| BackgroundAlzheimer’s disease (AD) is prevalent in the elderly more than 65 years old. The clinical features of AD are progressively cognitive impairment, nervous-mental disorders and the loss of activity of daily life. The etiology and pathogenesis of AD are still unknown nowadays. Compelling evidence indicated that, in addition to the aggravation of beta-amyloid protein, neurofibrillary tangles and loss of neurons, insulin signaling impairment was one of the early pathological features of AD. Results in recent years have shown that insulin signaling plays a crucial role in the regulation of brain glucose metabolism, neurotransmission, learning and memory, synapse plasticity and anti-apoptosis. And more and more evidence indicated that hippocampus in brains of AD patients exhibited defective insulin signaling with altered levels and cellular distribution of insulin receptors, inhibitory serine phosphorylation of insulin receptor substrate-1 (IRS-1) and increased basal levels of phosphorylated Akt at serine 473. What’s more, results from epidemiological studies showed that compared with people without type two diabetes mellitus(T2DM), the risk of amnestic or non-amnestic mild cognitive impairment in the patients with T2DM increased 2.5 times. And T2DM may aggravate the impairment of cognition and the hyperinsulinemia and insulin resistance were associated with cognitive impairment in the elderly. In addition, there were hyperinsulinemia and decreased levels of insulin in cerebrospinal fluid in the early stage of AD. Therefor, it’s proposed that insulin resistance was associated with the progression of cognitive impairment in patients with AD. And this inference was further confirmed by the clinical effects of intranasal insulin delivery in the treatment of patients with AD. In recent years, several randomized, controlled studies about the effects of intranasal insulin delivery in the treatment of patients with AD indicated that insulin could improve the cognition, decrease the levels of plasma AP and improve the glucose metabolism in the brain of patients with AD. In addition, animal experiments found that insulin could reduce the deposition of Aβ in the brain.Therefor, there is no doubt that insulin signaling impairment is associated with the occurrence and development of Alzheimer’s disease. However, insulin receptors are expressed not only on neurons but also on astrocytes. But previous studies about insulin signaling impairment in AD were mostly focused on neurons, and the effect on insulin signaling in astrocytes is rarely reported.Astrocytes, which are 6 times more than neurons, are major component of the central nervous system (CNS) and account for 50% of the brain volume. They play crucial role in neurotransmitter regulation, energy metabolism, and anti-oxidation in the CNS. Recent evidence indicated that insulin signaling pathway participated in the regulation of all the functions in astrocytes. Glutamate transporter subtype-1, exclusively expressed on astrocytes, accounts for more than 90% of glutamate uptake in the CNS. Several studies indicated that insulin could increase the expression and membrane distribution of glutamate transporter subtype-1 on astrocytes and then increase the uptake of glutamate in the synaptic cleft. In addition, insulin signaling pathway was required for insulin-like growth factor-1/insulin mediated anti-oxidative function in astrocytes. Most importantly, astrocytes are the only nerve cells that could synthesis and storage glycogen in the CNS of the adult. And glycogen is the only form of energy stored in the brain. What’s more, accumulating evidence indicated that glycogen was the main energy source for neurons during hypoglycemia or intense activation. And glycogen was essential for the formation of learning and memory. It s evident that the storage of glycogen was regulated by insulin presumably through the classical phosphatidylinositol 3 kinase (PI3K)/Akt pathway. In consideration of the importance of insulin signaling in normal astrocytic function, it is necessary to investigate the changes and effects of insulin signaling of astrocytes in brains of patients with Alzheimer s disease.Amyloid β1-42, the main component of senile plaques, is generated from amyloid precursor protein. Amyloid β1-42 is aggregated into amyloid P1-42 oligomers or amyloid β1-42 fibrillars in brains of AD patients. In addition, it is known that amyloid β1-42 oligomers are the more toxic form that can induce insulin signaling impairment, synapse loss and memory impairment. Therefor, amyloid β1-42 oligomers were preferably used to establish models of Alzheimer s disease in vitro.Aims:1ã€To investigate the effect of amyloid β1-42 oligomers on insulin signaling;2ã€To investigate the underlying mechanisms of Aβ1-42 oligomers induced insulin signaling impairment in astrocytes.Methods:1. The culture of normal human astrocytes:the cell line of normal human astrocytes HA-1800 was purchased from ScienCell Research Laboratories in America. And cells were cultured in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with 10% fetal bovine serum(FBS) and 100U/mL penicillin/0.lmg/mL streptomycin at 37℃ inran atmosphere containing 5% CO2. Cells were passaged, cryopreserved or treated according to the growth of cells. Do not move the flask in the first 24h.To insure that the cells were in good conditions, the medium was changed in 24h before passage or cryopreservation. Commonly cells were passaged every three days and the culture medium was changed every two days.2. The preparation and verification of human synthetic Aβ1-42 oligomers:Aβ1-42 oligomers were prepared as described by Professor William J.Bowers in Rochester medicine center. In brief, human synthetic Aβ1-42 was suspended in hexafluoroisopropanol to reach 1 mM and stored as a dried film at-20℃ after evaporation of solvent using a Speed-Vac. To form Aβ1-42 oligomers, peptide films were re-suspended to 5 mM in dimethyl sulfoxide(DMSO), bath-sonicated for 10 min, diluted to 100 μM with cold PBS+0.05% SDS and vortexed for 30 sec. Samples generated above were allowed to proceed for 24 h at 4℃ and then further diluted with cold PBS to 11.1 nM before incubation for 2 weeks at 4℃. The oligomeric form of Aβ1-42 was verified by electron microscope. And prior to use, the oligomer solution was centrifuged at 4℃,13,000 rpm for 10 min to remove foreign substance.3. Western blotting was employed to determine the protein levels of total Akt, P-Akt Ser473, total IRS-1, P-IRS-1 Ser636/639, total ERK1/2 and P-ERK1/2. The concentration of primary antibody used in the trial were as follows:P-ERK1/2 (Thr202/Tyr204),1:6000; ERK1/2, 1:6000; P-IRS-1 Ser636/639,1:1000; IRS-1,1:1000; P-Akt Ser473,1:1000; Akt,1:1000; a-tubulin,1:2000.4. Immunofluorescence was performed to detect the expression of phosphorylated GSK3β at seririe 9 and tyrosine 216 and the expression and distribution of phosphorylated IRS-1 at serine 636/639 under a fluorescent microscopy and confocal microscopy, respectively. The concentration used in the trial was 1:50 for all primary antibodies in immunofluorescence analysis.5. Periodic Acid-Schiff (PAS)staining and glycogen assay kit were used to detect glycogen content in different intervention groups.6. Statistical analysis methods:The results were analyzed using SPSS 17.0 software. Image J software was used to analysis gray scale of western blot bands. Protein content was normalized by internal reference. Levels of phosphorylated protein were normalized by total protein. And levels of phosphorylated protein normalized by total protein was set to 1 in normal control group. Data were expressed as means ± SEM. One way ANOVA was used to compare differences between multiple groups. And t-test was used to compare differences between two groups. Differences were considered significant when P< 0.05.Results:1. Compared with NC group, levels of P-Akt Ser473 stimulated by insulin with different concentration were all increased. And this effect was dose dependent, reached a significantly higher level in 100 nM group(p<0.05). But with no changes in total levels of protein Akt. Levels of P-GSK3β Ser9, as well as glycogen storage were increased, while levels of P-GSK3β Tyr216 were significantly decreased in astrocytes in the presence of 100nM insulin for 30min.2. Compared with insulin group, levels of P-Akt Ser473 was significantly reduced after treatment with Aβ1-42 oligomers for 6,12 and 24 h, which was time dependent, and decreased most significantly in the 24h treatment group(P< 0.05). But with no changes in total levels of protein Akt. Treated with Aβ1-42 oligomers for 24h, levels of P-GSK3β Ser9, as well as glycogen storage were significantly decreased, while levels of P-GSK3β Tyr(216) were significantly increased in astrocytes.3. Compared with NC group, levels of P-ERK1/2 were increased in the Aβ1-42 group, peaked after 3 h of treatment (P< 0.05), and returned to basal levels within 24 h. But with no changes in levels of total ERK1/2. Compared with NC group, levels of P-ERK 1/2 accompanied with P-IRS-1 Ser636/639 were significantly increased after treatment with Aβ1-42 oligomers for 3h(P< 0.05), but with no changes in levels of total ERK1/2 and IRS-1. In addition, similar results were found in immunofluorescent analysis, but more interestingly, P-IRS-1 Ser636/639 was mainly distributed in the nucleus.4. Compared with Aβ1-42 oligomers group, levels of P-ERK1/2 accompanied with P-IRS-1 Ser636/639 were significantly decreased in the presence of specially upstream inhibitor of ERK1/2 PD98059(P<0.05), but with no changes in levels of total ERK1/2 and IRS-1.5. Compared with Aβ1-42 oligomers group, levels of P-Akt Ser473 and P-GSK3β Ser9, as well as glycogen storage were significantly increased(P<0.05), while levels of P-GSK3β Tyr216 were significantly decreased in astrocyets in the presence of PD98059.Conclusions:Our results demonstrated that insulin could stimulate the activation of insulin signaling in astrocytes. But Aβ1-42 oligomers impaired insulin signaling and suppressed insulin-induced glycogen storage in human astrocytes. In view of the effects of PD98059, it indicated that the inhibitory effects of Aβ1-42 oligomers on insulin signaling were probably due to ERKl/2-dependent serine phosphorylation of IRS-1 at 636/639 which may disturb the interaction between IRS-1 and PI3K. But further studies are needed to clarify the changes and effects of insulin signaling impairment in astrocytes in the development of AD.BackgroundAlzheimer’s disease (AD) is prevalent in the elderly more than 65 years old. The clinical features of AD are progressively cognitive impairment, nervous-mental disorders and the loss of activity of daily life. The etiology and pathogenesis of AD are still unknown nowadays. Compelling evidence indicated that, in addition to the aggravation of beta-amyloid protein, neurofibrillary tangles and loss of neurons, insulin signaling impairment was one of the early pathological features of AD. Results in recent years have shown that insulin signaling plays a crucial role in the regulation of brain glucose metabolism, neurotransmission, learning and memory, synapse plasticity and anti-apoptosis. And more and more evidence indicated that hippocampus in brains of AD patients exhibited defective insulin signaling with altered levels and cellular distribution of insulin receptors, inhibitory serine phosphorylation of insulin receptor substrate-1 (IRS-1) and increased basal levels of phosphorylated Akt at serine 473. What’s more, results from epidemiological studies showed that compared with people without type two diabetes mellitus(T2DM), the risk of amnestic or non-amnestic mild cognitive impairment in the patients with T2DM increased 2.5 times. And T2DM may aggravate the impairment of cognition and the hyperinsulinemia and insulin resistance were associated with cognitive impairment in the elderly. In addition, there were hyperinsulinemia and decreased levels of insulin in cerebrospinal fluid in the early stage of AD. Therefor, it’s proposed that insulin resistance was associated with the progression of cognitive impairment in patients with AD. And this inference was further confirmed by the clinical effects of intranasal insulin delivery in the treatment of patients with AD. In recent years, several randomized, controlled studies about the effects of intranasal insulin delivery in the treatment of patients with AD indicated that insulin could improve the cognition, decrease the levels of plasma AP and improve the glucose metabolism in the brain of patients with AD. In addition, animal experiments found that insulin could reduce the deposition of Aβ in the brain.Therefor, there is no doubt that insulin signaling impairment is associated with the occurrence and development of Alzheimer’s disease. However, insulin receptors are expressed not only on neurons but also on astrocytes. But previous studies about insulin signaling impairment in AD were mostly focused on neurons, and the effect on insulin signaling in astrocytes is rarely reported.Astrocytes, which are 6 times more than neurons, are major component of the central nervous system (CNS) and account for 50% of the brain volume. They play crucial role in neurotransmitter regulation, energy metabolism, and anti-oxidation in the CNS. Recent evidence indicated that insulin signaling pathway participated in the regulation of all the functions in astrocytes. Glutamate transporter subtype-1, exclusively expressed on astrocytes, accounts for more than 90% of glutamate uptake in the CNS. Several studies indicated that insulin could increase the expression and membrane distribution of glutamate transporter subtype-1 on astrocytes and then increase the uptake of glutamate in the synaptic cleft. In addition, insulin signaling pathway was required for insulin-like growth factor-1/insulin mediated anti-oxidative function in astrocytes. Most importantly, astrocytes are the only nerve cells that could synthesis and storage glycogen in the CNS of the adult. And glycogen is the only form of energy stored in the brain. What’s more, accumulating evidence indicated that glycogen was the main energy source for neurons during hypoglycemia or intense activation. And glycogen was essential for the formation of learning and memory. It s evident that the storage of glycogen was regulated by insulin presumably through the classical phosphatidylinositol 3 kinase (PI3K)/Akt pathway. In consideration of the importance of insulin signaling in normal astrocytic function, it is necessary to investigate the changes and effects of insulin signaling of astrocytes in brains of patients with Alzheimer s disease.Amyloid β1-42, the main component of senile plaques, is generated from amyloid precursor protein. Amyloid β1-42 is aggregated into amyloid P1-42 oligomers or amyloid β1-42 fibrillars in brains of AD patients. In addition, it is known that amyloid β1-42 oligomers are the more toxic form that can induce insulin signaling impairment, synapse loss and memory impairment. Therefor, amyloid β1-42 oligomers were preferably used to establish models of Alzheimer s disease in vitro.Aims:1ã€To investigate the effect of amyloid β1-42 oligomers on insulin signaling;2ã€To investigate the underlying mechanisms of Aβ1-42 oligomers induced insulin signaling impairment in astrocytes.Methods:1. The culture of normal human astrocytes:the cell line of normal human astrocytes HA-1800 was purchased from ScienCell Research Laboratories in America. And cells were cultured in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with 10% fetal bovine serum(FBS) and 100U/mL penicillin/0.lmg/mL streptomycin at 37℃ inran atmosphere containing 5% CO2. Cells were passaged, cryopreserved or treated according to the growth of cells. Do not move the flask in the first 24h.To insure that the cells were in good conditions, the medium was changed in 24h before passage or cryopreservation. Commonly cells were passaged every three days and the culture medium was changed every two days.2. The preparation and verification of human synthetic Aβ1-42 oligomers:Aβ1-42 oligomers were prepared as described by Professor William J.Bowers in Rochester medicine center. In brief, human synthetic Aβ1-42 was suspended in hexafluoroisopropanol to reach 1 mM and stored as a dried film at-20℃ after evaporation of solvent using a Speed-Vac. To form Aβ1-42 oligomers, peptide films were re-suspended to 5 mM in dimethyl sulfoxide(DMSO), bath-sonicated for 10 min, diluted to 100 μM with cold PBS+0.05% SDS and vortexed for 30 sec. Samples generated above were allowed to proceed for 24 h at 4℃ and then further diluted with cold PBS to 11.1 nM before incubation for 2 weeks at 4℃. The oligomeric form of Aβ1-42 was verified by electron microscope. And prior to use, the oligomer solution was centrifuged at 4℃,13,000 rpm for 10 min to remove foreign substance.3. Western blotting was employed to determine the protein levels of total Akt, P-Akt Ser473, total IRS-1, P-IRS-1 Ser636/639, total ERK1/2 and P-ERK1/2. The concentration of primary antibody used in the trial were as follows:P-ERK1/2 (Thr202/Tyr204),1:6000; ERK1/2, 1:6000; P-IRS-1 Ser636/639,1:1000; IRS-1,1:1000; P-Akt Ser473,1:1000; Akt,1:1000; a-tubulin,1:2000.4. Immunofluorescence was performed to detect the expression of phosphorylated GSK3β at seririe 9 and tyrosine 216 and the expression and distribution of phosphorylated IRS-1 at serine 636/639 under a fluorescent microscopy and confocal microscopy, respectively. The concentration used in the trial was 1:50 for all primary antibodies in immunofluorescence analysis.5. Periodic Acid-Schiff (PAS)staining and glycogen assay kit were used to detect glycogen content in different intervention groups.6. Statistical analysis methods:The results were analyzed using SPSS 17.0 software. Image J software was used to analysis gray scale of western blot bands. Protein content was normalized by internal reference. Levels of phosphorylated protein were normalized by total protein. And levels of phosphorylated protein normalized by total protein was set to 1 in normal control group. Data were expressed as means ± SEM. One way ANOVA was used to compare differences between multiple groups. And t-test was used to compare differences between two groups. Differences were considered significant when P< 0.05.Results:1. Compared with NC group, levels of P-Akt Ser473 stimulated by insulin with different concentration were all increased. And this effect was dose dependent, reached a significantly higher level in 100 nM group(p<0.05). But with no changes in total levels of protein Akt. Levels of P-GSK3β Ser9, as well as glycogen storage were increased, while levels of P-GSK3β Tyr216 were significantly decreased in astrocytes in the presence of 100nM insulin for 30min.2. Compared with insulin group, levels of P-Akt Ser473 was significantly reduced after treatment with Aβ1-42 oligomers for 6,12 and 24 h, which was time dependent, and decreased most significantly in the 24h treatment group(P< 0.05). But with no changes in total levels of protein Akt. Treated with Aβ1-42 oligomers for 24h, levels of P-GSK3β Ser9, as well as glycogen storage were significantly decreased, while levels of P-GSK3β Tyr(216) were significantly increased in astrocytes.3. Compared with NC group, levels of P-ERK1/2 were increased in the Aβ1-42 group, peaked after 3 h of treatment (P< 0.05), and returned to basal levels within 24 h. But with no changes in levels of total ERK1/2. Compared with NC group, levels of P-ERK 1/2 accompanied with P-IRS-1 Ser636/639 were significantly increased after treatment with Aβ1-42 oligomers for 3h(P< 0.05), but with no changes in levels of total ERK1/2 and IRS-1. In addition, similar results were found in immunofluorescent analysis, but more interestingly, P-IRS-1 Ser636/639 was mainly distributed in the nucleus.4. Compared with Aβ1-42 oligomers group, levels of P-ERK1/2 accompanied with P-IRS-1 Ser636/639 were significantly decreased in the presence of specially upstream inhibitor of ERK1/2 PD98059(P<0.05), but with no changes in levels of total ERK1/2 and IRS-1.5. Compared with Aβ1-42 oligomers group, levels of P-Akt Ser473 and P-GSK3β Ser9, as well as glycogen storage were significantly increased(P<0.05), while levels of P-GSK3β Tyr216 were significantly decreased in astrocyets in the presence of PD98059.Conclusions:Our results demonstrated that insulin could stimulate the activation of insulin signaling in astrocytes. But Aβ1-42 oligomers impaired insulin signaling and suppressed insulin-induced glycogen storage in human astrocytes. In view of the effects of PD98059, it indicated that the inhibitory effects of Aβ1-42 oligomers on insulin signaling were probably due to ERKl/2-dependent serine phosphorylation of IRS-1 at 636/639 which may disturb the interaction between IRS-1 and PI3K. But further studies are needed to clarify the changes and effects of insulin signaling impairment in astrocytes in the development of AD. |
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