Characteristic Studies On The Degradation Of Hepatic Lipid Droplets In The Chinese Soft-shelled Turtle(Pelodiscus Sinensis)

Hibernation is a survival strategy that some animals take to change their energy requirements by adjusting their morphology,physiological functions and behaviors when they are facing cold weather and food shortage.Previous studies have shown that some mammalian hibernating animals(such as American black bears,bats and thirteen ground squirrels)can reduce heat loss by lowering metabolic rate.However,current studies are focused on homothermic mammalian hibernating animals.As for ectotherms whose body temperature varies with the ambient temperature,the molecular mechanism of how the animals go through the entire hibernation period has not yet been elucidated.The Chinese soft-shelled turtle(Pelodiscus sinensis)is a typical kind of ectotherm and its body temperature will vary with the ambient temperature.Its hibernation period generally starts from November of the previous year and goes on until March or April of the next year.However,the mortality of Pelodiscus sinensis is still high during hibernation in the process of ecological breeding.Therefore,elucidating the molecular mechanism underlying the changes in energy metabolism of the Chinese soft-shelled turtle during hibernation is of vital importance to improving the survival rate of Pelodiscus sinensis and the development of Pelodiscus sinensis breeding industry.In this study,RNA sequencing(RNA-Seq)and digital gene expression profiling(DGE)analysis were performed on the liver of Pelodiscus sinensis between hibernation and non-hibernation.Differentially expressed genes(DEG)were identified and found related with changes of energy metabolism in the liver.Moreover,the changes of subcellular structure in the liver of Pelodiscus sinensis between hibernation and non-hibernation were compared using transmission electron microscopy(TEM)and light microscopy techniques.These results demonstrated the role of lipid droplets in the liver of Pelodiscus sinensis at the cellular level.To further explore the molecular mechanism underlying lipid droplet degradation in the liver of Pelodiscus sinensis during hibernation,analysis of RNA-Seq results and detection of lipolysis-related indicators revealed that lipolysis could promote the degradation of lipid droplets in the liver of Pelodiscus sinensis during hibernation.Simultaneously,we also found that lipid droplets in the liver of Pelodiscus sinensis during hibernation could be degraded via the autophagy pathway.To clarify the relationship between lipolysis and lipophagy in the breakdown of lipid droplets in the liver of Pelodiscus sinensis,we performed the reverse verification experiment in primary hepatocyte from the liver of Pelodiscus sinensis.The results of this experiment clarified the foundation of Pelodiscus sinensis survival during hibernation at cellular and molecular level,and thus provided the theoretical basis for improving the survival rate of animals.The main findings are as follows:Experiment ? Identification and analysis of differentially expressed genes(DEG)in the liver of Pelodiscus sinensis between hibernation and non-hibernationRNA-Seq and digital gene expression profiling(DGE)analysis in the liver of Pelodiscus sinensis between hibernation and non-hibernation was performed using the Illumina Hiseq 2500 high-throughput sequencing platform.Totally 8,730 DEGs were successfully identified which include 3,991 remarkably up-regulated and 4,739 remarkably down-regulated genes in the two libraries.GO and KEGG analysis revealed that DEGs are mainly involved in carbohydrate metabolism,lipid and fatty acid metabolism,and immune function.To substantiate these identified DEGs from the two libraries,9 genes were randomly chosen for analysis.RT-qPCR and Western Blot results further verified the accuracy of RNA-Seq results at gene and protein levels.The above results indicated that energy metabolism in the liver of Pelodiscus sinensis during hibernation involved an important shift,that the energy generation was changed from carbohydrate oxidation(non-hibernation)to lipid catabolism(hibernation).Experiment ? Morphological changes of liver from the Chinese soft-shelled turtle(Pelodiscus sinensis)between hibernation and non-hibernationThe changes of hepatic subcellular structures in the Chinese soft-shelled turtle(Pelodiscus sinensis)between hibernation and non-hibernation were observed using transmission electron microscopy and light microscopy.Chinese soft-shelled turtle a typical kind of reptile hibernating animals,and its body temperature will vary with the ambient temperature.Based on annual temperature changes in Nanjing,we could know that its hibernation period generally starts from November of the previous year and goes on until March or April of the next year.Periodic Acid-Schiff staining(PAS)showed that hepatic glycogen content in the Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernation is significantly higher than that in non-hibernation.Simultaneously,Oil Red O and BODIP Y 493/503 staining results indicated that lipid droplet content in the liver of Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernation is significantly higher than that in non-hibernation.Moreover,the decrease of triglycerides and cholesterol esters(the main component of lipid droplets)content in the liver of Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernation further verified the changes in lipid droplet content.Transmission electron microscopy results indicated that the size or diameter of lipid droplets did not change significantly between the two periods,the number of lipid droplets in the liver or single hepatocyte during hibernation is significantly lower than that in non-hibernation.The above results manifested that lipid droplets in the liver are an important energy source for the survival of Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernation,and thus provide material guarantee for improving its survival rate during hibernation.Experiment ? Lipolysis promotes the degradation of lipid droplets in the liver from Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernationTo explore the molecular mechanism underlying lipid droplet degradation in the liver of Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernation,RNA-Seq analysis found that lipid droplets in the liver during hibernation could be degraded through lipolysis.RT-qPCR and "Western Blot results further verified the accuracy of RNA-Seq sequencing results at gene and protein levels.RT-qPCR results indicated that the mRNA expression levels of lipolysis-related genes were increased,whereas the mRNA expression levels of lipogenesis-related genes were decreased in the hibernation group compared with those in the non-hibernation group.Simultaneously,the contents of non-esterified free fatty acids(NEFAs)in the liver and the mRNA levels of regulatory enzymes for mitochondrial?-oxidation during hibernation is significantly higher than that in non-hibernation.Under TEM,we observed that most mitochondria were circular or oval and maintained a certain distance from LDs in the non-hibernation group.By contrast,in the hibernation group,the shapes of partial mitochondria were transformed from round or oval to a crescent shape and the mitochondria were tightly associated with the LDs.To further illustrate the regulation mechanism of lipid droplet decomposition,the sucrose density gradient centrifugation was applied in isolating lipid droplets from the liver.Western Blot results revealed that the accumulation of ATGL on the surface of lipid droplets promotes hepatic LD degradation during hibernation.The above results indicated that the accumulation of ATGL on the surface of LDs enhanced lipolysis in the liver of Pelodiscus sinensis during hibernation,and thus provided material guarantee for going through hibernation.Experiment ? Lipophagy promotes the degradation of lipid droplets in the liver from Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernationTo explore the molecular mechanism underlying lipid droplet degradation in the liver of Chinese soft-shelled turtle(Pelodiscus sinensis)during hibernation,RNA-Seq analysis found that lipid droplets in the liver during hibernation could be degraded through lipophagy.RT-qPCR and Western Blot results further verified the accuracy of RNA-Seq sequencing results at gene and protein levels.To explore the relationship between lipophagy and lipid droplet degradation,the sucrose density gradient centrifugation was applied in isolating lipid droplets from the liver.Western blot results showed that autophagy level(LC3,p62 and LAMP1)in the liver and isolated LDs of Pelodiscus sinensis was increased during hibernation.Furthermore,under TEM,we observed that LD in the liver of Pelodiscus sinensis during hibernation was enwrapped or entirely encased by isolation membranes or autophagosomes.Transcriptome sequencing in the present study manifested that PI3K-AKT signaling pathway was significantly enriched.RT-qPCR and western blot analysis indicated that the mRNA and protein expression in PI3K-AKT signaling pathway was consistent with RNA-Seq results.The above results reveal that the autophagy pathway activated by PI3K/AKT/mTOR signaling pathway could promote the degradation of LD in the liver of Pelodiscus sinensis during hibernation.Experiment ? As the main pathway,lipolysis promote the degradation of lipid droplets in hepatocyte from Chinese soft-shelled turtle(Pelodiscus sinensis)During hibernation,lipid droplets stored in the liver of Pelodiscus sinensis are gradually consumed.Previous RNA-Seq and subsequent confirmatory experiment have demonstrated that both lipolysis and autophagy could promote the degradation of lipid droplets in the liver of Pelodiscus sinensis during hibernation.To further determine the relationship between lipolysis and autophagy in the process of LD breakdown,we applied the inhibitors of lipolysis and autophagy(DEUP and 3MA)in cultured primary hepatocytes from the liver of Chinese Soft-shelled turtle.Confirmatory experiments in vitro were performed in cultured primary hepatocytes from the liver of Chinese Soft-shelled turtle.DEUP and 3MA were applied in inhibiting lipolysis and autophagy.After suppressing autophagy with 3MA,the ratio of LC3II/LC3I was significantly decreased,whereas the expression of p62 was increased.Compared with the control group,the content of triglyceride(TG)in hepatocytes was significantly increased after 3MA treatment,while the content of free fatty acids(FFA)was significantly decreased,indicating that inhibition of autophagy would affect lipid droplet degradation.After DEUP treatment,the TG content in cultured cells increased significantly,while the FFA content decreased.At this point,the autophagy level in cultured hepatocytes was slightly increased(the ratio of LC3?/LC3? did not change,but the expression of p62 decreased),indicating that inhibition of lipolysis could also affect the degradation of lipid droplets.After treating with 3MA and DEUP simultaneously,TG content in hepatocytes increased significantly,while the FFA content decreased.However,the changes of TG and FFA content in co-treatment group has little difference with DEUP treatment,but has a significant difference with 3MA treatment.Similar changes in the number of lipid droplets in cultured hepatocytes were observed under TEM.The above results indicated that lipolysis and lipophagy could both promote the degradation of lipid droplets in the hepatocyte of Chinese soft-shelled turtle(Pelodiscus sinensis),and lipolysis is the main way for lipid droplet degradation.