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Experimental Research On Molecular Mechanism Of Berberine On Enhancing Insulin Secretion And Improving Insulin Resistance

Posted on:2010-08-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z S WangFull Text:PDF
GTID:1114360275986980Subject:Traditional Chinese Medicine
Abstract/Summary:PDF Full Text Request
Part 1 Berberine as a novel glucokinase activator modulatesinsulinoma cell function【Objective】To explore the effect of berberine on insulin secretion on NIT-1 cells line stimulatedwith different concentration of glucose, and to detect the effects of berberine on the activityand expression of glucokinase to reveal the potential mechanism of promoting insulinsecretion of berberine in vitro.【Methods】NIT-1 cells were stimulated with different concentration of glucose (high concentrationat 16.5 mM and low concentration at 5.5 mM) and were treated with different concentrarionof berberine, while biotin was used as positive control. The concentration of insulin wasmeasured with radioimmunoassay. The glucose utilization of NIT-1 cells was detected withliquid scintillation counting, while cellular ATP content with bioluminescence technique,glucokinase (GK) activity with enzymatic analysis respectively. GK and Glucokinaseregulatory protein (GKRP) expression were determined with Western blot, and GKtranslocation from nucleus to cytoplasm were measured with immunofluorescenece.【Results】Compared to blank group, berberine could promote GSIS, fercilitate glucose utilization,increase ATP content and GK activity, it could can improve GK expression, whereas theGKRP expression was inhibited, GK translocation from nucleus to cytoplasm was enhancedwhen cells were stimulated with high concentration of glucose, there were no significantdeviation for these effects when compared to biotin-a reagent which was been confirmed asGK activator. However, when NIT-1 cells were stimulated with low concentration ofglucose, all these activities of berberine or biotin were not been detected in comparison with blank group.【Conclusion】Berberine could promote GSIS, this action may be closely related to that berberineworked as GK activator, thus increasing the expression of GK but decreasing the expressionof GKRP, improving GK translocation from nucleus to cytoplasm thereby augmente GKactivity and promote insulin secretion ultimately when cells were stimulated with highconcentration of glucose. Part 2 Ameliorative Effect of berberine on endoplasmicreticulum stress in periphery organs of T2DM rats induced byhigh sucrose-fat diet and low-dose STZ【Objective】To observe the effects of berberine on glycolipids metabolism in rat with type 2diabetes mellitus (T2DM) induced by high sucrose-fat diet and low-dose STZ, and to detectthe effects of berberine on the expression of markers of endoplasmic reticulum stress andproteins related with insulin signal transduction in liver and adipose tissue to reveal themechanism of berberine on the therapeutic mechanism of type 2 diabetes mellitus.【METHODS】The rats with T2DM were induced by high sucrose-fat diet and the intravenousinjection of low-dose streptozotocin (STZ, 15mg/kg). Then modeled diabetic animals weredivided into model, berberine and 4-phenyl butyric acid (PBA) group, normal rats fed withcommon chow were designated as normal group. Six weeks later, the oral glucose tolerancetest (OGTT) was performed in all animals, the changes of routine body weight, fastingblood glucose (FBG), fasting serum insulin (FINS), total cholesterol (TC), triglyceride(TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) were measuredrespectively, the expression of JNK and c-Jun-phosphorylation,total IRS-I and AKT, IRS-1ser307 and AKT phosphorylation were detected with Western blot, the expression ofGRP78和ORP150 were measured with RT-PCR.【RESULTS】Compared with the model group, the result of OGTT was improved, the levels of thebody weights and FBG was decreased, FIN was increased, while TC, TG, LDL weredecreased and HDL was enhanced, meanwhile, the activity of JNK was reduced, GRP78and ORP150 transduction were down-regulated significantly after treated with berberine.Berberine could inhibite IRS-1 Ser307 after stimulation with insulin. It also enhanced the expression of AKT Ser473 obviously; there were no significant difference of these effectsas compared to PBA group.[CONCLUSION]It is suggested that the therapeutic effects of berberine on T2DM might be related to itsability of alleviating endoplasmic reticulum stress and improve insulin resistance. Part 3 Berberine reduces endoplasmic reticulum stress andimproves insulin signal transduction in Hep G2 cells【Objective】This study was carried out to investigate the effect of berberine on glucose metabolismon Hep G2 cells on the condition of endoplasmic reticulum stress induced withtunicamycine, and to detect the effects of berberine on markers of endoplasmic reticulumstress and insulin resistance to reveal the possible molecular mechanism of berberine onimprove insulin resistance.【Methods】Hep G2 cells were exposed to tumicamycin to induce tthe endoplasmic reticulum stress,the cells were pretreated with berberine or PBA. Glucose metabolism were measured withglucose oxidase method, the markers of ER stress-JNK were assessed with Western blot.The chaperones of ER stress -GRP78, ORP150 were detected with real time-PCR.Moreover, Ser307 phosphorylations of IRS-1, Ser473 phosphorylation of AKT weredetermined with Western blot.【Results】Berberine could enhance glucose uptake and inhibit glucose production when Hep G2cells were stimulated with insulin after the cells were exposed to tunicamycineo Meanwhile,the activity of JNK was blocked, Ser307 phosphorylations of IRS-1 was decreased whileasSer473 phosphorylation of AKT were increased, while GRP78 and ORP150 transductionwere down-regulated when cells wre treated with berberine.【Conelution】Berberine could alleviate the endoplasmic reticulum stress and insulin resistance inHep G2 cells induced with tunicamycine, the potential therapeutic mechanism of berberinemight be related with its inhibiting the activity of JNK and the transduction of GRP78 and ORP150. Therefore, it is suggested that berberine might improve ER folding capacity, andreduce IRS-1 Ser307 phosphorylation and enhance AKT Ser473 phosphorylation. Thus wepresume that the antidiabetic effect of berberine may be related to the reduction of ERstress and improvement of insulin signal transduction in Hep G2 cells.
Keywords/Search Tags:Berberine, Biotin, Islets cell, NIT-1, Glucokinase, Type 2 diabetes mellitus, Target tissues, Endoplasmic reticulum stress, Insulin resistance, Hep G2 cells, JNK, IRS-1 Ser307
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