The Role Of OPG In Browning Of White Adipose And Its Mechnism

PART I OPG KNOCKOUT AMELIORATES HIGH-FAT-DIET INDUCED OBESITY AND RELATED COMPLICATIONSObjective:To explore the effect of OPG on obesity and related complications in mice.Methods:High-fat diet last for 12 weeks from the age of 8 week was used to make obesity model in mice.The mice were divided into four groups,including standard-diet-fed wild-type mice group（SD-WT）,standard-diet-fed OPG knockout mice group(SD-OPG^-/-),high-fat-diet-fed wild-type mice group（HFD-WT）and high-fat-diet-fed OPG knockout mice group(HFD-OPG^-/-).The body weight were tested every week during making obesity model.At the age of 20 weeks,The energy metabolism assessment system was used to monitor heat in mice.Fasting glucose and food intake were tested.The tissues were weighted to calculate the ratio of tissue weight to body weight.HE staining was used to analyze the size of adipocyte.PCR was used to test the browning related genes and adipogenesis genes.Western blot was used to test protein levels of p-IR/IR and p-AKT/AKT in e WAT.Then,the m RNA expression levels of OPG in e WAT,i WAT and BAT of C57 male mice was detected by PCR,and the protein expression levels of OPG in three adipose tissues were detected by western blot.The m RNA expression levels of OPG in subcutaneous adipose tissue of C57 mice fed with standard diet,C57 mice fed with high fat diet,and db/db mice were analyzed by PCR.The protein expression level of OPG in subcutaneous adipose tissue was detected by western blot.Results:Compared with wild-type mice,OPG knockout mice had significantly lower body weight and no difference in food intake after standardized by body weight.Under high-fat diet,fasting blood glucose level in OPG knockout mice was lower than that of WT group.Compared with HFD-WT,The protein levels of P-IR/T-IR and P-AKT/T-AKT in e WAT increased in HFD-OPG^-/-.HFD-OPG^-/-showed lower ratio of tissue weight to body weight,reduced volume of fat cells,and enhanced m RNA level of UCP1,decreased m RNA level of AP2,unchanged m RNA level of PPARγ.The m RNA and protein expression levels of OPG in e WAT and i WAT were higher than in BAT.Compared with C57 mice fed with standard diet,the expression m RNA and protein levels of OPG in i WAT of high-fat-fed mice and db/db mice were significantly increased.Conclusions:OPG knockout can enhance energy expenditure,improve obesity and visceral fat insulin resistance induced by high-fat diet.OPG is highly expressed in e WAT and i WAT,and the expression of OPG is increased in i WAT of obese model.OPG knockout reduces obesity induced by high-fat diet,which may be related to increased energy expenditure and increased browning of white adipose tissue.Part II THE EFFECT OF OPG ON WHITE ADIPOSE BROWNING IN VIVOObjective: To clarify the effect of OPG on the browning of white adipose in vivoMethods: In vivo,with or without CL316,243 injection,12-week-old WT and OPG-/-were divided into four group: WT + Vehicle,OPG-/-+ Vehicle,WT + CL,OPG-/-+ CL.And energy metabolism assessment system monitors food intake,oxygen consumption,carbon dioxide production,respiratory entropy,heat and activity.i WAT sections were stained.The expression of browning-related genes such as UCP1,Prdm16,Pgc-1α and related signaling pathway proteins m TOR and AMPK in i WAT were detected by real-time quantitative PCR and western blot.8-week-old C57 mice were injected with Ad-GFP or Ad-OPG in i WAT situ,stimulated by CL316,243 injections,and divided into four groups: Ad-GFP + Vehicle,Ad-OPG + Vehicle,Ad-GFP + CL,and Ad-OPG + CL.The energy metabolism assessment system monitors food intake,oxygen consumption,carbon dioxide production,respiratory entropy,heat and activity.And i WAT sections were stained.The expression of browning-related genes such as UCP1,Prdm16,Pgc-1α and related signaling pathway proteins m TOR and AMPK in i WAT were detected by real-time quantitative PCR and western blot.Results: In vivo,after stimulated by CL316,243,the oxygen consumption,carbon dioxide production and heat of WT mice increased.UCP1 staining of i WAT and protein expression were enhanced.Under the stimulation of CL316,243,compared with WT mice,OPG-/-mice had increased oxygen consumption,carbon dioxide production and heat,while no significant difference in food intake was observed.i WAT UCP1 stainingand protein expression were enhanced.i WAT m RNA detection found that the expression levels of UCP1,Tmem26,and Pgc-1α were enhanced,and further protein detection results showed that p-m TOR/m TOR,p-S6K/t-S6 K proteins of OPG-/-mice were increased after CL316,243 stimulation.And there was no significant difference in p-AMPK/AMPK protein expression level.The results of in situ overexpression of OPG in i WAT showed that under the stimulation of CL316,243,OPG overexpression reduced the oxygen consumption,carbon dioxide production and heat of mice,and the UCP1 staining and protein expression of i WAT decreased.The m RNA test and the protein detection results showed that,after CL316,243 stimulation,OPG overexpression decreased the m RNA levels of UCP1 and Pgc-1α,and protein levels of p-m TOR/m TOR,p-S6K/t-S6 K in i WAT,while pAMPK/AMPK and p-ERK/t-ERK expression levels had no significant difference.Conclusions: In vivo,OPG knockout can enhance energy consumption and white fat browning under β-adrenergic receptor activation;i WAT overexpression of OPG in situ decrease energy consumption and white fat browning caused by β-adrenergic receptor activation;OPG may play this role through the m TOR/S6 K signaling pathway.Part III THE EFFECT OF OPG ON WHITE ADIPOSE BROWNING IN VITROObjective: To clarify the effect of OPG on the browning of white fat in vitroMethods: In vitro,SVF of i WAT from wild-type mice and OPG knockout mice was extracted.After SVF was induced and stimulated by CL316,243 to primary adipocytes,oil red staining was used to test lipid formation,and real-time quantitative PCR was used to detect the expression of browning-related genes,immunofluorescence was used to detect the expression of UCP1,and Western blot was used to detect the changes of browning-related proteins UCP1 and related signaling pathway proteins.On the other hand,SVF of i WAT from C57 mice was extracted,After SVF was induced and stimulated by CL316,243 to primary adipocytes,oil red staining was used to observe the formation of lipid droplets,and real-time quantitative PCR was used to detect brown Related gene expression,immunofluorescence detection of UCP1 expression,western blot detection of browning-related protein UCP1 and related signaling pathway protein changes.Results: In vitro,the expression levels of browning-related genes UCP1,Pgc-1α,Tmem26 m RNA and UCP1 protein in primary adipocytes from OPG-/-i WAT were increased compared with WT mice after induction and stimulation by CL316,243.Lipid droplet formed successfully in SVF from WT and OPG-/-.The protein levels of p-m TOR/t-m TOR and p-S6K/tS6 K increased,and there was no significant difference between pAMPK/AMPK and p-ERK/t-ERK.Compared with the control group,the expression of browning-related genes UCP1,Pgc-1α,Tmem26 m RNA and UCP1 protein of the SVF in the OPG overexpression group after induction and stimulation by CL316,243 were reduced,p-m TOR/t-m TOR and pS6K/t-S6 K protein levels were reduced,while p-AMPK/AMPK had no significant difference.Conclusion: In vitro,under induction and stimulation of CL316,243,OPG knockout enhances browning,enhances the m TOR/S6 K signaling pathway;OPG overexpression inhibits browning,inhibits m TOR/S6 K signaling pathway.Part IV THE MECHANISM OF WHITE FAT BROWNING BY OPGObjective: To explore the molecular mechanism of OPG on regulation of white fat browningMethods: SVF derived from i WAT of WT or OPG-/-mice,induced and stimulated by CL316,243,treated with rapamycin,and then detected on browning-related proteins and genes by PCR and western blot.Furthermore,ad-Cre was used in the SVF from Raptorloxp / loxp i WAT to knock out Raptor.And the SVF was induced and stimulated by CL316,243,with or without OPG inhibition,PCR was used to detect browning related genes,western blot to browning related proteins.Verify that OPG regulates white fat browning through m TORC1 signaling pathway.Results: In primary adipocytes stimulated by CL316,243 from OPG-/-mice,the m RNA levels of browning-related genes UCP1,Pgc-1α,Tmem26 and UCP1,Pgc-1α protein expression increased,but after treatment with Rapamycin,the m RNA levels of UCP1,Pgc-1α and Tmem26 and the protein levels of UCP1,Pgc-1α decreased.Similarly,primary adipocytes stimulated by CL316,243,after knockout of Raptor,the m RNA levels of UCP1,Pgc-1α,and UCP1,Pgc-1α protein in OPG inhibition group were reduced.Conclusion: Rapamycin or Raptor knockout can reverse the browning enhancement caused by OPG inhibition.OPG may regulate white fat browning mediated by β-adrenergic signaling through the m TORC1/S6KPgc-1α signaling pathway.