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 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 200 mmol/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.

No obvious pathological symptoms such as inflammation were observed in fatty goose liver, suggesting that there is a unique mechanism to inhibit the development of inflammation during the goose fatty liver formation. Previous studies have shown that high glucose activated the ubiquitin­proteasome. Protein kinase A (PKA) can interact with a key protein in the nuclear factor-kappaB pathway to activate the pathway and trigger inflammatory response. To further understand how inflammation is suppressed during goose fatty liver formation. The present study showed that inflammation and PKA protein level were reduced in goose fatty liver. Meanwhile, PKA can be modified by ubiquitination in goose liver and hepatocytes. The result of the study implied that glucose deposited during goose fatty liver formation may reduce the PKA protein content by increasing the PKA ubiquitination level, thereby inhibiting the inflammatory response. Our study not only contributes to elucidate the new mechanism for suppressed inflammation in goose fatty liver but also provides a reference for the study of fatty liver in other animals.

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.

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.

No significant pathological symptoms were observed in the liver of goose after overfeeding, suggesting that a specific protection mechanism exists in goose liver. Previous studies have shown that mitochondria may participate in the formation of goose fatty liver by improving its energy metabolism and the production of precursor metabolites. To further understand the role of mitochondria in the formation of goose fatty liver, the present study investigated the changes of mitochondrial function, anti-oxidant capacity, and apoptosis in goose fatty liver. There were found that the level of mitochondrial membrane potential was increased, no apoptosis was observed and anti-oxidant capacity was improved in goose fatty liver, no apoptosis was observed and anti-oxidant genes expressions were increased in goose primary hepatocytes after 40 mM glucose treatment. Our findings imply that apoptosis is inhibited by glucose-induced enhanced anti-oxidant activity in goose fatty liver. Our study not only contributes to revealing the protective mechanism in goose fatty liver but also providing new references for the study of nonalcoholic fatty liver in mammals.