The liver is the most metabolically active organ in the human body and is responsible for many vital functions, including lipid metabolism. Triglycerides (TGs) accumulate within hepatocytic lipid droplets. Notably, both the lipolysis and synthesis of TG can produce FAs and specific toxic lipid intermediates that activate intracellular inflammatory pathways. FA oxidation provides a major source of reactive oxygen species (ROS). During the process of hydrolysis, TG expands the availability of FA to metabolic pathways, such as peroxidation; increases ROS; and, subsequently, increases levels of oxidative stress. Oxidative stress results in the activation of several key pro-inflammatory signaling pathways, including the nuclear factor-kappa B (NF-κB) pathway. Conversely, although TG synthesis decreases cellular FA concentrations, this process also generates other potentially toxic lipid intermediates. Therefore, dysfunctional FA metabolism influences the cellular inflammatory state and is involved in the pathogenesis of liver disorders, such as non-alcoholic fatty liver disease (NAFLD).
The present study demonstrates that the exposure of HepG2 cells to OA as well as TNF-α can result in the increased expression of apoO. To account for this phenomenon, we examined gene expression in apoO-silenced HepG2 cells with the use of microarrays. According to our microarray data, silencing apoO in HepG2 cells leads to the differential expression of several important lipid signaling and inflammation genes.
In comparison with the negative control cells, ACSL4 was up-regulated in apoO-silenced HepG2 cells. ACSL4 encodes an isoform of the long-chain acyl-CoA synthetase (ACSL), which catalyzes acyl-CoA synthesis by converting long-chain FA to acyl-CoA. African-American NAFLD patients over-express ACSL4; ACSL4 mRNA levels have been positively associated with liver TG concentrations. Thus, increased ACSL4 expression could indicate an up-regulation of TG synthesis.
Mitochondria are the major site of FA oxidation. RGS16 encodes a protein which inhibits G protein-coupled receptor (GPCR)-stimulated FA oxidation in liver mitochondria. Its expression increased after transfection. CROT and CYP4F11 encode two key FA oxidation enzymes, respectively. In the peroxisome, storage of medium chain acyls slows down peroxisomal beta oxidation. When CROT activity increases, the level of medium chain acyls decreases as they are converted into acyl-carnitines. In the microsome, another FA oxidation site, CYP4F11 is the predominant catalyst of FA omega hydroxylation. Within this context, silencing of apoO with altered RGS16, CROT and CYP4F11 expression would modulate not only FA oxidation rates but also cellular TG content.
The NF-κB protein family includes transcription factors that regulate crucial cellular processes, such as the inflammation response. NFKBIZ encodes a novel member of the IκB family, IκB zeta. IκB zeta associates with both the p65 and p50 subunits of NF-κB and inhibits the transcriptional activity and DNA binding of NF-κB. USP2 is a ubiquitin-specific protease which is required for the phosphorylation of IκB and functions as an additional positive regulator of TNF-α-induced NF-κB signaling. The protein encoded by TNFSF15 gene is a cytokine that belongs to the tumor necrosis factor ligand family and is capable of activating NF-κB. In addition, the two cytokines IL-17 and CCL23 may induce inflammatory gene expression by interaction with the NF-κB pathway[15, 16]. As apoO silencing in HepG2 cells resulted in down-regulation of NFKBIZ and up-regulation of the pro-inflammatory molecules mentioned above, it is possible that apoO may exert anti-inflammatory effects through suppressing NF-κB pathway.
Notch signaling is involved in the inflammatory response. There is complex crosstalk between the Notch 2 and NF-κB pathways as both pathways can exert either synergistic or antagonistic effects depending on different cellular contexts[18–20]. APH-1 is one of the four components of γ-secretase complex, which is responsible for the release of the notch intracellular domain (NICD) into the cytoplasm. These subunits are sufficient and required for γ-secretase activity. The product of N2N gene is homologous to Notch 2. In vitro, N2N repressed the transcriptional activity of the Notch 2 protein in a dose-dependent manner. Our microarray data showed that activation of NF-κB pathway was accompanied by impaired expression of Notch2, APH-1B and enhanced expression of N2N, suggesting the antagonistic effect between the NF-κB and Notch 2 signaling pathways in apoO-silenced HepG2 cells.
Uncoupling protein (UCP) 2 and UCP3 are members of a mitochondrial carrier protein superfamily that controls the level of respiration coupling. The UCP2 and UCP3 genes are located together in a gene cluster, but the pattern of their expression is very different. UCP3 is primarily expressed in skeletal muscle tissue, whereas UCP2 is expressed widely. Although these two uncoupling proteins are thought to have similar physiological functions, the changes of UCP2 expression levels in liver cells may be more significant. UCP2 can dissipate the proton gradient across the mitochondrial inner membrane to prevent the proton-motive force from becoming excessive, thus limiting mitochondrial ROS production. It acts as a sensor for mitochondrial oxidative stress and protects against oxidative damage by controlling the production of ROS. UCP2 is also implicated in fat oxidation and the regulation of fat content. It functions as a metabolic switch that is involved in the choice of substrate oxidized by mitochondria which promotes FA metabolism over glucose utilization. It could be speculated that reduction of UCP2 expression in apoO-silenced HepG2 cells would lead to mitochondrial dysfunction accompanied by elevated ROS production and oxidative stress in hepatocytes. Subsequently, mitochondria might regulate the generation of ROS by altering the activity levels of enzymes that can affect FA oxidation. Therefore, reduction of UCP2 provides a possible mechanism in which FA metabolism and ROS-induced inflammatory responses can be simultaneously modulated in HepG2 cells after the silencing of apoO.
Some limitations of this study should be considered. Firstly, although the HepG2 hepatoma cell line is frequently used to study apolipoprotein metabolism, they are not the gold standard model for the study of the native liver. Therefore, gene expression changes related to apoO in our study may not reflect those that occur in liver in vivo. Secondly, we have only reported the result of an experiment that illustrated that apoO expression was dramatically affected by OA and TNF-α, but changes in inflammation and lipid metabolism genes in apoO-silenced HepG2 cells pretreated either with OA or TNF-α were not explored. Hence, the exact mechanisms involved in apoO-dependent changes in gene expression have yet to be elucidated. In addition, apoO, besides being secreted, could reside within cells where it is expressed. It may affect hepatocytes as a paracrine and/or autocrine factor, or even as an intracellular protein. This study did not differentiate whether changes of gene expression are due to the secreted apoO, or due to the intracellular apoO.