Research On Co-effect Of Insulin And Oligomeric Amyloid Beta On Phosphorylated Camp Response Element Binding Protein

Part OneImpact of different concentrations of oligomeric amyloid beta on phosphorylated cAMP response element binding proteinObjectiveTo investigate the impact of different concentrations of oligomeric amyloid beta on the level of phosphorylated cAMP response element binding protein.MethodsPC12 cells were cultured for 3h,6h,12h,24h,36 and 48h with different concentrations of Aβ1-42 oligomers(Aβ-derived diffusible ligands, ADDLs)(0.5μmol/L, 1μmol/L, 1.5μmol/L, 2μmol/L, 2.5μmol/L and 3μmol/L). The activation of calpain and protein level of catalytic subunits of PKA(PKA-C)and phosphorylated cAMP response element binding protein(pCREB) were analysed by western blot. ResultsAll concentrations of ADDLs could decrease pCREB(P<0.05), but had no impact on PKA-C levels(P>0.05).While 2μmol/L, 2.5μmol/L and 3μmol/L ADDLs decreased pCREB through activation of calpain, 0.5μmol/L, 1μmol/L and 1.5μmol/L ADDLs decreased pCREB through other mechanisms.ConclusionsDifferent concentrations of ADDLs decreased pCREB though different ways: high concentrations through activation of calpain, while low concentrations through other mechanisms. However, neither decreased pCREB levels through impact on the levels of PKA-C.Part TwoCo-effect of insulin and oligomeric amyloid beta on phosphorylated cAMP response element binding proteinObjectiveTo investigate the co-effect of insulin and oligomeric amyloid beta on phosphorylated cAMP response element binding protein.MethodsPC12 cells were cultured for 12h with different concentrations of Aβ1-42 oligomers(Aβ-derived diffusible ligands, ADDLs)(0.5μmol/L and 2μmol/L) and insulin(100nmol/L and 1μmol/L). The activation of calpain and protein level of catalytic subunits of PKA(PKA-C)and phosphorylated cAMP response element binding protein(pCREB) were analysed by western blot. All these parameters were compared with those of PC12 cells treated with ADDLs or insulin alone.Results(1) When PC12 cells were cultured with insulin alone, compared with controls, calpain activation was not altered by insulin treatment. However, the level of pCREB was increased at about 30min(P<0.05) and soon backed to normal state.(2) When PC12 cells were cultured with ADDLs alone, the results were previously described.(3) When PC12 cells were cultured for 12h with different concentrations of ADDLs(0.5μmol/L and 2μmol/L) and insulin(100nmol/L and 1μmol/L), compared with cells cultured with ADDLs alone, 100nmol/L insulin had no impact on calpain activation and the level of pCREB(P>0.05);When PC12 cells were cultured for 12h with 0.5μmol/L ADDLs and 1μmol/L insulin, insulin could partially reverse the downregulation of pCREB (P<0.05), though it had no influence on calpain activation(P>0.05);When PC12 cells were cultured for 12h with 2μmol/L ADDLs and 1μmol/L insulin, insulin could exacerbate the activation of calpain by ADDLs(P<0.05) and further downregulate the level of pCREB(P<0.05).(4) The level of PKA-Cα, PKA-Cβwas not altered under any of the circumstances described above when compared with controls(P>0.05).ConclusionsWhether insulin could exaggerate or reverse the neurotoxicities of ADDLs is dependent on the mechanisms of how different concentrations of ADDLs downregulate pCREB. When insulin acted with a relatively low concentration of ADDLs, which decreased pCREB without the activation of calpain, it showed a protective effect as it partially reversed the downregulation of pCREB; when acting with a relatively high concentration of ADDLs, which decreased pCREB through the activation of calpain, insulin exacerbated the neurotoxicities of ADDLs by enhancing the activation of calpain and downregulation of pCREB.