| Berberine(BBR),the major alkaloid of rhizoma coptidis,has been used for medicinal purposes in Chinese medicine since long.In diabetic mice and diabetic patients,BBR was shown to lower blood glucose,lose weight,improve glucose tolerance and insulin resistance.The biological action and its targets related to glucose and lipid metabolism is similar to those of silent information regulator 1(Sirtl),a deacetylase enzyme which also exerts anti-inflammatory effects and improves insulin sensitivity.The study on the role of Sirtl in berberine' regulating effects on glucose and lipid metabolism will provide a clue for research on diabetes in Chinese medicine and for potential mechanism of berberine' s multiple pharmalogical actions in 3T3-L1 adipocytes.Objective:To observe the role of Sirtl in berberine'anti-inflaimnatory property and regulating effects on glucose metabolism will be explored in Sirtl knockdown of 3T3-L1 adipocytes.Furthermore the effects of berberine on signaling molecules or transcription factors related to glucose metabolism which are controlled by Sirtl will be investigated.Methods:l)The WB and real time PCR method was used to detect the effect of Sirtl after treatment with different concentrations of berberine(1,5,10?Mo1/L)on the 3T3-L1 adipocytes.2)The 3T3-L1 adipocytes were divided into four groups:control group,BBR group,Ex527(or Iv-Sirtl)group and Ex527(or 1v-Sirtl)+ BBR group,treatment with at 24 hours,the medium was changed to DMEM containing 0.2%BSA and InM insulin for 12 h,The medium was removed and its glucose concentrations were determined by the glucose oxidase method.The amount of glucose consumption was calculated by the glucose concentrations of blank wells subtracting the remaining glucose in the cell-plated wells.3)The 3T3-L1 adipocytes were divided into four groups:control group,BBR group,Ex527(or lv-Sirtl)group and Ex527(or lv-Sirtl)+ BBR group,the LKB1 Ser428,AMPK-aThr172?ACCser79 phosphorylation protein and PGC-laacetylization detected by IP and WB method.4)The 3T3-L1 adipocytes were divided into six groups:control group,TNF-agroup,TNF-a+BBR group,Ex527 group,Ex527+TNF-agroup,Ex527+TNF-a+BBR group,treatment with at 24 hours,the medium was changed to DMEM containing 0.2%BSA and 1nM insulin 15min,replaced by PBS containing 30?M 2-NBDG and 1?pg/mlDAP2 Fluorescent probe for 2h,take pictures by high intension cell imaging and calculate the average fluorescence intensity of 3T3-L1 adipocytes.5)The 3T3-L1 adipocytes were divided into six groups:control group,TNF-agroup,TNF-a+BBR group,Ex527 or lv-Sirtl group,Ex527 or lv-Sirtl+TNF-agroup,Ex527 or lv-Sirtl+TNF-a+BBR group,the AKT ser473,IRS-1 Y612phosphorylation protein detected by WB method.Results:1)Berberine had up-regulated Sirtl on 3T3-L1 adipocytes at 1 and 5 ?Mol/L and berberine at 5?Mol/L were more significant(P<0.05);2)Berberine significantly increased basal glucose consumption of 3T3-L1 adipocytes,(P<0.05),the action of berberine increased basal glucose consumption was decreased when Sirtl were suppressed by Ex527 or knockdown by lv-Sirtl,(P<0.05).3)Berberine significantly increased the phosphorylation of LKB1 ser428,AMPK-?Thr172?ACC ser79,and decreased the acetylization of PGC-1? however,when the Sirtl were suppressed by Ex527 or knockdown by lv-Sirt1,the action of berberine was decreased.4)Berberine stimulated glucose uptake in 3T3-L1 adipocytes compared with the basal level,(P<0.05),and the action of berberine was decreased when Sirtl were suppressed by Ex527,(P<0.05).5)The proinflammatory TNF-a decreased phosphorylation of AKTser473,berberine caused the recovery of the decreased phosphorylation of AKT ser473,but the action of berberine was decreased when the Sirtl were suppressed by Ex527 or knockdown by lv-Sirtl.However,the phosphorylati.on of IRS-1 at the site of tyrosine 612 was not be affected before and after treatment by berberine.Conclusion:Berberine through a AMPK/AKT pathway improve glucose metabolism depending on Sirtl in 3T3-L1 adipocytes... |
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