High Glucose Exacerbates β-amyloid Production In HEK293APP Swedish Mutant Overexpression Cells And Beneficial Role Of Benfotiamine

Alzheimer’s disease (AD) and diabetes mellitus (DM) are two of the most common conditions that affect the elderly. Recent evidence from a population-based study shows a link between DM and AD. The prevelance rate of AD is high as much as2to5times in population suffered from DM compared with non-DM control subjects. Decreased cerebral glucose utilization and energy metabolism in the patients with AD represent very early abnormalities that precede cognitive impairment and pathological alterations. Experimental investigations also suggest that the abnormality of cerebral glucose metabolism is the key etiological factor. Conversely, the patients with AD also have a high risk of developing type2diabetes mellitus.The disturbance of glucose metabolism in brain increases the risk for cognitive dysfunction or dementia, especially AD-type dementia. However, the underlying mechanism remains to be clarified.AD is a neurodegenerative disease characterized clinically by progressive cognitive impairment and pathologically by the presence of extracellular senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs) in the brain. Senile plaques are largely composed of β-amyloid (A(3), a4kDa peptide derived from the β-amyloid precursor protein (APP). Aβ is toxic to neurons, and the deposition of Aβ and the formation of senile plaques are considered to be the primary cause of AD. Glycogen synthase kinase3(GSK-3) is a serine/threonine protein kinase highly abundant in brain, involved in the regulation of glucose metabolism and glycogen synthesis. Now, GSK-3is implicated in many different physiological and pathological processes. Interestingly, recent evidence shows that GSK-3also contributes to AD pathogenesis. Inhibiting GSK-3activity has been demonstrated to reduce the Aβ production and accumulation in APP overexpressing mice. In our previous study, we evaluated the effect of benfotiamine on Aβ generation using an APP/presenilin-1(APP/PS1) double transgenic mouse model. Our previous results indicated that benfotiamine significantly enhanced the phosphorylation levels and reduced enzymatic activities of both GSK-3a and GSK-3β in the APP/PS1mice, suggesting that a GSK-3dependent pathway may be involved in the beneficial effects of benfotiamine. Here, we utilize HEK293APPsw overexpression cell line to study whether high glucose exacerbates Aβ secretion and benfotiamine reduces Aβ production and their possible mechanism in vitro. Part1:High glucose exacerbates β-amyloid production in HEK293APP Swedish mutant overexpression Cells.一、The influence of different glucose concentration on β-amyloid production of HEK293APP swedish mutant overexpression cells and HEK293cells.Objective:To explore the influence of different glucose concentration on P-amyloid production of HEK293cells and HEK293APP swedish mutant overexpression cells.Methods:The HEK293cells and HEK293APP swedish mutant overexpression cells were chosen as the experimental cells. We cultured the HEK293APP swedish mutant overexpression cells in different glucose concentrations (the final concentrations of glucose in the culture medium were (1,3,4.5,5.5,6.5,7.5,8.5,10.5g/L).The cells were also incubated for different time, including6,12and24h. The supernatant liquid of cells was collected, which was used for ELISA to observe β-amyloid production in different glucose concentrations and different time. Also, the HEK293APP swedish mutant overexpression cells and HEK293cells were cultured in different glucose concentration (1g/L and5.5g/L) for12h. The supernatant liquid of both cells was collected, which was used for ELISA to observe β-amyloid production in different glucose concentration.Result:The AP production of HEK293APPsw mutant overexpression cells was hiegher than that of HEK293cells. The trend of the Aβ production of HEK293APPsw mutant overexpression cells was first rising and then dropping, along with the increased concentrations of glucose in the cell culture medium and the prolongation of the incubated time. Compared with cells incubated in cell culture medium with lg/L, the Aβ production of cells incubated in cell culture medium with5.5g/L for12h was higher significantly(p<0.01), and for6h was also higher significantly (p<0.05). Although the Aβ production in cell culture medium with5.5g/L for24h tend to rise, there was no significant difference compared with that in the culture medium with1.0g/L for24h(p>0.05).Conclusion:Different glucose concentration can enhance β-amyloid production in HEK293APP swedish mutant overexpression cell line but does not affect HEK293cell line.While the optimal incubated condition to significantly enhance Aβ production was that the concentration of glucose in cell culture medium was5.5g/L and the incubation time was12h.二、The effect of different glucose concentrations on the proliferation and survival of HEK293APP Swedish mutant overexpression CellsObjective:To explore the influence of different glucose concentrations on the proliferation and survival of HEK293APP Swedish mutant overexpression cellsMethods:The HEK293APP Swedish mutant cells were chosen as the experimental cells, we cultured HEK293APP Swedish mutant overexpression cells in different glucose concentrations (final concentrations of glucose were(1,3,4.5,5.5g/L).The HEK293APP Swedish mutant overexpression cells were incubated for12h. The supernatant liquid of HEK293APP Swedish mutant overexpression cells were collected, which were used for CCK-8assay to observe cell prolifereation and survival in different glucose concentrations.Result:There was no significant difference of the proliferation and survival of HEK293APP Swedish mutant overexpression Cellss in different glucose concentrations(1,3,4.5,5.5g/L)(p>0.05). Conclusion:The glucose concentration does not affect the proliferation and survival of HEK293APP Swedish mutant Cells.Part2:Benfotiamine decreases Aβ production of HEK293APP Swedish mutant overexpression cells under high glucoseObjective:To investigate whether benfotiamine reduces Aβ productionof HEK293APP Swedish Mutant cells.Methods:The HEK293APP swedish mutant cells were chosen as the experimental cells. We cultured HEK293APP Swedish mutant overexpression cells in the culture medium with glucose concentration of5.5g/L for12h. The HEK293APP Swedish mutant overexpression cells were divided into four groups randomly:control group (5.5g/L glucose concentration), benfotiamine group (10μg/ml), benfotiamine group (20μg/ml), benfotiamine group (40μg/ml).The supernatant liquid of HEK293APP swedish mutant overexpression cells was collected, which was used for ELISA to observe A(3production.Result:Benfotiamine significantly reduced Aβ production compared with control group (20μg/ml group P<0.05;40μg/ml group P<0.01), but10μg/ml group showed no significant difference compared with control group.Conclusion:Benfotiamine siginificantly reduces Aβ production in HEK293APP Swedish mutant overexpression cells.Part3:The mechanisms of high glucose increasing Aβ production and benfotiamine reducing Aβ productionObjective:To explore the mechanisms of high glucose and benfotiamine altered Aβ production of HEK293APP Swedish mutant overexpression cells.Methods:We chosen HEK293APP Swedish mutant overexpression cells as the testing cells. The cells were divided into three groups randomly:low glucose control group (1g/L glucose concentration), high glucose control group (5.5g/L glucose concentration), and benfotiamine group (40μg/ml). Each group was incubated for12h, then the protein of cells was collected, which was used for assaying enzyme activity and western blot. The ratios of phospho-GSK-3a/GSK-3a and phospho-GSK-3β/GSK-3β were investigated by western blot. The activities of GSK-3a, and GSK-3β were determined by kits according to the manufacturer’s instructions.Result:The results showed that the ratios of p-GSK-3a/GSK-3a and p-GSK-3p/GSK-3p in high-dose control group were significantly decreased compared those of low-dose control group (p-GSK-3a/GSK-3a, p<0.01; p-GSK-3p/GSK-3β, p<0.05). Furthemore, the activities of GSK-3a and GSK-3β in high-dose control group were significantly increased compared to low glucose control group (p<0.05). Also, we found that benfotiamine group(40μg/ml) significantly decreased the enzyme activities of GSK-3a and GSK-3β compared to high glucose control group (p<0.05)Conclusion:The results indicate that the GSK-3activities are associated with increasing Aβ production induced by high glucose and benfotiamine decreases Aβ production through inhibiting the activities of GSK-3.