Palmitic acid is a major type of SFAs in the liver and plays essential roles in maintaining liver health. Previous study reported that 0.2 mM palmitic acid markedly decreased the cell viability in HepG2 cells ; in contrast, our laboratory demonstrated that the cell viability of goose primary hepatocytes was not changed by treatment with 0.6 mM palmitic acid , suggesting that there may be significant differences in the palmitic acid tolerance between goose and human hepatocytes. In this study, the palmitic acid of 0.2-0.9 mM was used to treat goose primary hepatocytes and LO2 cells cultured in vitro. Our results showed that the maximum tolerant concentration to palmitic acid of goose primary hepatocytes was 0.6 mM, which was consistent with the results of Pan et al. ; however, the maximum tolerance concentration of palmitic acid in LO2 cells is less than 0.2 mM, which was consistent with the results of the study on HepG2 cells . The above results showed that goose primary hepatocytes have a higher tolerance to palmitic acid than LO2 cells, which may be one of the reasons why goose liver has an excellent capacity for fat accumulation.
To fully explore palmitic acid tolerance of goose primary hepatocytes, we then compared the mRNA levels of ER stress-, inflammation-, and apoptosis- related genes in goose primary hepatocytes and LO2 cells. ER stress, inflammation, and apoptosis are important basis for evaluating the tolerance of fatty acids in hepatocytes. ER stress occurs extensively in the livers of individuals with steatohepatitis . Previous studies indicated that addition of palmitic acid increased the expression of ER stress-related genes in mammalian hepatocytes [21, 22]. In this study, we found that treatment with 0.6 mM palmitic acid had no significant effect on the mRNA expression of ER stress-related genes (XBP, BIP and ATF6) in goose primary hepatocytes. However, in LO2 cells, levels of ER stress-related genes were markedly increased (P<0.05), and mRNA levels of BIP was increased more than 10-fold. XBP, ATF6 and BIP are located on three pathways that cause the UPR response of ER stress , the mRNA expression of the three genes did not change significantly in goose primary hepatocytes, indicating that goose primary hepatocytes have a strong tolerance to palmitic acid in ER stress response. In addition, increasing evidence indicated that treatment of mammalian hepatocytes with palmitic acid can increase the content of cellular pro-inflammatory factors [23, 24]. Inflammatory factors IL-6, IL-1, and IFN-γ are used as biomarkers of hepatitis in mammals, and their expression has significantly increased in mammalian fatty hepatitis . Both ER stress and inflammatory response lead to massive apoptosis of cells. Our results showed that addition of palmitic acid had no significant effect on the mRNA levels of the inflammatory response-related genes (IL-6, IL-1β and IFN-γ) and apoptosis-related genes (Bax, Bcl-2, Caspase-3 and Caspase-9) in goose primary hepatocytes, but in LO2 cells their mRNA levels markedly increased compared to the control group (P<0.05). Remarkably, the mRNA levels of Bax were increased more than 3-fold, which is consistent with the results in HepG2 cells . Therefore, we conclude that goose hepatocytes were more tolerant to palmitic acid than LO2 cells in ER stress, inflammation and apoptosis.
Results from previous studies reveled that overfeeding significantly increased expression of genes related to fat synthesis in the goose liver [13, 27]. In the liver, palmitic acid elongation the carbon chain is mainly catalyzed by ELOVL6 enzyme, and desaturation under the action of SCD1, FADS1, and FADS2, and finally forms non-toxic triglycerides under the action of DGAT2 . And the above five genes mRNA levels significantly increased in the goose liver after overfeeding . In our study, the levels of fatty acid desaturation-related genes (SCD1 and FADS2) mRNA was markedly enhanced by addition of 0.6mM of palmitic acid (P<0.05) in goose primary hepatocytes, while no significant differences were seen in LO2 cells. We also found that the expression of the fatty acid elongate enzyme-related gene (ELOVL6) in both cells significantly increased (P<0.05), whereas that of triglyceride synthesis-related gene (DGAT2) remained statically unchanged in these two cells. Together, these results indicated that palmitic acid has an important effect on the fatty acid desaturation process in goose hepatocytes, and also indicates that the fatty acid desaturation process may play an important role to palmitic acid tolerance of goose liver.
SCD1 is the key regulatory enzyme responsible for the desaturation of SFAs, which can desaturation palmitic acid into MUFAs. Moreover, increasing studies have shown that the mRNA levels of SCD1 was increased in overfeeding-induced goose fatty liver [1, 12, 13]. Therefore, this study was to study the effects of SCD1 on palmitic acid tolerance of goose primary hepatocytes. In our study, under the treatment of 0.6 mM palmitic acid, downregulation of SCD1 was able to decrease the tolerance of goose primary hepatocytes to palmitic acid (P<0.05), indicating that SCD1 play crucial role in palmitic acid tolerance of goose hepatocytes. Then, we further investigated the palmitic acid tolerance of goose primary hepatocytes in ER stress, inflammatory and apoptosis after downregulating with SCD1. Our data showed that downregulation of SCD1 had no significant effect on the mRNA expression of ER stress-related genes (XBP, BIP and ATF6)，indicating that SCD1 did not affect the palmitic acid tolerance of goose primary hepatocytes in ER stress response. Furthermore, SCD1 downregulation resulted in the mRNA levels of inflammatory factors (IL-6 and IL-1β) were significantly increased, suggesting that SCD1 can regulate palmitic acid tolerance of goose primary hepatocytes in inflammation pathway where IL-6 and IL-1β are located. Moreover, our results showed that downregulation of SCD1, the mRNA levels of apoptosis related genes (Bax, Bcl-2, Caspase 3 and Caspase 9) were not significantly changed, but the protein expression of cytochrome C and the apoptosis rate significantly increased, which indicated that SCD1 increases palmitic acid tolerance goose primary hepatocytes by inhibiting apoptosis. These data demonstrated that SCD1 plays a critical role in mediating palmitic acid tolerance of goose primary hepatocytes through inflammation and apoptosis response.
Finally, to further explore the specific pathways of SCD1 regulating palmitic acid tolerance of goose primary hepatocytes. Then, we tested the mRNA levels of key genes in the SCD1 regulatory pathways. In our study, we found that under the treatment of 0.6mM palmitic acid, downregulation of SCD1 significantly increased the mRNA expression of AKT1, AKT2, FOXO1 and SIRT1, while that of mTOR and AMPK were not significantly altered, indicating SCD1 can regulate the AKT/FoxO1 pathway at the transcription level. In addition, SIRT1 can regulate FoxO1 through acetylation, and early studies have demonstrated that FoxO1 play an important role in regulating inflammation and apoptosis [29-31], but the regulatory relationship between SCD1 and SIRT1 has not been reported. Therefore, we speculated that SCD1 may regulate the AKT/FoxO1 pathway through SIRT1/FoxO1 pathway or increase the ratio of SFAs/MUFAs to inhibit the occurrence of inflammation and apoptosis response. Together, these data support a conclusion that SCD1 may increase the palmitic acid tolerance of goose primary hepatocytes by regulating the AKT/FoxO1, SIRT1/FoxO1 pathways to inhibit the occurrence of inflammatory and apoptosis.
In summary, data from the present study suggested that goose primary hepatocytes have a higher tolerance to palmitic acid than LO2 cells and that SCD1 has a crucial role in enhancing the palmitic acid tolerance of goose primary hepatocytes by regulating inflammation- and apoptosis-related genes expression.