Role of the stearyl-coenzyme A desaturase 1 gene in regulating palmitic acid tolerance of goose primary hepatocytes

Background Unlike mammals, goose fatty liver shows a strong tolerance to fatty acids without obvious injury. Stearyl-coenzyme A desaturase 1 (SCD1) serves crucial role in desaturation of saturated fatty acids (SAFs), but its role in the SAFs tolerance of goose hepatocytes has not been reported. This study was conducted to explore the role of SCD1 in regulating palmitic acid tolerance of goose primary hepatocytes. To evaluate the palmitic acid tolerance of cultured hepatocytes, MTT was examined to reflect the effect of palmitic acid on cell viability, and quantitative PCR was used to detect the mRNA expression levels of several genes related to ER stress, inflammation, and apoptosis, and the role of SCD1 in palmitic acid tolerance of goose hepatocytes was explored using RNA interfere. Our results indicated that goose hepatocytes exhibited a higher tolerant capacity to palmitic acid than human hepatic cell line (LO2 cells). Furthermore, the mRNA levels of fatty acid desaturation-related genes ( SCD1 and FADS2 ) and fatty acid elongate enzyme-related gene ( ELOVL6 ) were significantly upregulated in goose primary hepatocytes treated with 0.6 mM palmitic acid. However, in cultured LO2 cells, expression of ER stress-related genes ( XBP , BIP and ATF6 ), inflammatory response-related genes ( IL-6 , IL-1β and IFN-γ ) and apoptosis-related genes ( Bax , Bcl-2 , Caspase-3 and Caspase-9 ) was significantly enhanced by the addition of 0.6 mM palmitic acid. Additionally, siRNA-mediated downregulation of SCD1 significantly reduced the palmitic acid tolerance of goose primary hepatocytes under the treatment of 0.6 mM palmitic acid; meanwhile, the mRNA expression of inflammatory-related genes ( IL-6 and IL-1β and several key genes involved in the PI3K/AKT, FoxO1, mTOR and AMPK pathways ( AKT1 , AKT2 , FOXO1 and as well as the cytochrome and ( 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


Introduction
In mammals, excessive fat deposition in hepatocytes causes ER stress, inflammation and apoptosis, consequently leading the non-alcohol fatty liver to exacerbate into fatty hepatitis [1]. Goose (Anser anser), as a descendant of migrant birds, has an excellent capacity to deposit fat in the liver. In poultry production, this capacity is exploited for producing the fatty liver that is 5-to 10-fold larger than normal liver after 2-3 weeks of overfeeding [2,3]. Interestingly, its liver can develop serious steatosis without overt injury [4], showing minimal inflammation and other immune-related responses, which suggests that goose has different physiological and metabolic characteristics from mammals. In a previous study, the mRNA levels of tumor necrosis factor alpha (TNFα) was significantly downregulated by overfeeding in the goose liver [5]. Moreover, during the formation of goose fatty liver, expression of ER stress marker genes, 78 kDa glucose-regulated protein (Grp78) and X-box binding protein 1 (Xbp1), was also inhibited [6]. These results demonstrated that goose liver has a strong tolerance to fatty acids so as to deposit a large amount of fat without the occurrence of lesions.
Stearoyl-CoA desaturase (SCD) is recognized as a pivotal enzyme in the biosynthesis of monounsaturated fatty acids (MUFAs) by catalyzing the insertion of the cis double bond at the delta-9 position of stearoyl-CoA (C16:0) and palmitoyl-CoA (C18:0), which are subsequently converted into corresponding MUFAs, respectively [7]. In the past several decades, studies on SCD1 were mainly related to fat synthesis, as SCD1-knocked mice showed reduced TG content, inhibited de novo synthesis of fatty acids, and less hepatic steatosis [8]. Several recent reports supported the notion that the SCD1 is critical for regulating the ratio of SFAs to MUFAs. Janikewicz et al. [9] reported that SCD1 had an effect on ER stress response of pancreatic β-cells by altering the SFAs to MUFAs ratio. In another study, Iwai et al. [10] found that SCD1 affected apoptosis in mouse proximal tubular cells by regulating the MUFAs/SFAs ratio. Moreover, it was found that in overfeeding-induced goose fatty liver, the MUFAs/SFAs ratio significantly increased while the content of both palmitic acid and stearic acids decreased [4,11]. Thus, it can be generalized that SCD1 exerts an important role in regulating 4 MUFAs/SFAs ratio.
In goose, SCD1 is ubiquitously expressed in metabolic tissues and shows higher expression levels in liver [1,12]. In addition, goose SCD1 has higher genomic copy number than that of humans and chickens [13]. Therefore, we hypothesized that SCD1 could be involved in the regulation of goose hepatic steatosis. To test our hypothesis, this study aims to 1) compare the effects of palmitic acids at different concentrations on cell viability using MTT as well as expression of genes related to ER stress, inflammation, and apoptosis between goose primary hepatocytes and LO2 cells, 2) examine the effects of downregulating SCD1 expression via RNA interference on cell viability as well as expression of ER stress, inflammation, and apoptosis-related genes in goose hepatocytes under the treatment palmitic acid, and 3) investigate the effects of downregulating SCD1 expression via RNA interference on expression of key genes involvement of PI3K/AKT, FoxO1, mTOR and AMPK pathways in goose hepatocytes under the treatment palmitic acid. These data may provide a better understanding of the molecular mechanisms underlying SCD1 regulating the tolerance of SFAs in goose primary hepatocytes.

Isolation and Culture of Goose Primary Hepatocytes
Hepatocytes were isolated from ten 20-day-old Tianfu meet geese (Anser anser) that were hatched at the same time and raised under the condition of natural light and temperature at the Experimental Farm for Waterfowl Breeding of Sichuan Agricultural University (Sichuan, China) according to the methods introduced by Seglen [14], and were then cultured with Dulbecco's modified Eagle's medium (DMEM; Hyclone, Utah, USA) containing 10% fetal bovine serum (Gibco, USA). The cells were incubated at 40°C in a humidified atmosphere containing 5% CO 2 , and the medium was renewed after 3 hours of culture. 24 hours later, the medium was replaced with serum-free DMEM medium.

MTT Assay
The assay for cell viability was performed to Natali et al. [15]. Goose primary hepatocytes and LO2 cells were plated at a density of 1×10 4 cells/well in a 96-well culture dish. After 24 hours, cells were then incubated for 4 hours with 1 mg/mL MTT in a 37℃ incubator, which is converted from the yellow tetrazolium compound to the purple formazan derivative by mitochondria of living cells. After removal of the unconverted MTT, the formazan product was dissolved in DMSO and the formazan dye absorbance was measured at 490 nm.

Oil Red O staining
Cells were stained with Oil-Red-O to examine the amount of fat accumulation in the cells. Goose primary hepatocytes and LO2 cells were cultured on 6-well culture slides, fixed in formalin and stained using a method described by Ramirez-zacarias et al. [16]. Briefly, the wells were fixed

Cytochrome c Immunofluorescence and DAPI Assay
The cyt-c protein expression in goose primary hepatocytes was detected by using Cytochrome c (Cytochrome C antibody) Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instruction. DAPI staining was used with DAPI Staining Solution (Beyotime Biotechnology, Shanghai, China), and cells were incubated for 10 min with 0.5 ug/mL DAPI Solution in a 37℃ incubator.

RNA Extraction and cDNA Synthesis
Total RNA was extracted from cultures cells using TRIzol Reagent (Invitrogen) according to the manufacturer's instruction. The quality and quantity of total RNA were checked by electrophoresis on a 1.5% agarose gel. The cDNA was obtained by a cDNA Synthesis Kit (Takara, Japan) under the manufacturer's protocol with 1 ug of total RNA as a template.

Quantitative Real-time PCR (qRT-PCR)
The primers (Table 1)  were used as the internal controls. The mRNA expression levels of target genes were measured by qRT-PCR. The qRT-PCR was performed in a 96-well Bio-Rad iQ5 (Bio-Rad Laboratories, USA) using a Takara ExTaq RT-PCR Kit and SYBR Green as the detection dye (Takara, Japan). Real-time PCR was carried out under the following condition: 1 cycle of pre-denaturation at 95°C for 10 seconds; 40 cycles of 95°C for 5 seconds, and 60°C for 40 seconds, and starting at a temperature of 55°C and increasing by 0.5°C every 10 seconds to determine primer specificity. All cDNA samples were tested three times, and the results were normalized to the levels of goose GADPH and β-actin expression.

Data Analysis
The relative mRNA expression of target gene was calculated by the comparative Ct method (2 −ΔΔCt 7 methods) [17]. All data were subjected to analysis of variance (ANOVA), and the means were compared for significance using Tukey's test. The ANOVA and t-tests were performed using the SPSS 23.0 software (IBM, USA). Differences were considered statistically significant at P<0.05.

Comparative analysis of the palmitic acid tolerance of goose and human hepatocytes
The results showed that goose primary hepatocytes had a higher tolerant capacity to palmitic acid than LO2 cells (Fig. 1). When treated with 0.2-0.6 mM palmitic acid, the cell viability of goose primary hepatocytes was not change, but under 0.7-0.9 mM treatment, the cell viability significantly decreased (P<0.05). For LO2 cells, when treated with 0.2-0.9 mM palmitic acid, the cell viability significantly decreased (P<0.05), and with the increase of palmitic acid concentration, the cell viability decreased continuously.

Effects of palmitic acid on the mRNA levels of ER stress-, inflammation-and apoptosisrelated genes in goose primary hepatocytes and LO2 cells
To further verify palmitic acid tolerance of goose primary hepatocytes, we then compared the mRNA levels of ER stress-, inflammation-, and apoptosis-related genes in both cells. As shown in Fig.2, when treated with 0.6 mM palmitic acid, the mRNA levels of ER stress-related genes (XBP, BIP and ATF6), inflammatory response-related genes (IL-6, IL-1β and IFN-γ) and apoptosis-related genes (Bax, Bcl-2, Caspase-3 and Caspase-9) were not significantly different from the control group in goose primary hepatocytes, but in the LO2 cells their expression levels significantly increased compared to the control group (P<0.05).

Effects of palmitic acid on lipid accumulation and involved gene expression in goose primary hepatocytes and LO2 cells
As shown in Fig.3, under the treatment of 0.6 mM palmitic acid, the lipid accumulation of goose primary hepatocytes was significant higher than LO2 cells (P<0.05). As shown in Fig. 4, the mRNA levels of fatty acid desaturation-related genes (SCD1and FADS2) was significantly promoted (P<0.05) in goose primary hepatocytes, while no significant differences were seen in LO2 cells. In addition, the mRNA expression of the fatty acid elongate enzyme-related gene (ELOVL6) in both cells significantly 8 increased (P<0.05), whereas that of triglyceride synthesis-related gene (DGAT2) remained statically unchanged in these two cells.

Effects of SCD1 downregulation on cell viability and the mRNA levels of ER stress-, inflammation-and apoptosis-related genes in goose primary hepatocytes
The expression profile of SCD1 promoted us to investigate its function in palmitic acid tolerance of goose primary hepatocytes. Then, we interfered SCD1 in goose primary hepatocytes by using siRNA. and SIRT1), and the expression of PI3K was decreased (P<0.05), while that of mTOR and AMPK were not no significantly altered (Fig.7).

Discussion
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 [18]; in contrast, our laboratory demonstrated that the cell viability of goose primary hepatocytes was not changed by treatment with 0.6 mM palmitic acid [19], 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. [19]; 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 [18]. 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 [20]. Previous studies indicated that addition of palmitic acid increased the expression of ER stressrelated 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 [20], 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 [25]. 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 apoptosisrelated 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 [26]. 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 [28]. And the above five genes mRNA levels significantly increased in the goose liver after overfeeding [13]. 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 11 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][30][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.       Effects of SCD1 downregulation on the mRNA levels of PI3K/AKT, FoxO1, mTOR and AMPK pathway-related genes in goose primary hepatocytes under palmitic acid treatment. * indicates a significant difference at P<0.05.