The Impact Of Sidt2 Knockout On Hepatic Lipid Metabolism In Mouse Model

Part 1: The study of hepatic lipid metabolism in Sidt2 knockout miceObjectives: To explore the effect of Sidt2 on mouse hepatic lipid Metabolism.Methods: Experiments were conducted when the male Sidt2 knockout mice and its control were 6 and 12 weeks of age.Additionally,Sidt2 knockout and control mice were fed high saturated fat(HF)diet for 12 weeks since the age of 6 weeks.Serum triglycerides and total cholesterol were measured by using enzymatic methods.The livers were weighted,and the sections were stained with hematoxylin/eosin or oil red O.Electron microscopy were performed in liver blocks.Lipids were extracted from livers and determination of TG was carried out using enzymatic methods.ADFP was determined by Western blotting.Food consumption were measured for 4 weeks starting at the age of 5 week in mice fed mouse standard chow diet.Serum ketone levels and TG secretion were measured by enzymatic methods.Hepatic expression of genes involved in lipogenic and lipolytic enzymes were performed by Real time quantitative PCR analysis.Results:(1)The TG level between these two groups before and after the high fat diet were statistically significant(P<0.05).(2)Enlarged size and weight of liver from Sidt2^-/-mice were found in the chow-diet and HF-diet groups.Histological studies(liver sections were stained with both H–E and oil red O)show the liver of the Sidt2^-/-mice contained much more lipid droplets than that of wild-type mice.Increased LD number and size wre found in the Sidt2^-/-mice by Electron microscopy.Consistent with the increased LDs in histological studies,both liver TG content and ADFP level in Sidt2^-/-mice were higher than those in wide type.Elevated serum ALT and AST levels were found in Sidt2^-/-mice.Above mentionded difference were amplified between Sidt2^-/-and WT mice after 12 weeks of an HF diet.(3)We found decreased serum ?-hydroxybutyrate levels in Sidt2^-/-mice compaired to control levels.However,there was no significant diffence in hepatic TG secretion and expression of genes involved in lipogenic and lipolytic enzymes between Sidt2^-/-and WT groups.There was no significant difference in food consumption for 4 weeks starting at the age of 5 week between Sidt2^-/-and WT groups.(4)There was measurable difference in liver TG content,ADFP level and serum ?-hydroxybutyrate levels between 6-week-old Sidt2^-/-and WT mice.Conclusions: The knockout of Sidt2 gene in mice was associated with increases of serum TG levels and abnormal lipid accumulation in the liver,the phenomenon may be realized by decreasing hepatocellular ?-oxidation of fatty acids.Part 2:The impact of Sidt2 on autophagy in Sidt2 knockout mouseObjectives: To study the effect of Sidt2 on autophagy,explore the relation of autophagy and hepatic steatosis in Sidt2 knockout mouse.Methods: LC3-II which is the phosphatidylethanolamine-conjugated form of LC3 and p62/SQSTM1 which is known to be selectively degraded via the autophagic pathway,from livers of Sidt2^-/-and WT mice,were investigated by Western blotting.Furthermore,we evaluated the autophagic flux by immunoblot and densitometric analysis of LC3-II and p62 levels in primary cultured fibroblasts treated with a control or rapamycin.The endogenic LC3 dots were determinded by immunofluorescence assay,and autophagic vacuoles were observed by electron micrographs under induction of autophagy conditions.Results:(1)LC3-II levels were significantly decreased and p62 protein levels was increased in livers derived from Sidt2^-/-mice compared with that from WT mice.This showed autophagic flux was inhibited.(2)Consistent with the result in vivo,attenuate autophagic flux emerged in fibroblasts from Sidt2^-/-mice.Decreased numbers of LC3-positive dots and autophagic vacuoles were found by immuno-fluorescence and Electron microscopy assay,respectively.Conclusions: Decreased TG breakdown and ?-oxidation of fatty acids in Sidt2^-/-mice was primarily attributed to autophagy inhibition,further led to abnormal lipid accumulation in the liver.