| BackgroundThe Suprachiasmatic nucleus(SCN)of the hypothalamus,as the core pacemaker of the mammalian circadian clock,synchronizes the circadian clock of peripheral tissues by integrating light and non-light signals,so as to regulate the physiological processes and behavior of the body to better adapt to the changes of the day and night cycle.Energy metabolism includes the absorption,conversion,storage,and decomposition of nutrients.These processes are synchronized by the SCN via neural and humoral regulation to ensure the absorption,decomposition,and utilization of nutrients at the best time of day.Adipose tissue realizes periodic autonomic oscillation through the transcriptional and translational negative feedback loop of molecular clock,and the SCN synchronizes the autonomic oscillation of adipose tissue with the day and night,so that the physiological function of adipose tissue shows a nearly 24-hour rhythm.Adipose tissue is divided into White adipose tissue(WAT)and Brown adipose tissue(BAT)according to the function and structures.At high energy levels,white adipose tissue stores energy in the form of triglycerides and uses it as fuel at low energy levels.White adipose tissue also has endocrine functions,releasing leptin,fatty acids,tumor necrosis factor in a circadian rhythm to regulate food intake,energy turnover,inflammatory response and other physiological processes.Brown adipose tissue generates heat and maintains body temperature through non-shivering thermogenesis,a process that is also controlled by the circadian clock.Although there is a neuroregulatory relationship between the SCN and adipose tissue,it is still unclear how the SCN participates in the body’s thermogenesis and decomposition of fat in the low temperature environment.In particular,our previous study found that SCN lesion could counteract the death of mice induced by time restricted feeding at 21℃ ambient temperature and maintain body temperature homeostasis,indicating that the function of SCN on adipose tissue may not be limited to circadian regulation,but also the regulation of function.This study focuses on the scientific issues about the influence of SCN on the function of adipose tissue under time restricted feeding at 21℃.By intervening the function of SCN,the phenotypes of adipose tissue from morphology to function were analyzed,the relationship between the changes of molecules and metabolites were explored in the maintenance of body temperature homeostasis.Furthermore,the key molecules of SCN affecting the function of adipose tissue were identified.This study provides a basis for further understanding the relationship between SCN and adipose tissue.Scientific issues1)Whether the SCN regulates the function of brown adipose tissue under ZT16-20 time restricted feeding at 21℃?2)How does the SCN alter the energy metabolism of white adipose tissue under ZT1620 time restricted feeding at 21℃?Methods1.To explore whether SCN affects brown adipose tissue,the following experimental methods were used:(1)The SCN of the mice was lesioned by the damage electrode.After the mice recovered for a week,the Clock Lab circadian rhythm recording system was used to screen the arrhythmic mice to obtain the successful SCN lesioned mice.After the completion of subsequent experiments,histological staining was used to verify whether the SCN was lesioned and whether the peripheral brain area was intact.(2)The core body temperature and brown adipose tissue temperature of SCN lesioned or sham SCN lesioned mice were recorded.After treated with ZT 16-20 time restricted feeding at 21℃,the core body temperature and brown adipose tissue temperature of mice were measured again,and the changes of body temperature between SCN lesioned and sham SCN lesioned mice were compared.(3)The brown adipose tissue samples of SCN lesioned and sham SCN lesioned mice were collected after ZT 16-20 time restricted feeding at 21℃.HE staining and transmission electron microscopy were performed to observe the changes in internal structure of brown adipose tissue.(4)RNA-seq sequencing was performed to analyze the gene expression changes in brown adipose tissue of SCN lesioned and sham SCN lesioned mice after ZT16-20 time restricted feeding at 21℃,and to find the key pathways affected by SCN.2.To explore how SCN affects white adipose tissue energy metabolism,the following experimental methods were used:(1)The white adipose tissue samples of SCN lesioned and sham SCN lesioned mice after ZT16-20 time restricted feeding at 21℃ were collected,and HE staining was performed to observe whether the white adipose tissue changes during this process.(2)The metabolic parameters of SCN lesioned and sham SCN lesioned mice were detected by metabolic cages to study the effect of SCN on energy metabolism in mice.(3)The white adipose tissue samples of SCN lesioned and sham SCN lesioned mice after ZT16-20 time restricted feeding at 21℃ were collected for RNA-seq to study the gene expression in white adipose tissue.(4)Validation and intervention for the candidate pathways.Results and Conclusions(1)Under AD libitum,the circadian rhythm of core body temperature and brown adipose tissue temperature of SCN lesioned mice were disappeared.Under the stimulation of ZT16-20 time restricted feeding at 21℃,the SCN lesioned mice were better able to maintain core body temperature and brown adipose tissue temperature than sham SCN lesioned mice.(2)Principal component analysis(PCA)analysis was performed on the gene expression data of brown adipose tissue under ZT16-20 time restricted feeding at 21℃,and the top 100 genes with the largest contribution value were selected for pathway enrichment analysis.It was found that SCN lesion mainly affected ion channels,cytoplasmic and membrane structural pathways.Transmission electron microscopy showed that the inner mitochondrial membrane was intact in SCN lesioned mice,while the mitochondrial membrane gap was enlarged and mitochondrial cristae was sparse in sham SCN lesioned mice.(3)The brown adipose tissue of sham SCN lesioned mice was denser than that of SCN lesioned mice under AD libitum conditions,and the difference was further increased after ZT 16-20 time restricted feeding at 21℃.(4)Under AD libitum,SCN lesioned mice had more amount of fat than sham SCN lesioned mice.Under ZT16-20 time restricted feeding at 21℃.SCN lesioned mice showed lower fat consumption rate.(5)Metabolic cage analysis showed that the metabolic rhythm of SCN lesioned mice was lost compared with sham SCN lesioned mice.With the extension of fasting time,the metabolism of sham SCN lesioned mice decreased,while that of SCN lesioned mice remained stable.(6)RNA-seq analysis of white adipose tissue showed that the pathways related to fatty acid metabolism were significantly inhibited in SCN lesioned mice.Correspondingly,phos-HSL protein level in brown adipose tissue of SCN lesioned mice was significantly lower than that of sham SCN lesioned mice.(7)The sham SCN lesioned mice were fed with high fat diet to increase the fat content,and 30%calorie restricted feeding was treated with SCN lesioned mice to reduce the fat content,and then stimulated with ZT16-20 time restricted feeding at 21℃.The difference in core body temperature between the two groups was disappeared,but the brown adipose tissue temperature and tail temperature of SCN lesioned mice were higher than those of sham SCN lesioned mice.These results suggested that SCN lesioned mice stored more fat under AD libitum,although there was no difference in food intake between SCN lesioned and sham SCN lesioned mice;under ZT16-20 time restricted feeding at 21℃,compared with SCN lesioned mice,the decomposition rate of fat stores in brown adipose tissue and white adipose tissue of sham SCN lesioned mice was higher,and the white adipose tissue had a trend of browning,but the thermogenesis did not increase,which eventually led to the destruction of body temperature homeostasis;SCN lesioned mice had more fat reserves with a lower breakdown rate,and the brown adipose tissue had a more stable thermogenesis,so they could maintain body temperature homeostasis.When the initial body fat content of sham SCN lesioned and SCN lesioned mice were balanced and then subjected to ZT16-20 time restricted feeding at 21℃,the difference in core body temperature between the two groups was near equal,but the brown fat temperature of the lesioned mice was still higher than that of sham SCN lesioned mice,and the heat dissipation ability of the tail was stronger in SCN lesioned mice.This research has some limitations.This study only observed the phenotypes after SCN lesioned in response to ZT16-20 time restricted feeding at 21℃.at individual level,tissue level and molecular level without carrying cause and effective experiments.Further works will be needed to answer the key questions how the SCN regulates the performance of adipose tissue function. |
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