Goose Primary Hepatocytes Research Articles

Glucose inhibits the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA.

Protein kinase A (PKA) plays an important role in cellular life activities. Recently, PKA was found to bind to the inhibitor of nuclear factor-kappaB (IκB), a key protein in the nuclear factor-kappaB (NF-κB) pathway, to form a complex involved in the regulation of inflammatory response. However, the role of PKA in the anti-inflammatory of goose fatty liver is still unclear. A total of 14 healthy 70-d-old male Lander geese were randomly divided into a control group and an overfeeding group. Inflammation level was analyzed by histopathological method in the liver. The mRNA and protein abundance of PKA and tumor necrosis factor-alpha (TNFα), as well as the ubiquitination level of PKA, were detected. Moreover, goose primary hepatocytes were cotreated with glucose, harringtonine, and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132). Finally, the co-immunoprecipitated samples of PKA from the control and overfeeding group were used for protein mass spectrometry. The results showed that no difference in PKA mRNA expression was observed (P > 0.05), while the PKA protein level in the overfed group was significantly reduced (P < 0.05) when compared with the control group. The ubiquitination level of PKA was higher than that of the control group in fatty liver. The mRNA expression of PKA was elevated but protein abundance was reduced in goose primary hepatocytes with 200mmol/L glucose treatment (P < 0.05). The PKA protein abundance was dramatically reduced in hepatocytes treated with harringtonine (P < 0.01) when compared with the glucose-supplemented group. Nevertheless, MG132 tended to alleviate the inhibitory effect of harringtonine on PKA protein abundance (P = 0.081). There was no significant difference in TNFα protein level among glucose-treated groups and control (P > 0.05). Protein mass spectrometry analysis showed that 29 and 76 interacting proteins of PKA were screened in goose normal and fatty liver, respectively. Validation showed that PKA interacted with the E3 ubiquitination ligases ring finger protein 135 (RNF135) and potassium channel modulatory factor 1 (KCMF1). In summary, glucose may inhibit the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA. Additionally, RNF135 and KCMF1 may be involved in the regulation of PKA ubiquitination level as E3 ubiquitination ligases.

Goose Hepatic IGFBP2 Is Regulated by Nutritional Status and Participates in Energy Metabolism Mainly through the Cytokine-Cytokine Receptor Pathway.

Changes in the nutritional status of animals significantly affect their health and production performance. However, it is unclear whether insulin-like growth factor-binding protein 2 (IGFBP2) mediates these effects. This study aimed to investigate the impact of changes in nutritional and energy statuses on hepatic IGFBP2 expression and the mechanism through which IGFBP2 plays a mediating role. Therefore, the expression of IGFBP2 was first determined in the livers of fasting/refeeding and overfeeding geese. The data showed that overfeeding inhibited IGFBP2 expression in the liver compared with the control (normal feeding) group, whereas the expression of IGFBP2 in the liver was induced by fasting. Interestingly, the data indicated that insulin inhibited the expression of IGFBP2 in goose primary hepatocytes, suggesting that the changes in IGFBP2 expression in the liver in the abovementioned models may be partially attributed to the blood insulin levels. Furthermore, transcriptome sequencing analysis showed that the overexpression of IGFBP2 in geese primary hepatocytes significantly altered the expression of 337 genes (including 111 up-regulated and 226 down-regulated genes), and these differentially expressed genes were mainly enriched in cytokine-cytokine receptor, immune, and lipid metabolism-related pathways. We selected the most significant pathway, the cytokine-cytokine receptor pathway, and found that the relationship between the expression of these genes and IGFBP2 in goose liver was in line with the findings from the IGFBP2 overexpression assay, i.e., the decreased expression of IGFBP2 was accompanied by the increased expression of LOC106041919, CCL20, LOC106042256, LOC106041041, and IL22RA1 in the overfed versus normally fed geese, and the increased expression of IGFBP2 was accompanied by the decreased expression of these genes in fasting versus normally fed geese, and refeeding prevented or attenuated the effects of fasting. The association between the expression of these genes and IGFBP2 was verified by IGFBP2-siRNA treatment of goose primary hepatocytes, in which IGFBP2 expression was induced by low serum concentrations. In conclusion, this study suggests that IGFBP2 mediates the biological effects induced by changes in nutritional or energy levels, mainly through the cytokine-cytokine receptor pathway.

Study on the Mechanism of MC5R Participating in Energy Metabolism of Goose Liver.

Nutrition and energy levels have an important impact on animal growth, production performance, disease occurrence and health recovery. Previous studies indicate that melanocortin 5 receptor (MC5R) is mainly involved in the regulations of exocrine gland function, lipid metabolism and immune response in animals. However, it is not clear how MC5R participates in the nutrition and energy metabolism of animals. To address this, the widely used animal models, including the overfeeding model and the fasting/refeeding model, could provide an effective tool. In this study, the expression of MC5R in goose liver was first determined in these models. Goose primary hepatocytes were then treated with nutrition/energy metabolism-related factors (glucose, oleic acid and thyroxine), which is followed by determination of MC5R gene expression. Moreover, MC5R was overexpressed in goose primary hepatocytes, followed by identification of differentially expressed genes (DEGs) and pathways subjected to MC5R regulation by transcriptome analysis. At last, some of the genes potentially regulated by MC5R were also identified in the in vivo and in vitro models, and were used to predict possible regulatory networks with PPI (protein-protein interaction networks) program. The data showed that both overfeeding and refeeding inhibited the expression of MC5R in goose liver, while fasting induced the expression of MC5R. Glucose and oleic acid could induce the expression of MC5R in goose primary hepatocytes, whereas thyroxine could inhibit it. The overexpression of MC5R significantly affected the expression of 1381 genes, and the pathways enriched with the DEGs mainly include oxidative phosphorylation, focal adhesion, ECM-receptor interaction, glutathione metabolism and MAPK signaling pathway. Interestingly, some pathways are related to glycolipid metabolism, including oxidative phosphorylation, pyruvate metabolism, citrate cycle, etc. Using the in vivo and in vitro models, it was demonstrated that the expression of some DEGs, including ACSL1, PSPH, HMGCS1, CPT1A, PACSIN2, IGFBP3, NMRK1, GYS2, ECI2, NDRG1, CDK9, FBXO25, SLC25A25, USP25 and AHCY, was associated with the expression of MC5R, suggesting these genes may mediate the biological role of MC5R in these models. In addition, PPI analysis suggests that the selected downstream genes, including GYS2, ECI2, PSPH, CPT1A, ACSL1, HMGCS1, USP25 and NDRG1, participate in the protein-protein interaction network regulated by MC5R. In conclusion, MC5R may mediate the biological effects caused by changes in nutrition and energy levels in goose hepatocytes through multiple pathways, including glycolipid-metabolism-related pathways.

Glucose-induced enhanced anti-oxidant activity inhibits apoptosis in goose fatty liver.

The development of mammalian nonalcoholic fatty liver disease is associated with oxidative stress, reduced mitochondrial function, and increased apoptosis in hepatocytes; however, the expressions of mitochondria-related genes are elevated in goose fatty liver, suggesting that there may be a unique protective mechanism in goose fatty liver. The aim of the study was to investigate this protective mechanism in terms of anti-oxidant capacity. Our data showed no substantial differences in the mRNA expression levels of the apoptosis-related genes including B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax), cysteinyl aspartate-specific proteinase-3 (Caspase-3), and cysteinyl aspartate-specific proteinase-9 (Caspase-9) in the livers of the control and overfeeding Lander geese groups. The protein expression levels of Caspase-3 and cleaved Caspase-9 were not markedly different between the groups. Compared with the control group, malondialdehyde content was significantly lower (P < 0.01), glutathione peroxidase (GSH-Px) activity, glutathione (GSH) content, and mitochondrial membrane potential levels were higher (P < 0.01) in the overfeeding group. The mRNA expression levels of the anti-oxidant genes superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), and glutathione peroxidase 2 (GPX2) were increased in goose primary hepatocytes after 40 mM and 60 mM glucose treatment. Reactive oxygen species (ROS) levels were significantly reduced (P < 0.01), whereas the mitochondrial membrane potential was maintained at normal levels. The mRNA expression levels of the apoptosis-related genes Bcl-2, Bax, and Caspase-3 were not substantial. There were no significant differences in the expression levels of Caspase-3 and cleaved Caspase-9 proteins. In conclusion, glucose-induced enhanced anti-oxidant capacity may help protect the function of mitochondria and inhibit the occurrence of apoptosis in goose fatty liver.

Involvement of IGFBP5 in the Development of Goose Fatty Liver via p38 Mitogen-Activated Protein Kinase (MAPK)

Previous studies showed that insulin-like growth factor-binding protein 5 (IGFBP5) plays a role in non-alcoholic fatty liver disease; however, its expression and function in goose fatty liver remain unknown. To address this, we obtained a full-length mRNA sequence of the goose IGFBP5 gene using a 5′-rapid amplification of cDNA ends assay and nested polymerase chain reaction (PCR). Additionally, using the newly acquired sequence of 5’-untraslated region, we determined the missing sequence of the first intron. Bioinformatics analysis revealed three exons and three introns in the goose IGFBP5 gene. Quantitative PCR analysis indicated that the mRNA abundance of IGFBP5 was significantly lower in goose fatty liver than in the normal liver. Comparison of transcriptomes of goose primary hepatocytes transfected with IGFBP5 overexpression vector versus those transfected with empty vector identified 777 differentially expressed genes (DEGs). The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated the focal adhesion, ECM-receptor interaction, regulation of the actin cytoskeleton, mitogen-activated protein kinase (MAPK) signaling, and GnRH signaling pathways. Immunoblotting revealed the induction of the p38 MAPK pathway by IGFBP5 overexpression, which is in line with the suppressed expression of IGFBP5 and p38 MAPK in goose fatty liver than in normal liver. These findings suggest that IGFBP5 is involved in the development of goose fatty liver via the p38 MAPK pathway.

Mitochondrial-bound hexokinase 1 can affect the glucolipid metabolism and reactive oxygen species production in goose fatty liver

To investigate the functions of hexokinase 1 (HK1) in the formation of goose fatty liver, a total of 40 healthy 63-day-old male Landes geese were selected and randomly assigned to a control group and an overfeeding treatment. In addition, the overexpression or RNA interference assay of HK1, and transcriptome analysis after HK1 overexpression were performed in the goose primary hepatocytes. Data showed that the mRNA expression of hepatic HK1 was upregulated in overfed treatment compared to control on the 19 days of overfeeding. The expression of HK1 was increased in 50 mM glucose treatment in hepatocytes. Moreover, overexpression of HK1 tended to increase the relative lipid accumulation level and had weakened fluorescence intensity of reactive oxygen species (ROS), while knockdown of HK1 resulted in a tendency of relative lipid content decrease and had enhanced fluorescence intensity of ROS in cells in comparison to the control. The verification of transcriptome analysis indicated that the expression of ceruloplasmin (CP), acyl-CoA dehydrogenase medium-chain (ACADM), phosphoglucomutase 2 (PGM2), and phospholipase A2 group IVA (PLA2G4A) was significantly induced by HK1 overexpression, while that of enoyl-CoA hydratase, short chain 1 (ECHS1), cytochrome P450 family 2 subfamily C member 19 (CYP2C19), carnitine palmitoyltransferase 1 A (CPT1A), and oxidative stress-induced growth inhibitor 1 (OSGIN1) was inhibited. In summary, HK1 could promote fat deposition by affecting the process of glucolipid metabolism in the formation of goose fatty liver. Additionally, HK1 might decrease the ROS level in hepatocytes by regulating the expression of redox-related genes. HIGHLIGHTS The HK1 gene may promote the goose fatty liver production. The HK1 gene may be used as a research target to decrease the reactive oxygen species level in fatty liver.

Forkhead box protein O1 (FoxO1) regulates lipids metabolism and cell proliferation mediated by insulin and PI3K-Akt-mTOR pathway in goose primary hepatocytes

In order to explore the role of forkhead box protein O1 (FoxO1) in the lipid metabolism and cell proliferation, goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVP-BEZ235, and then transfected with FoxO1 interference plasmid. The related parameters of lipid metabolism and cell proliferation were measured. The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration (P < 0.05); and increased the proliferative index (PI), cell DNA synthesis, protein expression of Cyclin D1 in goose primary hepatocytes (P < 0.05). Secondly, the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1 (P < 0.05); however, there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI (P > 0.05). Lastly, the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly (P < 0.05). In summary, FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes. Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.

The PI3K/Akt/mTOR signaling pathway regulates lipid metabolism mediated by endoplasmic reticulum stress in goose primary hepatocytes

Abstract We have previously demonstrated that the PI3K/Akt/mTOR signaling pathway was closely linked to the lipid metabolism homeostasis in goose primary hepatocytes. Recently, it was demonstrated that endoplasmic reticulum stress (ERS) can not only affect lipogenesis, but also is related to the PI3K/Akt/mTOR signaling pathway, so it was hypothesised that ERS can affect the lipid metabolism through the PI3K/Akt/mTOR signaling pathway. The ERS model was established by treating goose primary hepatocytes with tunicamycin (TM), and then incubated with PI3K/Akt/mTOR signaling pathway inhibitors LY294002 (LY), Rapamycin (Ra) or NVP-BEZ235 (NVP). Hepatocytes were assigned into 5 groups, including control group, TM group, TM+LY group, TM+Ra group, and TM+NVP group. The results showed that compared with the control group, the mRNA expression and the protein content of BIP/GRP78 significantly increased ( P<Â 0.05) in the TM group, indicating that the ERS model was established successfully. The result of Oil Red O staining revealed that TM caused an increase ( P<Â 0.05) in lipid accumulation and in the mRNA expression of PI3K and S6K, implying that ERS could activate the PI3K/Akt/mTOR signaling pathway. Compared with the TM group, the mRNA expression of genes involved in the PI3K/Akt/mTOR signaling pathway ( PI3K, Akt1, mTOR and S6K), lipogenesis ( ACCα, FAS and SREBP-1), VLDL-TG assembly and secretion ( MTTP, DGAT1 and DGAT2) significantly decreased ( P<Â 0.05) in the TM+LY group, the TM+Ra group, and the TM+NVP group, whereas the protein content of CPT1 and MTTP significantly increased ( P<Â 0.05) in the TM+LY group, the TM+Ra group, and the TM+NVP group. These findings suggest that inhibition of the PI3K/Akt/mTOR signaling pathway can effectively decrease the steatosis mediated by ERS in goose hepatocytes.

Role of forkhead box protein O1 and insulin on cell proliferation mediated by sirtuin 1 in goose primary hepatocytes

Summary To explore the role of insulin and forkhead box protein O1 (FoxO1) on cell proliferation regulated by sirtuin 1 (Sirt1) in goose primary hepatocyte, goose primary hepatocytes were isolated and incubated with Sirt1 inhibitor and nicotinamide, and then, some hepatocytes were transfected with FoxO1 interference plasmid or treated with insulin. The indexes related with cell proliferation were measured. The results first showed Sirt1 inhibition induced by nicotinamide increased cell proliferation in goose primary hepatocytes (P 0.05), protein content of p21 (P > 0.05), and cell proliferative index (PI) (67.40 vs. 69.00%). Finally, the PI of cotreatment with nicotinamide and insulin (56.65%) was lower than the single nicotinamide treatment (67.40%); the DNA synthesis rate in cotreatment of nicotinamide and insulin was lower than the single nicotinamide treatment (P

Complement C3 participates in the development of goose fatty liver potentially by regulating the expression of FASN and ETNK1.

Non-alcoholic fatty liver disease (NAFLD) occurs in humans, domestic animals and poultry. Different from upregulation of complement C3 in human NAFLD, C3 expression is inhibited in goose fatty liver (GFL), implying a specific role of C3 in GFL. This study was mainly focused on uncovering the uniqueness of goose liver cells in the regulation of C3 expression and identifying the downstream genes of C3 to improve understanding on the specific role of C3 in GFL. The results showed that C3 expression was inhibited in the liver, muscle and fat tissues of the overfed versus control (normally fed) geese. Oleate and insulin could inhibit C3 expression in goose primary hepatocytes but induce it in mouse primary hepatocytes. A total of 1,123 differentially expressed genes (DEGs) were affected by C3 overexpression and were mainly enriched in immune response/inflammation and catabolism-related KEGG pathways. Additionally, the representative downstream genes (FASN and ETNK1) of C3 could mediate the role of C3 in the development of GFL. In conclusion, the suppression of C3 in GFL is at least partially attributed to hyperinsulinemia, hyperlipidemia and uniqueness of goose liver cells. Complement C3 does not only affect hepatic steatosis but also affect inflammation/immune response in GFL.

Differential regulation on C5 expression in goose versus mammals by glucose/palmitate provides a potential protection for goose fatty liver.

Goose fatty liver is a specific type of nonalcoholic fatty liver that is protected from harmful effects associated with severe steatosis. Our previous findings suggest that suppression of the complement C5 may be relevant, but the mechanism is unclear. Therefore, in this study, we first verified the expression pattern of complement genes (including C5) during goose fatty liver formation and then determined the liver fat content and fatty acid composition by high-performance liquid chromatography (HPLC), followed by selecting the differential metabolites to treat HepG2, goose and mouse primary hepatocytes, aiming to explore the mechanism of C5 and inflammation suppression in goose fatty liver. The data confirmed the suppression of complement genes (including C5) in goose fatty livers. Moreover, fat content was significantly higher in fatty liver versus normal ones, with oleic acid and palmitic acid dominantly accounting for the difference. In line with this, high concentration of palmitate led to down regulation of C5 expression in goose primary hepatocytes whereas upregulation in mouse primary hepatocytes and HepG2 cells. In conclusion, regulation on C5 expression by fatty liver related factors including high level of palmitic acid may contribute to the protection of goose liver from severe hepatic steatosis.

GC-TOF-MS-Based Metabolomics Analyses of Liver and Intestinal Contents in the Overfed vs. Normally-Fed Geese.

Simple SummaryNon-alcoholic fatty liver disease has been considered as one of the most important causes of liver disease, and it is a threat to human and animal health worldwide. Interestingly, goose fatty liver can reach 8–10 times the weight of normal liver with no overt pathological symptoms, suggesting that there are some protective mechanisms. Scientists have indicated that gut microbiota participate in the formation of non-alcoholic fatty liver disease in human and mammalian animals. However, it is unclear whether gut microbiota and their metabolites contribute to goose fatty liver. The aim of the present study was to investigate the metabolomic analyses of liver and intestinal contents in overfed vs. normally fed geese. The results showed that the formation of goose fatty liver is accompanied by obvious changes in the metabolic profiles of liver and intestinal contents. The intestinal metabolites can affect the formation of goose fatty liver by affecting the metabolisms of glucose and fatty acid, oxidative stress, and inflammatory reactions. These findings provide a basis for future work addressing the relationship between intestinal metabolites and the development of non-alcoholic fatty liver disease.No overt pathological symptoms are observed in the goose liver with severe steatosis, suggesting that geese may host unique protective mechanisms. Gas chromatography time-of-flight mass spectrometry-based metabolomics analyses of liver and intestinal contents in overfed vs. normally fed geese (26 geese in each treatment) were investigated. We found that overfeeding significantly changed the metabolic profiles of liver and intestinal contents. The differential metabolites mainly belong to fatty acids, amino acids, organic acids, and amines. The differential metabolites were involved in glycolysis/gluconeogenesis, glycerolipid metabolism, the pentose phosphate pathway, fatty acid degradation, the sphingolipid signaling pathway, and the biosynthesis of unsaturated fatty acids. Moreover, we determined the biological effects of arachidonic acid (ARA) and tetrahydrocorticosterone (TD) in goose primary hepatocytes and intestinal cells. Data showed that the mRNA expression of arachidonate 5-lipoxygenase (ALOX5) in goose primary intestinal cells was significantly induced by 0.50 mM ARA treatment. Cytochrome P-450 27A1 (CYP27A1) mRNA expression was significantly inhibited in goose primary hepatocytes by 1 µM TD treatment. In conclusion, the formation of goose fatty liver is accompanied by significant changes in the metabolic profiles of liver and intestinal contents, and the changes are closely related to the metabolisms of glucose and fatty acids, oxidative stress, and inflammatory reactions.

Fasting and overfeeding affect the expression of the immunity- or inflammation-related genes in the liver of poultry via endogenous retrovirus

It is known that nutrition and immunity are connected, but the mechanism is not very clear. Endogenous retroviruses (ERV) account for 8 to 10% of the human and mouse genomes and play an important role in some biological processes of animals. Recent studies indicate that the activation of ERV can affect the expression of the immunity- or inflammation-related genes, and the activities of ERV are subjected to regulation of many factors including nutritional factors. Therefore, we hypothesize that nutritional status can affect the expression of the immunity- or inflammation-related genes via ERV. To verify this hypothesis, the nutritional status of animals was altered by fasting or overfeeding, and the expression of intact ERV (ERVK18P, ERVK25P) and immunity- or inflammation-related genes (DDX41, IFIH1, IFNG, IRF7, STAT3) in the liver was determined by quantitative PCR, followed by overexpressing ERVK25P in goose primary hepatocytes and determining the expression of the immunity- or inflammation-related genes. The data showed that compared with the control group (no fasting), the expression of ERV and the immunity- or inflammation-related genes was increased in the liver of the fasted chickens but decreased in the liver of the fasted geese. Moreover, compared with the control group (routinely fed), the expression of ERV and the immunity- or inflammation-related genes was increased in the liver of the overfed geese. In addition, overexpression of ERVK25P in goose primary hepatocytes can induce the expression of the immunity- or inflammation-related genes. In conclusion, these findings suggest that ERV mediate the effects of fasting and overfeeding on the expression of the immunity- or inflammation-related genes, the mediation varied with poultry species, and ERV and the immunity- or inflammation-related genes may be involved in the development of goose fatty liver. This study provides a potential mechanism for the connection between nutrition and immunity.

Role of stearyl-coenzyme A desaturase 1 in mediating the effects of palmitic acid on endoplasmic reticulum stress, inflammation, and apoptosis in goose primary hepatocytes.

ObjectiveUnlike 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 (PA) tolerance of goose primary hepatocytes.Methods3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide was examined to reflect the effect of PA on hepatocytes viability, and quantitative polymerase chain reaction was used to detect the mRNA levels of several genes related to endoplasmic reticulum (ER) stress, inflammation, and apoptosis, and the role of SCD1 in PA tolerance of goose hepatocytes was explored using RNA interfere.ResultsOur results indicated that goose hepatocytes exhibited a higher tolerant capacity to PA than human hepatic cell line (LO2 cells). In goose primary hepatocytes, the mRNA levels of fatty acid desaturation-related genes (SCD1 and fatty acid desaturase 2) and fatty acid elongate enzyme-related gene (elongase of very long chain fatty acids 6) were significantly upregulated with 0.6 mM PA treatment. However, in LO2 cells, expression of ER stress-related genes (x box-binding protein, binding immunoglobulin protein, and activating transcription factor 6), inflammatory response-related genes (interleukin-6 [IL-6], interleukin-1β [IL-1β], and interferon-γ) and apoptosis-related genes (bcl-2-associated X protein, b-cell lymphoma 2, Caspase-3, and Caspase-9) was significantly enhanced with 0.6 mM PA treatment. Additionally, small interfering RNA (siRNA) mediated downregulation of SCD1 significantly reduced the PA tolerance of goose primary hepatocytes under the treatment of 0.6 mM PA; meanwhile, the mRNA levels of inflammatory-related genes (IL-6 and IL-1β) and several key genes involved in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), forkhead box O1 (FoxO1), mammalian target of rapamycin and AMPK pathways (AKT1, AKT2, FoxO1, and sirtuin 1), as well as the protein expression of cytochrome C and the apoptosis rate were upregulated.ConclusionIn conclusion, our data suggested that SCD1 was involved in enhancing the PA tolerance of goose primary hepatocytes by regulating inflammation- and apoptosis-related genes expression.

Increase of E3 ubiquitin ligase NEDD4 expression leads to degradation of its target proteins PTEN/IGF1R during the formation of goose fatty liver.

Goose fatty liver may have a unique protective mechanism as it does not show a pathological injury even in the case of severe steatosis. Although neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) participates in repair and regeneration of injured liver through its target proteins, its role in nonalcoholic fatty liver disease remains unknown. Using quantitative polymerase chain reaction (PCR) and immunoblot analyses, here, we found that the messenger RNA (mRNA) and protein expressions of NEDD4 were induced in goose fatty liver compared with normal liver. The mRNA expression of the gene of phosphate and tension homology deleted on chromosome ten (PTEN) and insulin-like growth factor 1 receptor (IGF1R) was also induced in goose fatty liver; however, their protein expression was or tended to be suppressed. Moreover, the co-immunoprecipitation analysis indicated that there was a physical association between NEDD4 and PTEN in goose liver, which was consistent with the ubiquitination of PTEN in goose fatty liver. Furthermore, NEDD4 overexpression in goose primary hepatocytes suppressed the PTEN and IGF1R protein levels without a significant effect on their mRNA expression. In conclusion, the increased expression of NEDD4 leads to the degradation of PTEN and IGF1R proteins through ubiquitination in goose fatty liver, suggesting that NEDD4 may protect goose fatty liver from severe steatosis-associated injury via its target proteins during the development of goose fatty liver.

Fatty acids modulate the expression of pyruvate kinase and arachidonate-lipoxygenase through PPARγ/CYP2C45 pathway: a link to goose fatty liver

Cytochrome P-450 2C45 (CYP2C45) is the most highly expressed cytochrome P-450 isoform in chicken liver, and may play an important role in avian liver biology. However, information regarding the function of CYP2C45 in fatty liver is generally limited. The aim of this study was to investigate the role of CYP2C45 during the development of goose fatty liver. Our result indicated that the transcription of CYP2C45, together with PK and ALOX5, was increased in goose liver upon overfeeding for 19 D (P < 0.05). In goose primary hepatocytes, CYP2C45 RNA expression was also upgraded by the treatment with various chemicals like insulin, the fatty acids, and PPAR agonists (P < 0.05). We also found that both CYP2C45 overexpression and troglitazone treatment could increase the expression of pyruvate kinase (PK) and arachidonate 5-lipoxygenase (ALOX5), and furthermore, showed that the up-regulation of PK and ALOX5 induced by troglitazone could be suppressed by small interfering RNAs targeting CYP2C45 (P < 0.05). These findings suggest that fatty acids treatment and the overfeeding can induce the up-regulation of CYP2C45 expression possibly via PPARγ and that the induction of PK and ALOX5 in goose fatty liver is at least partially attributed to fatty acid-induced expression of CYP2C45. Thus, our data provides an insight into the mechanism by which glycolysis and arachidonic acid metabolism are modulated in goose fatty liver.

Retraction notice to: "Role of Mammalian sirtuin 1 (SIRT1) in lipids metabolism and cell proliferation of goose primary hepatocytes" MCE Volume 382, Issue 1, 25 January 2014, Pages 282-291.

Retraction notice to: "Role of Mammalian sirtuin 1 (SIRT1) in lipids metabolism and cell proliferation of goose primary hepatocytes" MCE Volume 382, Issue 1, 25 January 2014, Pages 282-291.

Inhibition of PI3K-Akt-mTOR signal pathway dismissed the stimulation of glucose on goose liver cell growth.

In the formation of goose fatty liver induced by a high-carbohydrate diet, it is characterized by the quick cell growth of liver. The carbohydrate is mostly digested and absorbed in the small intestine by the form of glucose. Recent studies have suggested a crucial role for PI3K-Akt-mTOR pathway in regulating cell proliferation, and then we speculate that PI3K-Akt-mTOR pathway may mediate glucose-induced liver cell proliferation. Goose primary hepatocytes were isolated and incubated in either no addition as a control or glucose or PI3K-Akt-mTOR pathway inhibitors or cotreatment with glucose and PI3K-Akt-mTOR pathway inhibitors. The results firstly showed that 35mmol/l glucose stimulated the mRNA level and protein content of factors involved in PI3K-Akt-mTOR signal pathway in goose primary hepatocytes. Secondly, 35mmol/l glucose evidently changed the cell cycle PI index and protein expression of cyclin D1. Meanwhile, the upregulation of 35mmol/l glucose on the DNA synthesis rate, cell cycle PI index, the mRNA expression, protein content and protein expression of factors involved in the cell proliferation was decreased significantly by the inhibitors of PI3K-Akt-mTOR pathway, LY294002, rapamycin or NVP-BEZ235. In summary, glucose could stimulate the cell proliferation, and the PI3K-Akt-mTOR pathway inhibitors could dismiss glucose-induced the upregulation of cell proliferation in goose primary hepatocyte.

Insulin Promotes the Expression of the Gluconeogenic Rate-Limiting Enzymes Phosphoenolpyruvate Carboxykinase (Pepck) and Glucose 6-Phosphatase (G6pase) through PI3k/Akt/mTOR Signaling Pathway in Goose Hepatocytes

To identify what makes insulin have an activating or inhibiting role in gluconeogenesis in goose hepatocytes and whether insulin regulates PEPCK and G6Pase through the PI3k/Akt/mTOR pathway or not, goose primary hepatocytes were isolated and cultured in vitro. After 12h cultured in serum-free medium, hepatocytes were incubated for 24 h in the medium with no addition (control) or with the addition of50, 100, and 150 nM of insulin, 1000 nM NVP-BEZ235, or co-addition of 150nM insulin and 1000nM NVP-BEZ235. Glucose concentration and PEPCK and G6Pase expression were determined. The results showed that PEPCK and G6Pase mRNA levels and activities were up regulated in the 50, 100, and 150nM insulin treatments, while glucose concentration was not significantly altered (p> 0.05). Compared with the activation role of 150nM insulin alone, the co-treatment with1000nM NVP-BEZ235 and 150nM insulin significantly down regulated PEPCK mRNA level and G6Pase protein activity (p< 0.05). However, there is a different result on mRNA level of G6Pase. In conclusion, G6Pase and PEPCK are up regulated by insulin through PI3k/Akt/mTOR pathway in goose hepatocytes. However, G6Pase mRNA and protein levels may be regulated by insulin through different signaling pathways.

Supplementing dietary sugar promotes endoplasmic reticulum stress-independent insulin resistance and fatty liver in goose

It is known that endoplasmic reticulum stress (ERS) contributes to insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD) in mammals. However, we recently demonstrated that overfeeding with a traditional diet (mainly consisting of cooked maize) does not induce ERS in goose. As cellular studies show that high glucose and palmitate can trigger ERS in mammalian cells, we hypothesized that supplementing sugar to the traditional diet could induce ERS, thus promoting insulin resistance and fatty liver. To test the hypothesis, we first treated goose primary hepatocytes with high glucose (25 mM and 50 mM) and palmitate (0.5 mM) supplemented with or without 0.25 mM oleate. Data indicated that, as in mammalian cells, high glucose and palmitate indeed induced ERS in goose primary hepatocytes, and palmitate-induced ERS was suppressed by supplemental 0.25 mM oleate. We then tested the hypothesis with an in vivo study, in which Landes geese overfed with traditional or novel diets (i.e., the traditional diet supplemented with sugar) were compared with control geese (normally fed with cooked maize) for ERS, IR and fatty liver. The differences in glucose tolerance, insulin tolerance and postprandial blood glucose between the geese overfed with traditional and novel diets suggested that supplementing dietary sugar promoted IR. This promotion was accompanied with an increasing trend of liver weight and abdominal fat weight relative to body weight. Surprisingly, compared to overfeeding with the traditional diet, overfeeding with the novel diet did not induce ERS, even further suppressed ERS in goose fatty liver. Together, our findings suggest that supplementing dietary sugar promotes ERS-independent IR and fatty liver in goose. It is intriguing to discover the factor(s) protecting goose liver from ERS as well as the non-ERS mechanism underlying IR.