The Effects Of Central Over-expression BMP9 On Hepatic Glucose Metabolism And The Mechanism

PART ? THE HYPOTHALAMIC EXPRESSION OF BMP9 IN MICEAim: To detect the expression of hypothalamic BMP9 in mice on normal chow diet and high fat diet and db/db mice.Methods: Mice fed with normal chow diet and high fat diet for 12 weeks,and ob/ob mice fed with normal chow diet respectively were chosen and then randomly divied into these groups: normal chow diet group(NCD),high fat diet group(HFD)and db/db mice,n=4,respectively.The expression of BMP9 in hypothalamus was detected by Western blot.Results: The hypothalamic expression of BMP9 protein was decreased significantly in C57BL/6J HFD group compared to NCD group(p<0.01).Meanwhile,the BMP9 protein levels was decreased significantly in db/db mice compared with NCD group(p<0.01).Conclusion: The high fat diet and obese can result in a decreasing in hypothalamic BMP9.PART ? THE EFFECTS OF CENTRAL OVER-EXPRESSION BMP9 ON HEPATIC GLUCOSEAim: To investigate the specific role hypothalamic BMP9 in the regulation of hepatic glucose production.Methods: C57BL/6J mice were randomly divided into two groups fed with normal chow diet(NCD,n=40)or high fat diet(HFD,n=40).At the end of 12 th weeks,all mice were received intracerebroventricular(icv)Ad-BMP9 or Ad-GFP and divided into NCD+ Ad-GFP group(NCD+ Ad-GFP,n=20),NCD+ Ad-BMP9 group(NCD+Ad-BMP9,n=20),HFD+ Ad-GFP group(HFD+ Ad-GFP,n=20),and HFD+Ad-BMP9 group(HFD+Ad-BMP9,n=20).Hypothalamic BMP9 protein levels were measured by Western blot.The fasting glucose,fed glucose,body weight,food intake,blood pressure and plasma triglyceride(TG),total cholesterol(TC),free fatty acid(FFA),alanine aminotransferase(ALT)and aspartate aminotransferase(ALT)were detected.Glucose tolerance and insulin sensitivity were analyzed by intraperitoneal glucose tolerance test,inulin tolerance test and hyperinsulinemic-euglycemic clam technique.Results: In normal chow mice,after i.c.v administration of Ad-BMP9,hypothalamic BMP9 protein levels was significantly higher.Similarly,hypothalamic BMP9 protein levels was also significantly higher in high fat diet mice.After icv Ad-BMP9 or Ad-GFP,the fasting/fed glucose,body weight,food intake,blood pressure,and TG,TC,FFA,ALT,AST had no significant statistical differences between NCD+ Ad-BMP9 group and NCD+ Ad-BMP9 group(p>0.05).Compared with NCD+ Ad-GFP group,these indicators above significantly higher in HFD+ Ad-GFD group(p<0.05).However,these indicators above significantly lower in HFD+ Ad-BMP9 group compared to HFD+ Ad-GFP group(p<0.05),except for ALT and AST).There was no significant difference in RER and VO2 between NCD+Ad-BMP9 group and NCD+ Ad-GFP group,while VO2 were significantly higher in HFD+Ad-BMP9 group than that in the HFD+ Ad-GFP group(p<0.05),and RER was significantly lower in HFD+Ad-BMP9 group compared with HFD+ Ad-GFP group(p<0.05).In the clamp steady period,glucose infusion rate(GIR),glucose clearance rate(GRd)and hepatic glucose production(HGP)had no statistical difference between NCD+ Ad-BMP9 group and NCD+ Ad-GFP group(p>0.05);but HFD+Ad-BMP9 group had higher GIR and GRd compared to HFD+ Ad-GFP group(p<0.01),and lower HGP(p<0.01).There was no significant difference in adipose tissue and muscle glucose uptake between NCD+Ad-BMP9 group and NCD+ Ad-GFP group(p>0.05).There was no significant difference in adipose tissue glucose uptake between HFD+Ad-BMP9 group and HFD+ Ad-GFP group(p>0.05),while compared with HFD+ Ad-GFP group,HFD+Ad-BMP9 group had higher muscle glucose uptake(p<0.05).Conclusion: Central over-expression BMP9 reverses diet-induced insulin resistance and decrease hepatic glucose production.PART ? THE EFFECTS OF CENTRAL OVER-EXPRESSION BMP9 ON HYPOTHALAMIC REALTIVE SIGNALINGS AND HEPATIC GLUCONEOGENIS ENZYME IN MICE ON A HIGH FAT DIETAim: To detected whether intracerebroventricular Ad-BMP9 administration in the hypothalamus affects hypothalamic insulin signaling and mTOR pathway and controls glucose homeostasis by suppressing hepatic gluconeogenesis enzymes in mice fed with high fat diet.Methods: Twenty C57BL/6J mice were randomly divided into normal chow diet group(NCD,n=10)and high fat diet group(HFD,n=10)for 12 weeks.At the end of 12 th week,Ad-BMP9 or Ad-GFP was injected into right lateral ventricles using a microsyringe.And they were randomly divided into normal chow diet+ Ad-GFP group(NCD+ Ad-GFP,n=5),normal chow diet+ Ad-BMP9 group(NCD+ Ad-BMP9,n=5),high fat diet + Ad-GFP group(HFD+ Ad-GFP,n=5),and high fat diet +Ad-BMP9 group(HFD+Ad-BMP9,n=5).Hypothalamic NPY,Ag RP,POMC m RNA and hepatic PEPCK,G6 Pase m RNA were measured by realtime fluorescence quantitative PCR.Hypothalamic Ins R,AKT,mTOR,p70S6 K protein and hepatic PEPCK,G6 Pase protein were measured by Western blot.Hypothalamic c-fos expression levels were detected by.immunohistochemistry and hypothalamic ARC PIP3 information was measured by immunofluorescence.Results: Compared with NCD+ Ad-GFP group,NCD+Ad-BMP9 group showed no significant difference in hypothalamic NPY,Ag RP,POMC m RNA expression(p>0.05).HFD+Ad-BMP9 group had lower expression of hypothalamic NPY m RNA compared to HFD+ Ad-GFP group(p<0.01),and had higher expression of hypothalamic POMC m RNA compared to HFD+ Ad-GFP group(p<0.01).There was no significant difference in hypothalamic AKT,Ins R,mTOR,p70S6 K protein phosphorylation and hepatic PEPCK,G6 Pase m RNA and protein level between NCD+Ad-BMP9 group and NCD+ Ad-GFP group(p>0.05).However,HFD+Ad-BMP9 group had higher expression of hypothalamic AKT,Ins R,mTOR,p70S6 K protein phosphorylation levels,and had lower hepatic PEPCK,G6 Pase m RNA and protein compared to HFD+ Ad-GFP group(p<0.05 or p<0.01).NCD+Ad-BMP9 group and NCD+ Ad-GFP group had some Fos positive cells in the nucleus of hypothalamus,but there was no significant difference between the two groups(p>0.05).Compared with HFD+ Ad-GFP group,HFD+Ad-BMP9 group had more Fos positive cells(p<0.05).Qunatitiative analysis of basal PIP3 levels in hypothalamic ARC cells,there was no difference between HFD+ Ad-BMP9 group and HFD+ Ad-GFPgroup.After stimulation with insulin,moderate and high levels of immunoreactive PIP3 were significantly higher in HFD+ Ad-BMP9 mice compared with HFD+ Ad-GFP mice.Conclusion: On a normal chow diet,central BMP9 overexpression can not affects hypothalamic insulin signaling pathway and mTOR signaling pathway,and hepatic gluconeogenesis enzyme.While overexpression of BMP9 in the hypothalamus resulting from intracerebroventricular injection of adenov Ins Rus expression BMP9 improve hypothalamic insulin resistance and hepatic gluconeogenesis in mice fed with high fat diet and was accomplished by activates hypothalamic mTOR pathway and increases c-fos and PIP3 formation.PART IV CENTRAL OVER-EXPRESSION BMP9 IMPROVE DIET-INDUCES INSULIN RESISTANCE THROUGH A CENTRAL mTOR PATHWAYAim: To investigate whether central overexpression BMP9 improved hypothalamic insulin resistance and hepatic glucose production dependent on the hypothalamic mTOR pathway.Method: Twenty C57BL/6J mice fed with high fat diet for 12 weeks,and then a cannula was implanted in right lateral ventricles allowed for Ad-BMP9 and rapamycin injection.They were randomly divided into HFD+ Ad-GFP group(n=10)and HFD+Ad-BMP9 group(n=10).A 7-day postadenov Ins Ral injection,food was removed 6h and 2 ?l of i.c.v.rapamycin at a concentration of 25ug/2?l.These mice were divided into Ad-GFP+DMSO group(n=5),Ad-GFP+ rapamycin group(n=5),Ad-BMP9+DMSO group(n=5),Ad-BMP9+ rapamycin group(n=5).Glucose tolerance and insulin sensitivity were evaluated by glucose tolerance test and insulin tolerance test.Hepatic PEPCK,G6 Pase m RNA and were measured by PCR.Hypothalamic mTOR,p70S6 K protein and hepatic PEPCK,G6 Pase protein were measured by Western blot.Results: Glucose tolerance test and insulin tolerance test showed that Ad-BMP9+DMSO group had lower blood glucose and the area under the curve compared to Ad-GFP+ rapamycin group,Ad-GFP+DMSO group and Ad-BMP9+ rapamycin group,respectively(p<0.05).Meanwhile,Ad-BMP9+DMSO had significantly higher in expression of mTOR,p70S6 K protein in the hypothalamus that in other groups(p<0.05 or p<0.01),and significantly lower in expression of PEPCK,G6 Pase m RNA and protein in liver(p<0.05 or p<0.01).Conclusion: Central overexpression of BMP9 improves hypothalamic insulin resistance and decreases hepatic glucose production in mice on a high fat diet and that activate hypothalamic mTOR pathway.