| Fasting is the most commonly used method in clinical treatment of obesity.However,fat mobilization induced by fasting affects the changes of lipid metabolism in the liver,easily leading to the occurrence of fatty liver.Negative energy balance induced fatty liver has also been reported in livestock production.For example,high-yielding cows are prone to develop fatty liver during perinatal period.It is estimated that 40% to 60% of high-yielding cows(with a daily milk yield of more than 35kg)will develop moderate or high fatty liver during perinatal period,and the occurrence of fatty liver will reduce the production performance and service life of cows.The study of the molecular mechanism of fasting affecting lipid metabolism in liver has guiding significance for the treatment of obesity in humans and the reduction of the incidence of negative energy balance induced fatty liver in livestock and poultry.The effects of fasting on lipid and energy metabolism have been reported in some studies,but most of the studies focus on the metabolic changes of energy restriction at a certain point,and there is a lack of studies on continuous energy restriction.On the other hand,there is a lack of comparative studies on the influence of body condition(obesity degree)of animals before fasting on lipid metabolism after fasting.In this study,obese mice and normal mice were selected as research objects and treated with different fasting times.Changes in liver tissue anatomy,physiological and biochemical indexes were compared,and differences in liver lipid droplet morphology and lipid metabolism-related gene expression activity were analyzed.On this basis,r NA-SEQ technology was used to study the effect of fasting on liver transcription level in normal and obese mice,and Cidec,which plays a key role in lipid drop fusion,was further selected as the research object.The molecular mechanism of fasting’s regulation of liver Cidec expression was analyzed from the perspective of upstream transcription factors,signaling pathways and mirnas of Cidec.The main experimental results are as follows:1.C57BL/6J mice were fed with high-fat diet for 8 weeks,and an obese mouse model was constructed.The obese and normal mice were fasted for 0 h,8 h,16 h,24 h and 32 h.The anatomical changes of body weight,adipose tissue and liver were observed,and the results showed that the body weight and liver index decreased with the prolongation of fasting time,and the phenomenon was more obvious in the obese mice.The adipose tissue decreased significantly,and the color of liver became lighter gradually.The changes of blood physiological indexes in obese mice and ordinary mice after fasting were further measured,which showed that the blood indexes of obese mice fluctuated greatly after fasting compared with ordinary mice,with glucose content decreasing,LDL and cholesterol content showing a downward trend,and free fatty acid content and triglyceride content showing an overall upward trend.Through the detection of lipid transport,lipid synthesis and lipolysis in the liver of mice with different fasting times,it was found that the genes responsible for fatty acid transport,lipolysis and lipid synthesis were up-regulated and down-regulated in the liver.The results suggest that the energy source of the body undergoes a shift after fasting and lipid mobilization occurs,in which the liver plays a key role in the shift of energy source from glucose to fat.2.The liver tissues of mice with different fasting times were collected,and the morphological changes of the lipid droplets in the liver tissues were observed by the tissue sectioning technique and the tissue oil red O staining method.The results showed that lipid droplet volume increased in the liver sections of mice after fasting,and the increase in liver lipid droplet volume occurred at 8 h after fasting in ordinary mice.In addition,the color of liver lipid droplets was inconsistent after fasting,and the color of the droplets with increased volume was lighter.The above results were most obvious in obese mice fasting for 16 h and 24 h.In order to investigate the effect of the fasting on the hepatic lipid droplet size,we investigated the effect of the fasting on the liver.In order to investigate the reasons for the increase in the size of liver lipid droplets after fasting,we measured the changes of free fatty acids,triglycerides and other physiological indexes in liver tissues of obese mice at 16 h and 24 h of fasting,and found that the content of triglycerides,free fatty acids and cholesterol increased after fasting.The results showed that the expression of genes related to fatty acid breakdown and fatty acid synthesis in liver increased after fasting,while the expression of genes related to fatty acid synthesis tended to decrease.The above results suggest that the substances required for the increase of lipid droplet volume come from triglycerides and fatty acids outside of hepatocytes,rather than being synthesized by hepatocytes themselves.In order to further investigate the sources of the increased lipid in the liver of obese mice,we examined the changes in morphology,lipid content and expression of lipid metabolism-related genes in adipose tissue near the spleen and inguinal adipose tissue of obese mice after 16 h and 24 h fasting,respectively.The results showed that the inguinal fat content decreased to a greater extent after fasting,triglycerides and cholesterol decreased and free fatty acid content increased,while the expression of genes related to fat synthesis and lipolysis was down-regulated and up-regulated in the inguinal adipose tissue.This result suggests that fasting causes fat mobilization,but the order of adipose tissue mobilization differs in different parts of the body,and this study suggests that inguinal adipose tissue mobilization is preferential to the splenic part of the adipose tissue.3.In order to investigate the molecular mechanism of liver lipid droplet volume increase in different fasting times,RNA-Seq technology was used to analyze the transcriptome changes after fasting.A total of 3,556 differentially expressed genes,including 1,996 up-regulated genes and 1,560 down-regulated genes,were screened by constructing 30 m RNA libraries of obese and normal mice fasted for 0 h,8 h,16 h,24 h and 32 h,and analyzing the differentially expressed genes,their GO and KEGG enrichment by bioinformatics.Further analysis of the GO enrichment of differentially expressed genes showed that these differentially expressed genes were significantly enriched into relevant biological processes such as immune response,cytoskeleton and lipid metabolism.Specific analysis of changes in the expression of lipid deposition-related genes revealed that Cidec was extremely highly expressed in obese mice.In addition,the m RNA expression levels of the eight differentially expressed genes identified by RNA-Seq were validated using q RT-PCR,and the results showed that the expression trends of q RT-PCR and RNA-seq were consistent,indicating that the results of RNA-Seq sequencing are accurate and reliable.4.In order to study the mechanism by which fasting regulates the increase in lipid droplet volume in liver tissue,we selected Cidec,which plays a key role in lipid droplet fusion,as the object of this study.The results showed that the m RNA and protein levels of Cidec were significantly increased,and the m RNA level increased about 200-fold at 16 h of fasting.The cells were treated with different nutrient media and the expression of Cidec was found to be increased after energy restriction.In order to investigate the effect of Cidec on lipid droplet morphology,Cidec gene was overexpressed in the mouse hepatocyte cell line AML12.The results showed that the lipid droplet volume increased significantly and the number of lipid droplets decreased in Cidec overexpressing cells,while the lipid droplet volume was smaller and the number of lipid droplets was higher in control cells and cells that had not been successfully transfected.5,In order to further investigate the regulation of Cidec expression up-regulation during fasting,the expression levels of AMPK were detected in obese mice at different fasting times,and it was found that AMPK and its phosphorylation levels increased continuously with the prolongation of fasting time.Further analysis of transcription factors downstream of AMPK that promote lipid droplet fusion-related transcription factors revealed that PPARγ expression increased with the prolongation of fasting time,and PPARγwas found to increase the fluorescence activity of the Cidec promoter using the dual-fluorescein reporter system.By cellular simulation of fasting conditions,the expression patterns of AMPK,PPARγ and Cidec were found to be identical and increasing.These results suggest that under fasting conditions,the expression of Cidec through AMPK-PPARγ-Cidec signaling pathway regulates lipid droplet fusion.6,In order to further investigate the phenomenon of significantly elevated expression of Cidec after fasting,bioinformatics was used to predict candidate miRNAs that target the regulation of Cidec,and the prediction results showed that miR-7a-5p could potentially target Cidec.miR-7a-5p mimic was transfected into 293 T cells and the results showed that miR-7a-5p could target Combined with the 3’-UTR of Cidec,the 3’-UTR mutation technique using Cidec all showed that miR-7a-5p inhibited the expression of Cidec.By detecting the expression of miR-7a-5p in obese mice after fasting,it was found that its expression gradually decreased with the prolongation of fasting.The expression of lipid droplet-associated proteins was detected after transfection of cells with miR-7a-5p and Cidec,respectively,and the opposite trend was found.This result suggests that miR-7a-5p controls the formation of lipid droplet fusion by being able to target the 3’-UTR of Cidec and inhibit the expression of Cidec.In conclusion,this study analyzed the molecular regulatory mechanism of fasting in mice through physiological and biochemical parameters,histomorphology,transcriptomics and gene expression regulation,and provided theoretical evidence for the improvement of metabolism,especially lipid metabolism,in fasting.In addition,the results of this study demonstrate that the AMPK-PPARγ-Cidec signaling pathway regulates the fusion of lipid droplets after fasting,which provides a new approach for the study of liver fat deposition under fasting conditions. |
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