To Explore The Phenotype And Mechanism Of UTX Liver-specific Knockout Mouse

Histone modification is an important epigenetic modification,which plays an important role in regulation of gene expression.Until now,many histone modification styles have been found,including acetylation,methylation,ubiquitination and phosphorylation.Among these,the methylation of histone H3lysine residues on the N end is the most important.UTX,a demethylase of H3K27,was first reported in 2007,which has been found to participate in a variety of important biological functions,such as stem cell differentiation,embryonic development,hematopoiesis and cancer.Unfortunately,there is not a research associated with metabolic functions.So far,there have been a lot of histone methylation modification enzyme plays an important role in metabolism As reported,histone H3K36 demethylase JHDM1a,can be a negative regulator on gluconeogenesis of liver;Enzyme of histone modification G9a plays an important role in biological clock of liver to switch freely in Fasting and Refeeding loop;Demethylase JHDM2a can affect obesity and hyperlipidemia by influencing fatty acid oxidation of the brown adipose tissue and skeletal muscle.There are so many similar reports,suggesting close relationship between histone modification enzymes and metabolism.At the same time,as with UTX-1 knockdown or mutant,the C elegans could have a significantly prolonged life and this effect depended on the regulation of insulin signal pathway.Besides,the effect on insulin signal pathway was highly conservative in mammals.Literature results indicate that UTX may be associated with glucose and lipid metabolism of liver.Therefore,our study firstly observed the expression characteristics of UTX on normal and abnormal liver tissues.Then,we used primary liver cells form mice to simulate the body's response to insulin.All of these were to provide more evidence to illustrate the relationship between histone modification enzymes and metabolism.Fortunately,there existed UTX flox mouse on Model Animal Research Institute of Nan Jing University,so we constructed a liver-specificity UTX knockout mouse and analyzed its phenotype and possible mechanismsPart I Analysis of clues to indicate that UTX may have functions on glucose and lipid metabolism of liverObjective:To observe the expression characteristics of UTX on normal and abnormal liver tissues.To use primary liver cells form mice to simulate the body's response to insulin.Methods:Tissues of heart,liver,spleen,lung,kidney,brain,skeletal muscle,pancreas,brown fat and white fat from female mouse were separated,and total proteins from these tissues were used to detect the UTX protein expression by western blot;Primary liver cells from mice was separated and cultured,then stimulated by different concentrations of insulin or the same concentration at different time points respectively,to analyze UTX expression level in these induced cells;Constructed diet induced obese mice models,and got the liver tissue to test UTX expression by western blot.Results:1)Among these metabolism related tissues as brain,skeletal muscle,pancreas,brown fat,white fat and liver,the UTX expression of liver is higher,only falls behind the brain;2)The UTX mRNA level rises along with the increase of the concentrations of insulin,and reaches the highest at 100 nm;At the different time points stimulated by 100 nm concentration,the UTX expression level rapidly increases in the 10 min and then drops down,but still maintains a higher level between 30-120 min;3)UTX shows a higher expression in liver tissue of DIO mouse.Conclusion:The expression level of UTX was higher on normal and abnormal liver tissues.UTX could response to insulin in the primary liver cells from mice.That exploration of the relationship between UTX and glucose and lipid metabolism of liver provided theoretical and experimental basis to clarify UTX role in liver.Part II Construction of UTX liver-specific knockout mice and phenotypic analysisObjective:To construct a UTX liver-specific knockout mouse and observe its phenotypes about glucose and lipid metabolism under chow diet.Methods:To obtain female UTX^f/f;albcre mouse by mating the female UTX^f/f muse and the male albcre mouse.Knockout efficiency was validated by the qRT-PCR and Western Bolt.Choose UTX^f/f;albcre mouse and UTX^f/f/f mouse as control to monitor body weight every week from four weeks age old.Analyze of food intake and fecal excretion of mice by daily weight.Metabolic cage experiment indicated the thermogenesis,respiratory quotient and activity.Test the blood triglyceride,cholesterol,LDL-c,HDL-c,free fatty acids and glucose level by chemical methods.Serum insulin level,glucose tolerance test and insulin tolerance test indicated the insulin sensitivity.Pyruvate acid tolerance test was used to observe gluconeogenesis.H&E staining was used to observe the morphology change of liver tissue.Tissue analysis was used to analyze hepatic triglycerides,cholesterol and glycogen.Olive oil was gavaged to observe triglyceride clearance;Intraperitoneal injection of Poloxamer-407 or isotope was used to observe VLDL synthesis and secretion of liver;Extraction method was used to detect fecal cholesterol,triglyceride and bile acid levels from feces.Results:1)Knockout efficiency of UTX^f/f;albcre mouse could reach 70%,indicated that UTX liver-specific knockout mouse was constructed successfully;2)Body weight and fat mass of UTX^f/f;albcre mouse were normal.Daily food intake and feces excretion were with no significant difference.Metabolic cage experiments showed that respiratory quotient,energy consumption and activity of UTX^f/f;albcre mouse were not affected;3)UTX^f/f;albcre mouse showed significantly higher triglycerides and cholesterol level,with significantly higher LDL-c and relative lower HDL-c,but the blood glucose was normal;4)UTX^f/f;albcre glucose showed improved glucose tolerance and insulin sensitivity with normal insulin level,as well as decreased gluconeogenesis;5)Liver weight and pathology morphology was normal,and with normal cholesterol content,lower triglyceride and glycogen content in liver;6)Triglyceride clearance was with no change;7)VLDL secretion was increased.The ability of cholesterol and fatty acid synthesis of liver were obviously enhanced;8)Fecal cholesterol and fecal bile acid content were with no significant difference;9)Male UTX^f/y;albcre mouse did not appear the phenotype of hyperlipidemia.Conclusion:UTX liver-specific knockout mouse was constructed successfully.UTX^f/f;albcre showed hyperlipidemia and may be caused by enhanced cholesterol and fatty acid synthesis of liver.These suggested that UTX plays an important role on the regulation of lipid metabolism.Part III The mechanism of abnormal lipid metabolism of the UTX liver-specific knockout miceObjective:To study the possible molecular mechanisms about dyslipidemia of UTX liver-specific knockout miceMethods:qRT–PCR was used to analyze genes of key enzymes and transcription factors of synthesis of fatty acid and cholesterol;Detect the methylation level of H3K27 by Western blot and immunohistochemistry;Other histone modification enzymes associated with methylation of H3K27,such as JMJD3,EZH2 and SUZ12was observed by Western bolt.Results:1)Key enzyme genes of hepatic lipid synthesis,FAS,ACC,SCD1,ELOVL5 and ELOVL6 showed higher expressions,but the expression of gene Acly was normal in UTX^f/f;albcre mouse;2)Enzymes associated with cholesterol synthesis,MVK,MVD,IDI1,SQLE and Lss showed significantly increase and the key gene HMGCR showed normal expression;3)Transcription factors that regulate lipid metabolism,SREBP-1c and ChREBP were with no changes,but SREBP-2gene with lower expression;4)The methylation of H3K27,2mH3K27 and3mH3K27,was normal;5)Other histone modification enzymes that affected the level of H3K27 methylation,JMJD3,EZH2 and SUZ12 were with the same level with control mouse.Conclusion:The expression of key enzymes associated with fatty acid and cholesterol synthesis process increase obviously in UTX^f/f;Albcre mice,resulted in enhanced cholesterol and fatty acid synthesis.