Role Of AQP8 In The Formation Of Obesity Induced By High-fat Diet In Mice

Objective:Aquaporins（AQPs）are a series of homologous hydrophobic inner membrane proteins.AQP8,a member of aquaporin families,can transport water,hydrogen peroxide,ammonia and urea across membrane specifically.AQP8 are widely expressed in the digestive system,mainly in the duodenum,colon,jejunum,pancreas,parotid gland,salivary gland and liver.The liver is the organ with the highest expression of AQP8.There are two subtypes of AQP8 in the liver:one is N-glycosylated AQP8distributed on hepatocyte membrane and bile duct membrane with molecular weight of about 34 KD;the other is non-glycosylated protein with molecular weight of about 28KD,distributed in mitochondrial inner membrane.It has been reported that AQP8participates in the formation of bile by regulating the osmotic transport of bile duct water.AQP8 may also participate in the secretion of pancreatic juice and maintain the balance of cell osmotic pressure and mitochondrial volume during glycogen metabolism.Whether AQP8 participates in glycolipid metabolism and affects obesity caused by high-fat diet remains unclear.By using C57BL/6J wild-type and AQP8 knockout mice to prepare obesity model by high-fat diet,and examining the morphological changes of liver and fat tissues,and the related indicators of lipid metabolism in this study,.we make research on the role of AQP8 in the formation of obesity induced by high-fat diet and the mechanism of its action in liver.Methods:1.Prepration of obesity model:C57BL/6J wild type and AQP8^-/-mice were randomly divided into normal diet group（NC）,high fat diet group（HFD 8W）and high fat diet group（HFD 16W）.The normal group was fed with basic diet,and the high-fat diet group was fed with high-fat diet for 8 and 16 weeks.The weight of mice was measured weekly.2.Mice were dislocated to sacrifice after blood collection.Liver and adipose tissue were retained.Body fat rate,fasting blood sugar,insulin level,cholesterol（TC）,triglyceride（TG）,liver tissue and fat morphological observed were measured.The changes of serum total bile acid and intrahepatic bile acid in HFD 8W group were detected,and the effect of AQP8 on bile acid secretion was observed.The levels of TC and TG in liver were detected,and the expression levels of nucleic acid and protein related to lipid metabolism were detected by real-time quantitative PCR and Western blot.Results:1.The body weight and body fat rate of AQP8^-/-mice fed with high fat for 8W were significantly higher than those of wild type mice.In the normal diet group,the weight of AQP8^-/-mice was slightly higher than that of wild-type（C57BL/6J,C57）mice.When HFD 8 weeks,the weight gain of AQP8^-/-mice was significantly higher than that of wild-type mice,and the weight and body fat rate increased significantly（p<0.05 and 0.01,respectively）.After 8 weeks of high fat diet,the body weight of wild type mice increased rapidly.At 16 weeks,the body weight of wild type mice and gene knockout mice tended to be the same.At this time,the body fat rate of AQP8^-/-mice was lower than that of C57 mice,but there was no significant difference.Compared with C57 mice in NC group,the weight and body fat rate of C57 mice fed with high fat diet increased significantly（p<0.05 and 0.01,respectively）.2.Fasting blood glucose and insulin increased significantly in AQP8^-/-mice fed with high fat.Compared with C57 mice,the fasting blood glucose and insulin levels of AQP8^-/-mice in the normal diet group did not change significantly;the blood glucose of AQP8^-/-mice in the high fat diet group（8 weeks and 16 weeks）was significantly higher than that of C57 mice（p<0.01）,the insulin level increased,and HOME-IR increased significantly（p<0.01）.Compared with C57 mice,serum TC and TG of AQP8^-/-mice did not change significantly.3.Morphological changes of liver and adipose tissue in AQP8^-/-mice fed with high fat.HE staining showed that compared with wild-type mice,（1）Liver tissue:AQP8^-/-mice in normal diet group had slight swelling of intrahepatic cells.Fat droplets and balloon-like lesions appeared in the liver of AQP8^-/-mice in high-fat diet group for 8weeks.In 16 weeks of high-fat diet group,a large number of fat droplets appeared in the liver of AQP8^-/-mice,and fat droplets became larger with inflammation.（2）The number of adipocytes in AQP8^-/-mice decreased but and the area of lipid droplets increased in adipose tissue.4.High-fat diet down-regulates the expression of AQP8 in liver of wild-type mice Compared with the normal diet group,the expression of AQP8 in wild type mice decreased after 8 weeks of high fat feeding（p<0.001）,but still higher than that in AQP8^-/-mice（p<0.001）;after 16 weeks of HFD,the expression of AQP8 in liver of wild type mice decreased further（P<0.001）,and there was no significant difference between wild type mice and AQP8^-/-mice.5.Effects of High-fat Diet on Total Bile Acids in AQP8^-/-mice.Compared with C57 mice,after 8 weeks of high fat feeding,the total bile acid content in liver of AQP8^-/-mice increased significantly（p<0.05）,while the bile acid content in serum decreased（p<0.05）.6.Effects of AQP8^-/-mice fed with high fat diet on TC and TG in liver tissue.The levels of TC and TG in the liver of AQP8^-/-mice at 8 weeks of HFD were significantly higher than those of C57 mice after 8 weeks of high fat feeding（p<0.05）.7.Expression of FXR m RNA and Protein in Liver of AQP8^-/-mice after High Fat Feeding.Compared with C57 mice,the expression of FXR in liver of AQP8^-/-mice was significantly decreased（p<0.05）,and the protein expression of FXR was also significantly decreased（p<0.05）.8.Expression of SREBP-1C m RNA in the liver of AQP8^-/-mice fed with high-fat diet.Compared with C57 mice,the expression of SREBP-1c in AQP8^-/-mice liver was significantly increased after 8 weeks of high fat feeding（p<0.05）.9.Fatty acid synthase（FAS）gene transcription and protein expression in the liver of AQP8^-/-mice fed with high fat.Compared with C57 mice,after 8 weeks of high fat feeding,the expression of fas in AQP8^-/-mice hepatic m RNA increased（p<0.05）,and the protion expression of fas in AQP8^-/-mice was the same,with a significant increase（p<0.05）.10.The expression of hepatic X receptor alpha（LXRa）and cholesterol 7 alpha hydroxylase（CYP7A1）m RNA in AQP8^-/-mice was up-regulated.The levels of LXRa and CYP7A1 in AQP8^-/-mice were significantly increased after 8 weeks of high fat feeding（p<0.05）.11.Expression of aquaporin 9（AQP9）in the liver of AQP8^-/-mice fed with high-fat diet.Compared with C57 mice,the expression of AQP9 m RNA in liver of AQP8^-/-mice was significantly increased after 8 weeks of high fat feeding（p<0.05）.Conclusions:1.The decrease of AQP8 can promote the formation of obesity in mice,suggesting that AQP8 plays an important role in the formation of obesity.2.Long-term（especially more than 8-10 weeks）high-fat diet results in the down-regulation of AQP8 expression in the liver of mice,and the degree of obesity was more significant.3.AQP8 deletion promotes the expression of SREBP-1c and FAS by decreasing bile acid secretion,down-regulating FXR,and increases lipid synthesis in mice liver,thus then leading to obesity.4.Aquaporin 8 knockout promotes the expression of AQP9 in mice liver and alleviates the bile secretion disorder caused by AQP8 deficiency.