Zinc-α2-glycoprotein Relieved Seizure-induced Neuronal Glucose Uptake Impairment Via Insulin-like Growth Factor 1 Receptor-regulated Glucose Transporter 3 Expression

Part 1:The effect of seizure on neuronal glucose uptake and ZAG expressionObjective:To detect the effect of seizure on neuronal glucose uptake and ZAG expression in Mg²⁺-free artificial cerebrospinal fluid（ACSF）-induced neuronal seizure model.Methods:1.Primary cortical neurons extracted from newborn SD rats within 24hours were cultured with Mg²⁺-free ASCF to establish neuronal seizure model.2.Neuronal discharge in seizure model was verified by Fluo-4,AM calcium imaging experiments.3.The cell death rate of neuronal seizure model was detected by trypan blue staining.4.Neuronal glucose uptake in neuronal seizure model was detected by2-NBDG fluorescence method or Glucose Uptake Colorimetric Assay Kit.5.ZAG expression in neuronal seizure model was measured by western blot.Results:1.Fluo-4,AM calcium imaging shown the mean fluorescence intensity in Seizure group was higher than Controls（40.5730±7.4175 vs.23.2827±5.5209,p=0.036,n=3）.2.Trypan blue staining showed the cell death rate in Seizure group was not significantly different from that in Controls（0.2825±0.0140 vs.0.2985±0.0594,p=0.574,n=5）.3.The mean fluorescence intensity of 2-NBDG in Seizure group was significantly lower than Controls（28.3392±2.5323 vs.57.7486±11.9154,p=0.001,n=5）.Neuronal 2-DG6P level was also significantly decreased in Seizure group（23.8385±0.3544pmol vs.65.0876±3.1367pmol,p=0.002,n=3）.4.Western blot showed that ZAG protein level was significantly decreased in Seizure group（0.5151±0.0229 vs.1,p<0.001,n=5）.Conclusions:1.Mg²⁺-free ASCF treatment increases neuronal calcium and simulate neuronal discharge without affecting neuronal death rate.2.Seizure suppresses neuronal glucose uptake and ZAG protein expression.Part 2:The effect of ZAG on neuronal glucose uptake and GLUT-3 expressionObjective:To detect the effect of ZAG on neuronal glucose uptake and GLUT-3 expression.Methods:1.Primary cortical neurons extracted from newborn SD rats within 24hours were cultured.2.Cultured primary cortical neurons were transfected with ZAG-overexpression lentivirus（LV-ZAG）,ZAG-knockdown lentivirus（LV-RNAi）,Vector-ZAG（LV-ZAG Control）and Vector-RNAi（LV-RNAi Control）respectively.3.Mg²⁺-free ASCF treated cultured primary cortical neurons to establish neuronal seizure model.Neurons were divided into three groups:LV-ZAG Control group,LV-ZAG Control+Seizure group and LV-ZAG+seizure group.4.ZAG m RNA expression was detected by q RT-PCR.5.Neuronal ZAG,total GLUT-3（t GLUT-3）and membrane GLUT-3（m GLUT-3）expression were measured by western blot.6.Neuronal glucose uptake was assayed by Glucose Uptake Colorimetric Assay Kit.Results:1.q RT-PCR showed that ZAG m RNA level was increased in LV-ZAG treated neurons（170.0247±59.3013 vs.1,p=0.008,n=3）and decreased in LV-RNAi treated neurons（0.3710±0.0864 vs.1,p=0.006,n=3）.Western blot showed that ZAG protein expression was increased in LV-ZAG group（1.8524±0.4981 vs.1,p=0.004,n=7）and decreased in LV-RNAi group（0.4915±0.0952 vs.1,p<0.001,n=9）.2.LV-RNAi treatment decreased neuronal 2-DG6P level（43.1083±1.2138pmol vs.66.6940±2.3152pmol,p<0.001,n=3）.The neuronal 2-DG6P level in LV-ZAG+Seizure group was significantly higher than that in LV-ZAG Control+Seizure group（74.7602±2.4926pmol vs.23.8852±2.3108pmol,p<0.001,n=3）.3.Western blot shown that seizure suppressed neuronal m GLUT-3expression（0.4722±0.0343 vs.1,p<0.001,n=7）,t GLUT-3（0.7227±0.0950vs.1,p=0.001,n=7）expression and m GLUT-3/t GLUT-3 ratio（0.6602±0.0777 vs.1,p<0.001,n=7）,which was improved by LV-ZAG treatment（m GLUT-3:1.0968±0.1078 vs.0.4722±0.0343,p<0.001,n=7;t GLUT-3:0.9514±0.0770 vs.0.7227±0.0950,p=0.001,n=7;m GLUT-3/t GLUT-3 ratio:1.1640±0.1789 vs.0.6602±0.0777,p<0.001,n=7）.Similarly,the m GLUT-3 expression（0.5353±0.0338 vs.1,p<0.001,n=7）,t GLUT-3 expression（0.6407±0.0514 vs.1,p<0.001,n=7）and m GLUT-3/t GLUT-3 ratio（0.8405±0.0889 vs.1,p=0.003,n=7）were all decreased in LV-RNAi treated neurons.Conclusions:ZAG increased neuronal glucose uptake possibly by promoting neuronal GLUT-3 expression and its distribution on membrane.Part 3:The effect of IGF1R on neuronal glucose uptake and GLUT-3 expressionObjective:To investigate the effect of IGF1R on neuronal glucose uptake and GLUT-3 expression in primary cortical neurons.Methods:1.Primary cortical neurons extracted from newborn SD rats within 24hours were cultured.2.Cultured primary cortical neurons were treated with IGF1（agonist of IGF1R）and AXL1717（inhibitor of IGF1R）respectively.Neurons were divided into four groups:Controls,DMSO,IGF1 and AXL1717.3.Mg²⁺-free ASCF treated cortical neurons to establish neuronal seizure model.Neurons were divided into three groups:DMSO,DMSO+Seizure and IGF1+seizure.4.Neuronal t GLUT-3 and m GLUT-3 were measured by western blot.5.Neuronal glucose uptake was measured by Glucose Uptake Colorimetric Assay Kit.Results:1.Neuronal 2-DG6P level was increased in IGF1 treated neurons（71.6212±1.5726pmol vs.65.6981±0.8682pmol,p=0.027,n=3）and decreased in AXL1717 treated neurons（42.7565±0.8686pmol vs.65.6981±0.8682pmol,p<0.001,n=3）.2.Western blot shown increased neuronal t GLUT-3（2.1675±0.1739 vs.0.9824±0.1045,p<0.001,n=5）and m GLUT-3（1.8310±0.1803 vs.1.0600±0.0957,p<0.001,n=5）via IGF1 treatment,while m GLUT3/t GLUT-3 ratio（0.8512±0.1281 vs.1.0930±0.1777,p=0.032,n=5）was decreased.AXL1717 treatment decreased neuronal t GLUT-3（0.5950±0.0983 vs.1.1173±0.1267,p<0.001,n=5）and m GLUT-3（0.6612±0.0590 vs.1.0712±0.1003,p=0.001,n=5）,but m GLUT3/t GLUT-3ratio（1.2332±0.2879 vs.0.9679±0.1357,p=0.275,n=5）was not affected.3.Neuronal 2-DG6P level was increased in Seizure+IGF1 group compared to Seizure group（76.5301±5.9879pmol vs.23.8385±0.3544pmol,p=0.004,n=3）.IGF1 treatment also improved m GLUT-3（1.2730±0.0509vs.0.4408±0.1129,p<0.001,n=3）and t GLUT-3（1.5620±0.0783 vs.0.6599±0.0660,p<0.001,n=3）expression in seizure model neurons without affecting m GLUT-3/t GLUT-3 ratio（0.8174±0.0730 vs.0.6720±0.1890,p=0.536,n=3）.Conclusions:IGF1R increased neuronal glucose uptake through improving neuronal GLUT-3 expression rather than GLUT-3 distribution on membrane.Part 4:The role of IGF1R in regulation of neuronal glucose uptake and GLUT-3 expression by ZAGObjective:To study the role of IGF1R in regulation of neuronal glucose uptake and GLUT-3 expression by ZAG.Methods:1.Primary cortical neurons extracted from newborn SD rats within 24hours were cultured.2.Cultured cortical neurons were treated with lentivirus and drugs.Neurons were divided into six groups:LV-RNAi Control+DMSO,LV-RNAi+DMSO,LV-RNAi+IGF1,LV-ZAG Control+DMSO,LV-ZAG+DMSO and LV-ZAG+AXL1717.3.Neuronal t GLUT-3 and m GLUT-3 were measured by western blot.4.Neuronal glucose uptake was measured by Glucose Uptake Colorimetric Assay Kit.Results:1.IGF1 treatment increased neuronal 2-DG6P level（74.3259±0.9644pmol vs.43.1083±1.2138pmol,p<0.001,n=3）,m GLUT-3expression（1.8712±0.2292 vs.0.4309±0.0282,p<0.001,n=5）and t GLUT-3（2.4296±0.3112 vs.0.5293±0.0890,p<0.001,n=5）expression in LV-RNAi treated neurons,but did not affect the m GLUT-3/t GLUT-3 ratio（0.8174±0.0730 vs.0.6720±0.1890,p=0.536,n=5）in LV-RNAi treated neurons.2.AXL1717 treatment inhibited neuronal 2-DG6P level（32.2651±2.7077pmol vs.73.6646±0.8720pmol,p<0.001,n=3）,m GLUT-3expression（1.0551±0.1903 vs.2.6585±0.6528,p=0.010,n=3）and t GLUT-3（0.5385±0.0866 vs.1.4672±0.3638,p<0.001,n=3）expression in LV-ZAG treated neurons,but did not affect the m GLUT-3/t GLUT-3 ratio（1.9778±0.3422 vs.1.8962±0.6950,p=1.000,n=3）in LV-ZAG treated neurons.Conclusions:The effect of ZAG on neuronal glucose and GLUT-3expression was mediated by IGF1R.However,other mechanisms may be involved in the regulation of neuronal GLUT-3 distribution by ZAG.Part 5:The effect of ZAG on neuronal IGF1R expression and activityObjective:To detect the effect ZAG on neuronal IGF1R expression and activity.Methods:1.Primary cortical neurons extracted from newborn SD rats within 24hours were cultured.2.Lentivirus transfected cultured cortical neurons.Neurons were divided into four groups:LV-ZAG Control,LV-ZAG,LV-RNAi Control and LV-RNAi.3.The interaction between ZAG and IGF1R was verified using coimmunoprecipitation（CO-IP）.4.Neuronal membrane IGF1R（mIGF1R）,total IGF1R（t IGF1R）and phosphorylated IGF1R（p IGF1R）expression were measured by western blot.Results:1.CO-IP showed that ZAG bound to IGF1R.2.Western blot showed mIGF1R（0.5641±0.0786 vs.1,p<0.001,n=11）and p IGF1R（0.5583±0.0624 vs.1,p<0.001,n=11）expression were decreased in LV-RNAi treated neurons,but t IGF1R（1.0400±0.086 vs.1,p=0.155,n=11）level was not changed.LV-ZAG increased level of mIGF1R（1.7917±0.2945 vs.1,p<0.001,n=7）and p IGF1R（2.4018±0.2190 vs.1,p<0.001,n=7）,but did not affect t IGF1R level（1.0283±0.0675 vs.1,p=0.310,n=7）.3.Western blot showed that seizure decreased neuronal t IGF1R（0.5417±0.0205 vs.1,p<0.001,n=5）,mIGF1R（0.5716±0.0269 vs.1,p<0.001,n=5）,p IGF1R（0.3069±0.0609 vs.1,p=0.010,n=5）level and p IGF1R/t IGF1R ratio（0.5417±0.0205 vs.1,p<0.001,n=5）,but did not affected mIGF1R/t IGF1R ratio（1.0346±0.2259 vs.1,p=1.000,n=5）.LV-ZAG treatment did not change seizure-induced t IGF1R level（0.6158±0.2235 vs.0.5668±0.1124,p=0.958,n=5）decrease,but it reversed seizure-induced mIGF1R（1.5821±0.1657 vs.0.5716±0.0269,p<0.001,n=5）,p IGF1R（1.0311±0.5127 vs.0.3069±0.0609,p=0.007,n=5）level and p IGF1R/t IGF1R ratio（1.6141±0.2271 vs.0.5417±0.0205,p=0.001,n=5）decrease.Conclusions:ZAG increased neuronal IGF1R distribution on membrane and its phosphorylation possibly by binding,which was suppressed by seizure.