Study On The Mechanism Of Glucose Uptake By Microtubule System-based PKC ? Translocation Depending On SIN1

Objectives: For the improvement of human living quality,sedentary lifestyle and the outcome of aging society,the rate of type 2 diabetes mellitus is rising rapidly.Many factors interact together and lead to diabetes onset.It is generally believed that insulin resistance is the fundamental of type 2 diabetes mellitus.Insulin resistance generally refers to a pathology state that the normal dosage of insulin has lower effect than normal biological effect.Three kinds of situations can cause insulin resistance and backward acceptor flaw has closely relationship with the abnormal message delivery in insulin signaling pathways.In normal circumstances,after insulin stimulating,the signal message transfers into the cells step by step and activates a series of cascade process.Irregularity of insulin signal transduction pathway results in insulin resistance and reduced insulin biological effects.As glucose utilization reduction,organism appears glymetabolism disorder.Therefore,it is important to explore the molecular mechanism of insulin resistance in cells.Protein kinase C zeta(PKC ?)belongs to the family of atypical PKC.PKC ? located in downstream of PI3 K signaling pathways,under the stimulus of extracellular stimulation factors,PKC ? takes part in a variety of intracelluar processes and mediates the physiological function of cells.Under the stimulus of insulin,PKC ? regulates the downstream proteins AMPK and influences cells glucose uptake.Therefore,PKC ? is one of the important factors that affect cells glucose balance,it is important significance to explore the activation mechanism of PKC ?.Methods: 1.After siRNA transfection,glucose oxidase method was used to measure the glucose uptake rate after SIN1 knockdown;2.Immune coprecipitation and confocal fluorescence was used to explore whether there is a combination between SIN1 and PKC ?;plasmid transfection was used to knockout SIN1 pleckstrin homology domain,then the immune coprecipitation method was used to detect whether this union still exists;3.To make it clear of the effect of SIN1 on PKC ? activation,the western blotting was performed to detect the phosphorylation and translocation of PKC ? when SIN1 was knocked down;4.Plasmid tranfection was used to overexpress the SIN1,and the PKC ? phosphorylation and translocation was observed.5.Immune coprecipitation and confocal fluorescence were used to explore whether ?-Tubulin combines with PKC ?,the Hep G2 cells was stimulated in 0 min,1 min,5 min,10 min,20 min,30 min respectly,to observe the changed combination between SIN1 and PKC ?,and between Tubulin and PKC ?;6.After 4 hours intervention by paclitaxel or Nocodazole respectively,cytoplasmic membrane separation assay was used to study the effect of Tubulin destruction on PKC ? transposition,and coffin phosphorylation.Results: When SIN1 knockdown,glycemic uptake rate was decreased,glycemic uptake rate was decreased from 35.27%±2.77% to 30.83%±2.34%(p<0.05)with the stimulus of insulin and decreased from 39.95%±3.20% to 32.26%±3.61%(p<0.05)in control group,further data analysis showed that SIN1 knockdown cells were not sensitive to insulin stimulus;Both coprecipitation and immune-confocal datas suggest that SIN1 combined with PKC ?,the combination relationship keep the same before and after insulin stimulation.After SIN1 PH domian knockout,the combination was weakened;in SIN1 knockdown cells the PKC ? phosphorylation and transposition were reduced.Immune coprecipitation and confocal results hint that at Tubulin combined with PKC ? after insulin stimulation.After PTX and NDZ treatment,PKC ? transposition is restrained,coffin phosphorylation was reduced.Conclusions: Our research shows that in resting state,SIN1 has a stable combination relationship with PKC ? by PH functional domains;with the stimulus of insulin,the combination still exist,and co-locate in cells membrane.After SIN1 knockdown,the glycemic uptake rate was reduced;overexpress of SIN1 improved the activation of PKC ?,it implies that SIN1 influences the PKC ? phosphorylation and translocation and affects cell sugar uptake,Microtubule also participates in PKC ? translocation and regulate cofilin phosphlation based on tubulin assamble and disasemble.Under the stimulus of insulin,SIN1 may be active by PI3 K with the aid of microtubule.