The Role Of Endoplasmic Reticulum Transmembrane Protein Tm6sf2 E167K Mutation On Liver Lipid Metabolism In Mice

Objective:1.To establish transmembrane 6 superfamily member 2（Tm6sf2）E167K gene knock-in mouse model based on the CRISPR/Cas9 technology.2.Verify the relationship between endoplasmic reticulum transmembrane protein TM6SF2 E167K polymorphism and the development of NAFLD,and establish a new NAFLD genetically modified mouse model to provide a valuable animal model for exploring the mechanism in the occurrence and development of NAFLD and other lipid metabolic diseases.Methods:1.According to the mouse Tm6sf2 gene,sgRNA was designed and constructed,and connect the original expression of sg RNA and the original expression of Cas9 to get a plasmid that can express both the sgRNA and Cas9 at the same time.The Donor plasmid which carrying the Tm6sf2 E167K fragment was constructed,and the two plasmids were purified and injected into mouse fertilized eggs in vitro,and the eggs were then transplanted into pseudopregnant mouse uterus.The positive mice of F0 generation were obtained and the genotype was verified by PCR,Southern blot detection and sequencing.2.The Tm6sf2 ^167E/K heterozygous mice（HET）was obtained by mating between F0positive mice and wild mice,and 24 Tm6sf2 ¹67K/K67K/K homozygous male mice（KI）and 24Tm6sf2 ¹67E/E67E/E wild male mice（WT）were obtained by self-breeding strategy of HET mice.At the age of 8 weeks,the mice of each genotype were randomly divided into 3 groups with 8 mice in each group.The following experimental treatments were given:KI mice+CD（control diet）+0w group,KI mice+CD+16w group,KI mice+HFD（high fat diet）+16w group;WT mice+CD+0w group,WT mice+CD+16 w group,WT mice+HFD+16w group.The body weight of mice was recorded once a week from the first week of the experiment to the end of the experimental cycle.Oral glucose tolerance test（OGTT）was performed on the experimental mice.After anesthesia,the retroorbital vein blood was taken,and the plasma was separated to detect fasting blood glucose and fasting insulin levels and other glucose metabolism indicators.At the same time,the fasting plasma lipid metabolism indexes such as triglyceride,total cholesterol and transaminases were detected,and the experimental mice were dissected to observe the differences of the main organs.The liver of the mice was weighed and a small amount of liver tissue was taken for pathological sections.Results:1.The results of PCR verification and gene sequencing showed that the model of Tm6sf2 E167K gene knock-in mice was established successfully.The proportion of Wt,HET and KI genotypes produced by heterozygous mice self-compliance is in accordance with Mendel’s law of genetic separation.Tm6sf2 E167K knock-in mice do not have homozygous embryos.2.The fasting blood glucose and OGTT-2h blood glucose of Tm6sf2 ¹67K/K67K/K homozygous mice in both groups at 0 weeks（8 weeks old）and the control diet for 16weeks were higher than those of Tm6sf2 ^167E/E wild mice,and there were statistical differences（P<0.05）.The OGTT-0.5h blood glucose,OGTT-1h blood glucose,the area under OGTT-curve（OGTT-AUC）and insulin resistance index of Tm6sf2 ¹67K/K67K/K homozygous mice in the 16-week control diet group were higher than those of Tm6sf2¹67E/E67E/E wild mice,there were statistical difference（P<0.05）;The above-mentioned glucose metabolism indexes of the two genotype mice in the high-fat diet 16 week group were higher than those in the control diet 16 week group,but the above-mentioned glucose metabolism indexes of the two genotype mice in the high-fat diet 16 week group were not statistically different（P>0.05）.3.Tm6sf2 ¹67K/K67K/K homozygous mice in the high-fat diet 16-week group had increased body weight,liver weight,hepatic index,increased plasma TG and ALT level,increased liver TG content,and increased NAFLD activity score compared with Tm6sf2 ^167E/E wild mice,and there were statistical differences（P<0.05）;but there was no such difference in the two groups at 0 weeks and the control diet for 16 weeks.HE and Oil Red O staining results also showed that Tm6sf2 ^167K/K homozygous mice had more severe liver steatosis than Tm6sf2 ¹67E/E67E/E wild mice.Conclusion:1.The knock-in mouse model of Tm6sf2 E167K gene was established successfully.2.Under normal diet,Tm6sf2 E167K mutation may cause abnormal glucose metabolism in mice,mainly characterized by increased fasting blood glucose,decreased glucose tolerance and increased insulin resistance.3.Both Tm6sf2 E167K mutation and high-fat diet treatment can cause abnormal glucose metabolism in mice,but the effect of the latter may be stronger,when the two factors exist at the same time,the abnormal glucose metabolism in mice caused by high-fat diet treatment may mask the abnormal glucose metabolism caused by Tm6sf2E167K mutation in mice.4 Under the induction of high-fat diet,Tm6sf2 E167K mutation in mice can increase body weight and liver weight,increase plasma TG level and liver lipid deposition,and accelerate the progression of NAFLD.