| Objectives Obesity is the main reason to induce insulin resistance(IR),which is the core pathological mechanism in the procedure of obesity induced type 2Diabetes Mellitus(T2DM)and related disorders.It is necessary to intervene obesity in early stage,which can not only improve IR,but also prevent it from suffering T2 DM.Hypothalamic is the central of regulating food intake,the POMC and NPY neuros in arcuate nucleus(ARC)involve in the energy homeostasis by secrete peptides and hormones.As a deacetylase,SIRT1 plays a vital part in energy homeostasis by regulating the acetylation level of Fox O1 which is the substrate of SIRT1.In our study,we use the high‐fat diet induced IR and obesity rat(HIOR)as model,and investigated the effect of electroacupuncture(EA)on SIRT1 and Fox O1 of hypothalamic,observed the orexis‐related peptide which is regulated by Fox O1 in different acetylation levels in HIOR.Trying to illustrate the possible central mechanism of EA on improving the IR and obesity via regulating SIRT1/Fox O1 signaling pathway,and provide experimental evidence for application on obesity and IR‐related disorders by acupuncture clinically.Methods Ninety SPF Wistar rat,8 weeks,were obtained from certificated institution.Fifteen rat were selected randomly and feed with normal diet for 8 weeks,and 10 from 15 selected rats were allocated to normal group(NG).The rest of75 rats were feed with high‐fat diet for 8 weeks to building model.Fifty rats were randomly select from 61 rats which were build and identified as HIOR model,and allocated to model group(MG),electroacupuncture group(EAG),sham operation group(SOG),electroacupuncture and inhibitor group(EIG)and agonist group(AG).Each group have 10 rats.In the meantime,the euglycemic hyperinsulinemia clamp technique were performed to assess the level of insulin sensitivity.After allocation,rats in SOG,EIG,AG were permanently implanted with guide cannulas for further intervention.All the rats were recovered for 1 weeks after surgery.Rat in EAG and EIG received EA treatment at acupoint ST36,ST40,CV4 and CV12 for 3 times a week,frequency are 2Hz,electric current were set at 1m A.Rats in SOG were intracerebroventricular injected with artificial cerebrospinal fluid;Rat in EIG receive intracerebroventricular injection with EX‐527 which is an antagonist of SIRT1;Rats in AG receive intracerebroventricular injection with SIRT1720 which is an agonist of SIRT1.All the rat received intracerebroventricular injection at the same time with EA.Body mass,body length,food intake dose,Lee’s index,fast blood glucose(FBG)and postprandial blood glucose(PBF)were measured before and after2,4,6,8 weeks separately.IPITT and IPGTT were tested after 6 weeks.The euglycemic hyperinsulinemia clamp technique were measured in 3 rats selected randomly from each groups.Serum were collected form heart for insulin detecting before executed.Fresh hypothalamic tissue were deprived to detect the protein expression of SIRT1、Fox O1、Ac‐Fox O1、POMC and NPY by Western Blotting,detect m RNA level of SIRT1、Fox O1、Ac‐Fox O1、POMC and NPY by RT‐q PCR.Rat brain were perfusion fixed with paraformaldehyde before executed and then sliced into tissue section.Double‐ labeled immunofluorescence were apply to investigate the co‐expression of SIRT1/Fox O1,SIRT1/Ac‐Fox O1,Fox O1/POMC and Ac‐Fox O1/NPY protein.All the data collected above were analyzed statistically.Results1.The effect of EA on Body mass,body length,food intake dose,Lee’s index and insulin sensitivity of HIOR.Body mass in all groups increased persistently during intervention.Compared to NG,HIOR in MG have higher body mass before and after intervention.After 2 weeks,body mass of rats in AG were emerging lower than that in MG.After 6 weeks,body mass of rats in EAG were lower than that in MG.After 8 weeks,body mass of rats in EAG,AG,and EIG were all lower than that in MG.body mass in SOG did not show significant difference with MG during whole intervention.Compared to EAG,body mass in AG were lower and EIG did not show difference.Compared to NG,Lee’s index in MG were higher significantly before and after intervention.After 8 weeks treatment,Lee’s index in EAG and AG were decrease comparing to MG.Compared to EAG,Lee’s index in EIG were increased and AG did not show difference.Compared to NG,food intake dose were increased significantly in MG.Compared to MG,EAG,EIG and AG were decreased significantly.Compared to EAG,food intake dose in EIG were increased and AG were decreased.There were no significant difference in FBG during all groups.After 8weeks,comparing to NG,the PBG in MG were increased.Compared to MG,PBG in EAG,EIG and AG decreased significantly.There were no significant difference between SOG and MG.Compared to EAG,FBG in EIG and AG did not show difference.The serum insulin level in NG,EAG,EIG and AG were significantly lower than that in MG and SOG,while insulin level in EAG were lower than that in EIG and AG lower than EAG.In IPGTT,FBG in all group did not show significant difference.The increase value of PBG in EAG and AG were lower than that in MG and SOG after 30 min;while the difference were nonexistent after 120 min.In IPITT,the decrease value of PBG in NG,EAG and AG were significant lower than that in MG and SOG after 30 min,while PBG in NG,EAG and AG also lower than that in MG and SOG.Compared to NG,glucose infusion rate(GIR)in MG were significant decreased before treatment.There were no significant difference during the HIOR of each group.After 6 week’s treatment,GIR in rats of NG did not show alternation,while in EAG and AG were significant higher than that in MG and SOG.GIR in AG were higher than that in EAG.2.Effect of EA on protein expression of SIRT1/Fox O1 and orexis‐related peptide in HIOR hypothalamusCompared to NG,protein expression of SIRT1 and Fox O1 in HIOR hypothalamus decreased significantly.After 8 week’s intervention,compared to MG,protein expression of SIRT1 in AG were recovered to normal level,and the level of Fox O1 increased either.Protein expression of SIRT1 and Fox O1 in EAG were higher than that in MG.Protein expression of SIRT1 in EIG were higher than that in MG,and lower than that in EAG.Compared to MG,protein expression of SIRT1 and Fox O1 in SOG did not show significant alternation.Protein expression of Fox O1 in AG were higher than that in MG,and did not show difference with EAG.The protein expression of AC‐Fox O1 in HIOR hypothalamus of all groups show an opposite tendency to SIRT1.Compared to NG,protein expression of Fox O1 increased significantly in MG.Compared to MG,EA and agonist could decrease the protein expression of Fox O1 in HIOR.Protein expression of Fox O1 in EIG were lower than that in MG,and higher than that in EA.There are no significant difference between MG and SOG.Ac‐Fox O1 in AG were higher than that in NG and lower than that in EAG.Protein expression of POMC in HIOR hypothalamus were highest in all groups.Compared to NG,protein expression of POMC in EAG and AG increased significantly.Protein expression of POMC in EIG were lower than that in EAG,and higher than that in MG.There were no significant difference between MG and SOG.Compared to EAG,POMC in EIG were lower and did not show difference in AG.Compared to NG,protein expression of NPY in HIOR hypothalamus were increased significantly in MG.Compared to MG,protein expression of NPY in EAG and AG were decreased significantly and NPY in EIG and SOG did not show significant alternation.Compared to EAG,NPY in AG were lower and did not show difference in EIG.3.Effect of EA on protein co‐expression of SIRT1/FoxO1 and orexis‐related peptide in HIOR hypothalamus ARCDouble‐labeled IF of SIRT1/Fox O1 showed that cell counting of SIRT1 and Fox O1 had positive correlation.Compared to NG,expression of SIRT1 and Fox O1 in ARC decreased significantly in MG.Compared to MG,expression of SIRT1 and Fox O1 in ARC increased significantly in EAG,EIG and AG,while not altered in SOG.Compared to EAG,SIRT1 and Fox O1 in EIG were lower,and did not show difference in AG.Double‐labeled IF of SIRT1/Ac‐Fox O1 showed that cell counting of SIRT1 and Ac‐Fox O1 had negative correlation.Compared to NG,expression of Ac‐Fox O1 were increased significantly in MG and SOG.Compared to MG,expression of Ac‐Fox O1 were decreased significantly in EAG,EIG and AG,while not altered in SOG.Compare to EAG,Ac‐Fox O1 in EIG were higher and in AG were lower.Double‐labeled IF of Fox O1/POMC showed that cell counting of Fox O1 and POMC had positive correlation.Compare to NG,POMC in ARC decreased significantly in MG.Compared to MG,POMC in ARC increased significantly in EAG,EIG and AG,while POMC did not show alternation in SOG.Compared to EAG,POMC in EIG and AG did not show difference.Double‐labeled IF of Ac‐Fox O1/NPY showed that cell counting of Ac‐Fox O1 and NPY had positive correlation.Compared to NG,NPY in ARC were increased significantly in MG.Compare to MG,expression of NPY were decreased in EAG,EIG and AG,while NPY did not show alternation in SOG.Compared to EAG,NPY in EIG were lower and did not show difference in AG.4.Effect of EA on gene expression of SIRT1/Fox O1 and orexis‐related peptide in HIOR hypothalamus.After 8 week’s treatment,Compared to NG,SIRT1 m RNA decreased significantly in MG.Compared to MG,SIRT1 m RNA did not show significant alternation in EAG.SIRT1 m RNA in SOG were lower than that in MG,EIG were lower than SOG.SIRT1 m RNA in AG were higher than MG and did not show difference to NG.Compare to NG,Fox O1 m RNA decreased significantly in MG.Compared to MG,Fox O1 m RNA in EAG,SOG and AG did not show significant alternation.Fox O1 m RNA in EIG were lower than that in MG.Compared to NG,Ac‐Fox O1 m RNA increased significantly in MG.Compared to MG,Ac‐Fox O1 m RNA in EAG,SOG,EIG and AG did not show significant alternation.Compared to NG,POMC m RNA decreased significantly in MG.Compared to MG,POMC m RNA increased significantly in EAG,EIG and AG,while not altered in SOG.Compared to EAG,POMC m RNA in EIG were lower and in AG were higher.Compared to NG,NPY m RNA increased significantly in MG.Compared to MG,NPY m RNA decreased in EAG,EIG and AG,while not altered in SOG.Compared to EAG,NPY m RNA did not show difference in EIG and AG.Conclusions1.After 8 weeks interventions of EA,the body mass increasing,food intake dose and Lee’s index of HIOR were suppressed,which indicated that EA can improved the IR and obesity by inhibiting food intake.Meanwhile,the level of PBG and serum insulin were both decreased after EA treatment.Additionally,the IPGTT 、 IPITT and GIR were all improved after EA for 6weeks.The results suggested that EA can increase the insulin sensitivity besides controlling obesity.Combined with SIRT1 inhibitor,the effective of EA were antagonized partially.The phenotypes of controlling body mass,suppressing food intake and increasing insulin sensitivity also appeared after SIRT1 agonist intracerebroventricular injection.All the results suggested that EA have the function of controlling obesity and increasing insulin sensitivity via up‐regulating SIRT1.2.After 8 weeks interventions of EA,protein expression of SIRT1,Fox O1 and POMC in hypothalamus ARC were up‐regulated,while that of Ac‐Fox O1 and NPY were down‐regulated.Combined with SIRT1 inhibitor,EA induced up‐regulated protein expression of SIRT1,Fox O1 and POMC in hypothalamus ARC were antagonized partially,as well as the down‐regulated protein expression of Ac‐Fox O1 and NPY.The similar phenotypes that protein expression of SIRT1,Fox O1 and POMC in hypothalamus ARC were up‐regulated and Ac‐Fox O1 and NPY were down‐regulated existed after SIRT1 agonist intracerebroventricular injection.All the results suggested that EA can up‐regulate the protein expression of SIRT1 in hypothalamus ARC specifically,which involved in mechanism of improving IR and obesity.SIRT1 induced activity of deacetylation can reduce the acetylation level of Fox O1,which can suppress the protein expression of orexigenic peptide NPY and activate the protein expression of anorexigenic peptide POMC and reduce food intake,improve IR and obesity finally.3.After 8 weeks interventions of EA,m RNA level of SIRT1,Fox O1,Ac‐Fox O1 in hypothalamus did not altered comparing to MG,while the m RNA level of POMC were up‐regulated and NPY were down‐regulated.Combined with SIRT1 inhibitor,m RNA level of SIRT1 and Fox O1 were down‐regulated after EA treatment,while Ac‐Fox O1 did not altered.The EA induced up‐regulating in POMC m RNA and down‐regulating in NPY m RNA were antagonized partially.After the application of SIRT1 agonist intracerebroventricular injection,SIRT1 m RNA were up‐regulated and Fox O1,Ac‐Fox O1 did not show alternation,while the phenotypes of up‐regulating in POMC m RNA and down‐regulating in NPY m RNA were still existed.This results suggested that SIRT1 inhibitor and agonist can down or up‐regulated the gene expression of SIRT1,but EA cannot.The SIRT1 induced activity of deacetylation cannot affect the gene expressing of Fox O1 and Ac‐Fox O1,but the different levels of deacetylation Fox O1 can regulate the gene expression of downstream orexigenic or anorexigenic peptide such as POMC and NPY.The results suggested that the molecular biological target of EA are focus on post‐transcriptional control not on gene expression. |
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