features due to the energy loss and permeabilization of the plasma membrane [27].
Regulation of cell death by mitochondria is intimately tied with generation of reactive oxygen species (ROS) in the mitochondria [28]. Superoxide anion (O2−), which is the precursor of most ROS, is primarily generated at Complex I and Complex III in the mitochondria [28]. O2− itself is not a strong oxidant. Dismutation of superoxide by superoxide dismutase (SOD) can produce a stronger oxidant, hydrogen peroxide (H2O2), which can be partially reduced to generate one of the strongest oxidants, hydroxyl radical (HO) [29]. Together these oxidants can damage cellular components, proteins and resulted in cell death [29].
This study aims to evaluate how cAMP and FFAs affect hepatocyte survival and death behavior. Our results suggest that a high cAMP level potentiates mitochondrial fragmentation, mitochondrial ROS generation and necrotic cell death initiated by palmitate.
Section snippets
Cell culture and materials
Human hepatocellular carcinoma cell line HepG2 cells were obtained from American Type Culture Collection. Cells were cultured in DMEM (Dulbecco's modified Eagle's medium) (Invitrogen) containing 10% FBS (fetal bovine serum) (Invitrogen) and 2% PS (penicillin–streptomycin) (Invitrogen) at 37 °C in 10% CO2 atmosphere incubator. Saturated free fatty acid palmitate, monounsaturated free fatty acid oleate and polyunsaturated free fatty acid linoleate were purchased from Sigma in the form of sodium
Palmitate-induced cell death in HepG2 cells
Previous work had ascribed a lipotoxic effect to saturated FFAs, i.e. palmitate [1], [2]. We evaluated the effect of palmitate as well as unsaturated FFAs, oleate and linoleate, on HepG2 cells. The toxicity, as indicated by LDH release, was observed in palmitate-treated but not oleate- and linoleate-treated HepG2 cells (Fig. 1A). In addition, cell staining with propidium iodide (PI, labels dead cells and late apoptotic cells) and Alexa Fluor 488-conjugated annexin V (labels early and late
Discussion
Lipotoxicity induced by palmitate has been demonstrated in a number of different cell types. In human HepG2 cells, both apoptotic behaviors, such as caspase 3 activation (Fig. 8A) and annexin V labeling for phosphatidylserine (Fig. 1B), and necrotic behaviors, such as propidium iodide (PI) penetration (Fig. 1B), have been observed for palmitate-induced cell death. Many obese and diabetic patients have high plasma FFAs levels [18], [19], [20]. cAMP has been proposed as a potential drug target
Acknowledgments
The work was supported in part by National Institutes of Health (R01GM079688, R21CA126136, R21RR024439, and R21GM075838), National Science Foundation (CBET 0941055), and the MSU Foundation.
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