Study On The Role Of PNPLA3 I148M And M6SF2 E167K Double Mutations In Regulating Liver Lipid Metabolism In Mice

Objective:1.Construct PNPLA3 I148M and TM6SF2 E167K double mutant mouse models.2.Explore the role and molecular mechanism of PNPLA3 I148M and TM6SF2E167K double mutations in liver steatosis at the animal level,and reveal the combined role of double mutations at PNPLA3 I148M and TM6SF2 E167K in regulating liver lipid metabolism.Methods:1.PNPLA3 I148M and TM6SF2 E167K mutant mice were obtained from the Liver Disease Laboratory of Qingdao Municipal Hospital affiliated to Qingdao University. Single mutant homozygous mice were obtained from PNPLA3 and TM6SF2 heterozygous mice through self-cross.PNPLA3 mutant homozygous mice were mated with TM6SF2 mutant homozygous mice to obtain PNPLA3 and TM6SF2 double mutant heterozygous mice,PNPLA3 and TM6SF2 double mutant homozygous mice were obtained through self-cross of double mutant heterozygous mice.2.Adult male mice aged 8 weeks were randomly divided into 12 groups（7 mice in each group）with 4 genotypes.Divided into Wt-CD-0w、Wt-CD-16w、Wt-HFD-16w、Pnpla3^148I/ITm6sf2^167K/K-CD-0w、Pnpla3^148I/ITm6sf2^167K/K-CD-16w、Pnpla3^148I/ITm6sf2^167K/K-HFD-16w、Pnpla3^148M/MTm6sf2^167E/E-CD-0w、Pnpla3^148M/MTm6sf2^167E/E-CD-16w、Pnpla3^148M/MTm6sf2^167E/E-HFD-16w、Pnpla3^148M/MTm6sf2^167K/K-CD-0w、Pnpla3^148M/MTm6sf2^167K/K-CD-16w and Pnpla3^148M/MTm6sf2^167K/K-HFD-16w.Weigh and record your weight at a fixed time every week.At the end of the experiment,the liver of the mice was weighed and part of the liver tissue was taken for pathological sections.Glucose metabolism indexes such as oral glucose tolerance test,fasting plasma glucose and plasma insulin levels were detected.Plasma triglyceride（TG）,total cholesterol（TC）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,high density lipoprotein cholesterol（HDL）,low density lipoprotein cholesterol（LDL）and other lipid metabolism related indexes were detected.Results:1.The results of mouse gene testing proved that the double mutant mouse models of PNPLA3 I148M and TM6SF2 E167K were successfully constructed.2.The number of mice with different genes obtained by mating with PNPLA3 and TM6SF2 double mutant heterozygous mice was counted.Wt mice: PNPLA3 single mutant mice:TM6SF2 single mutant mice:Heterozygous(Pnpla3^148I/MTm6sf2^167E/K,Pnpla3^148I/MTm6sf2^167E/E,Pnpla3^148I/ITm6sf2^167E/KPnpla3^148M/MTm6sf2^167E/Kand Pnpla3^148I/MTm6sf2^167K/K)mice:Pnpla3^148M/MTm6sf2^167K/Kdouble mutant mice was approximately 1:1:1:12:1.3.Influence on growth and development:under normal diet,there was no significant difference in the body weight of mice with different genotypes（P>0.05）,while under high fat diet,the body weight of Pnpla3^148M/MTm6sf2^167K/Kmice was higher than Wt mice at 12 and 16 weeks（P<0.05）.The liver index and liver weight of Pnpla3^148M/MTm6sf2^167K/Kmice fed with high fat diet for 16w were higher than those of other three genotypes（P<0.05）.4.Effects on glucose metabolism of mice:when fed with normal diet for 16w,the fasting plasma glucose level of Pnpla3^148M/MTm6sf2^167K/Kmice was higher than that of the other three genotypes（P<0.05）,and the plasma insulin level was lower than that of the other three genotypes（P<0.05）;Fasting plasma glucose,OGTT-2h glucose,insulin resistance and OGTT-AUC of Pnpla3^148M/MTm6sf2^167K/Kmice were significantly higher than Wt mice（P<0.05）,and plasma insulin level was lower than Wt mice（P<0.001）when fed with high fat diet for 16w.5.Effects on liver lipid metabolism:High fat diet for 16w,plasma TG of Pnpla3^148M/MTm6sf2^167K/Kmice was lower than that of other three genotypes（P<0.05）,and plasma TC was higher than that of Wt mice and TM6SF2 single mutant mice（P<0.05）.The plasma ALT level of Pnpla3^148M/MTm6sf2^167K/Kmice was higher than that of Wt mice（P<0.05）;The plasma LDL level of Pnpla3^148M/MTM6SF2^167K/Kmice was higher than that of the other three genotypes（vs.Wt mice,P<0.01;vs.TM6SF2 single mutant mice,P<0.01;vs.PNPLA3 single mutant mice,P<0.05）;The plasma HDL level of Pnpla3^148M/MTM6SF2^167K/Kmice was higher than that of PNPLA3 single mutant mice and Wt mice（vs.PNPLA3 single mutant mice,P<0.01;vs.Wt mice P<0.05）;The liver TG of Pnpla3^148M/MTM6SF2^167K/Kmice was significantly higher than the other three genotypes（vs.Wt mice P<0.0001;vs.TM6SF2 single mutant mice,P<0.001;vs.PNPLA3 single mutant mice,P<0.01）.HE and oil red O staining showed that Pnpla3^148M/MTM6sf2^167K/Kmice had more severe steatosis than the other three genotypes.Conclusion:1.PNPLA3 I148M and TM6SF2 E167K double mutant mouse models were successfully constructed.2.Dual mutations of PNPLA3 I148M and TM6SF2 E167K led to abnormal glucose metabolism in mice under normal diet and high fat diet.3.Dual mutations of PNPLA3 I148M and TM6SF2 E167K produce superimposed effects on lipid deposition in liver of mice under the condition of high fat diet,accelerating the progression of NAFLD.