The Establishment Of ABCA6 Knockout Mice Model And Research On Its Phenotype

Objectives:ATP binding cassette(ABC)transporters are a group of transmembrane proteins mediating transmembrane transport of various molecules via ATP,including lipids,amino acids,peptides,glucoses and ions.Human ABC transporter protein contains seven subfamilies,ABCA A?G.ABCA6 is one of the ABCA subfamily members,with its mRNA detected in multiple tissues of human body.However,for now only little knowledge has been known to us,including the fact that ABCA6 mRNA expression was increased in macrophage differentiation process and decreased significantly after Ac-LDL stimulation,indicating the reaction of ABCA6 to cholesterol.Previous studies by our lab have suggested that the transcriptional regulation of ABCA6 can be attributed to FoxO.In the present study,preliminary study about the function of ABCA6 in energy metabolism was performed with in vitro and in vivo experiments.Methods:We first prepared polyclonal antibody anti-ABCA6.By using this antibody,samples of respective tissue were collected to detect the ABCA6 expression by Western Blot.The subcellular localization of ABCA6 in mouse primary hepatocytes was observed by immunofluorescence.For better understanding about the factors affecting ABCA6,the expression of ABCA6 has been detected with western blot in following four kinds of mice models:ApoE knockout mice,obese mice induced with high-fat diet,diabetic mouse model induced with STZ,and mice under fast condition.Primary mice hepatocytes were treated with AICAR the inhibitor of AMPK,aerobic respiration suppressor Olygomycin,glycolytic inhibitor 2-DG and ?-oxidation inhibitor Etomoxir,respectively.The ABCA6 expression was estimated by western blot.In order to further study about the effect of ABCA6 on energy metabolism,the ABCA6 knockout mice were established.Plasma was collected under condition of normal feed and fast of 24 hours,respectively,to detect the level of biochemical parameters,including total cholesterol(TCH),triglycerides(TG),ketone bodies,alanine aminotransferase(ALT)and aspartate aminotransferase(AST),respectively.To further study the glucose metabolism and energy consumption of ABCA6 KO mice,several examinations were carried to assess the glucose tolerance,insulin resistance and pyruvate metabolic tolerance.The TSE animal metabolism analysis was also preformed.Furthermore,the metabolism-related parameters were studied with mice liver sample under condition of normal feed and 24-hour fast,respectively.The mRNA levels of gene responsible for fatty acid oxidation and gluconeogenesis were tested.The protein expression of p-AMPK and HMGCS2 were analyzed as well by western blot.Moreover,the liver samples of ABCA6-KO mice were processed with paraffin-embedded H&E staining for observation of ultrastructure of mice hepatocytes with transmission electron microscopy.Results:The expression of ABCA6 was observed in heart,liver and skeletal muscle of mice,among which the most significant expression was found within liver.The fluorescent immunohistochemistry results indicated that,different from ABCA1 which mainly located on the membrane,ABCA6 located within cells and concentrated around the nuclear area,resembling the location of trans-Golgi network.The expression of ABCA6 within liver of ApoE knockout mice,high fat-induced obese mice and STZ-induced diabetic mice showed no significant change.The ABCA6 was up-regulated under fast condition,and ABCA6 expression within primary hepatocytes increased after administration with AICAR,2-DG or Olygomycin.The expression of ABCA6 was suppressed after Etomoxir treatment.The ABCA6 KO mouse was successfully established.No significant difference compared with normal mice was found for detected biochemical parameters,including total cholesterol,ALT and AST,after 24-hour fast.However,the content of triglycerides,ketone body was down-regulated in plasma and the triglycerides level within liver were also down-regulated.The ABCA-KO mice did not develop significant phenotype of diabetes or insulin resistance or dysfunction of energy consumption.The mRNA level of PDK4 responsible for fatty acid metabolism and G6Pase concerned with gluconeogenesis decreased in the liver of KO mice compared with wildtype after 24-hour fast.Meanwhile increased protein expression of p-AMPK and reduced HMGCS2 were also observed.The morphological study indicated about the slight swelling of mitochondria in ABCA6-KO mice,along with hypertrophy and dilatation of Golgi with numerous Golgi vesicles.Conclusion:ABCA6 may located within the Golgi apparatus within hepatocytes,with its expression level changed with oxidation of fat acids.No significant of change of glucose metabolism was observed in ABCA6-knockout mice,however,the ABCA6 knockout resulted in abnormality in mechanism of plasma ketone body and triglyceride.The results can be seen as basic evidence indicating that ABCA6 involves in the protein transportation and lipid metabolism,which may play its role on the secretion of triglyceride-rich VLDL from trans-Golgi network.