Study On The Mechanism Of Opg Regulating Liver Insulin Sensitivity

PARTⅠ THE EFFECTS OF OPG ON INSULIN SENSITIVITY IN HEPATOCYTES IN VITROObjective: Exploring the effects of OPG on hepatocyte glucose metabolism and insulin signaling pathway in vitroMethods: OPG overexpression and knockdown adenovirus were constructed,and insulin resistance model was established by treating hepal1-6 and L02 cell lines with high glucose and high fat FFAs or glucosamine（GlcN）.The glycogen content in the cells was detected by periodic acid Schiff staining.The glycogen content was quantitatively determined by a kit method.Cellular glucose uptake capacity was measured by using a 2-NBDG kit.The glucose metabolism related genes p-InsR,t-InsR,p-AKT,t-AKT,PEPCK,and G6 Pase were detected by western blot.Results: The OPG overexpression and knockdown adenovirus were constructed and successfully infected hepatocytes,and the insulin resistance model was also successfully established.Both glycogen stainingand quantitative detection showed that OPG can reduce the synthesis of glycogen synthesis.Moreover,the cell glucose uptake ability is decreased after overexpressed OPG.However,overexpression of OPG results in a significant decrease in p-AKT in western blot,a key factor in the insulin signaling pathway,but no change in p-InsR levels.Conclusion: OPG can regulate the glucose metabolism of hepatocytes,and may act on the regulation of the activity of AKT by affecting the downstream molecules of InsR.PARTⅡ OPG KNOCKOUT INCREASES INSULIN SENSITIVITY AND PROMOTES GLYCOGEN SYNTHESIS IN DIABETIC MICEObjective: Exploring the effects of OPG on hepatocyte glucose metabolism and insulin signaling pathway in vivo.Methods: The OPG heterozygotes mouse were extensively propagated to obtain sufficient male homozygous and wild type mice.At the 8th week,mice were randomly divided into four groups SD-WT,SD-OPG^-/-,HFD-WT and HFD-OPG^-/-,and fed with high-fat diet or normal diet.GTT and ITT tests were performed at 19 weeks,and the tests verified whether the mouse insulin resistance model was successful.At 20 weeks,blood samples were taken from the eyeball for the detection of various biochemical indicators.Liver tissue fixed with paraformaldehyde were used to do paraffin sectioning,and the change of liver glycogen content was observed by Periodic Acid-Schiff staining.The liver tissue was quantitatively tested for its glycogen content by a kit method.Real-time quantitative PCR was used to detect insulin signaling pathway related genes TRB3,ATF4,ATF6,SIRT1 etc.;Glycolysis related genes Gck,Pdk4 Changes in m RNA levels,etc.Western blotting to analyze glucose metabolism related genes p-Ins R,t-Ins R,p-AKT,t-AKT,PEPCK,G6 Pase,p-GSK3β,t-GSK3β,p-AMPK,t-AMPK,p-PKC,t-PKC,p-JNK,t-JNK,p-mTOR,t-mTOR,Raptor,p-S6K1,t-S6K1.Results: The area under the curve of GTT and ITT of the high fat diet fed mice were significantly lower than that of the normal diet groups,indicating that the insulin resistance model is successful.The area under the curve of GTT and ITT in OPG^-/-mice was significantly lower than that in WT,and the serum glucose level in OPG^-/-mice was significantly lower than that in the control group.These results indicate that OPG knockout can improve mice insulin sensitivity.The results of Periodic Acid-Schiff stain and hepatic glycogen content assay showed an increase in liver glycogen content in OPG^-/-mice.The m RNA levels of TRB3,ATF4,ATF6,SIRT1,Gck,Pdk4 did not change.Western blot results showed that p-AKT and p-GSK3β were significantly higher in the HFD-OPG^-/-group than in the HFD-WT group;whereas p-AMPK,p-PKC,p-JNK had no significant changes;compared with the control group PEPCK,G6 Pase,p-mTOR and p-S6K1 were significantly reduced in the HFD-OPG^-/-group,and the Raptor bands were found to have significant shift.Conclusion: The results in OPG knockout mice are consistent with the results of in vitro cell fractions.OPG knockout can improve insulin resistance in mice.And this process is likely to be regulated by the mTORC1 signaling pathway.PART Ⅲ UNDERLYING MECHNISM OF OPG REGULATING LIVER INSULIN SENSITIVITYObjective: To explore the molecular mechanism of OPG regulating hepatic insulin sensitivityMethods: The expression of mTOR,raptor,S6K1,IRS1,AKT and GSK3β was analyzed after overexpression of OPG in primary liver cells of c57 mice and OPG^-/-mice.The mTOR inhibitor rapamycin was used to verify whether OPG regulates the level of p-AKT by regulating mTORC1 negative feedback pathway.Subsequently,primary cells from Raptor KO mouse liver were isolated and treated with Ad-OPG to detect changes in AKT and GSK3β.It can further confirm whether OPG regulates liver insulin signaling pathway by regulating raptor.OPG protein and Raptor protein in cells were stained by double-labeled immunofluorescence,and then Co IP was used to verify whether OPG affects its function by directly binding to Raptor protein.CIAP（calf intestinal alkaline phosphatase）and phos-tag gel were used to verify whether OPG phosphorylate raptor that affects mTOR function.Results: Overexpressed OPG levels in primary hepatocytes resulted in a significant increase in p-mTOR,p-S6K1,p-IRS1 ser levels,and a significant decrease in p-AKT,p-GSK3β levels;Knockdown of OPG expression in cells resulted in significantly decreasing mTOR,p-S6K1,and p-IRS1 ser levels,while the levels of p-AKT and p-GSK3β were significantly increased.The addition of rapamycin abolished the effects of Ad-OPG.The similar results were observed in Raptor knockout cells to the results with addition of rapamycin.The double-labeled immunofluorescence assay combined with Co IP results showed that the OPG protein interacted with the Raptor protein.The results of the Phos-tag gel test showed that the Raptor was significantly shifted and the shift disappeared after the addition of CIAP.Conclusion: The interaction between OPG protein and Raptor protein increases phosphorylation level of Raptor and thereby increases the activity of mTORC1.OPG affects hepatic insulin sensitivity and glucose metabolism through the Raptor/ mTORC1/S6K1/ IRS1ser/ AKT/ GSK3βpathway.