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Introduction

Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders morpho-pathologically characterized by cellular Aβ amyloid plaque, intracellular neurofibrillary tangles and extensive neuronal death. Besides these pathological alterations, this disease is also associated with abnormalities in the colinergic, serotoninergic, noradrenergic and dopaminergic systems (1). These neurochemical changes may be related to the abnormal blood glucose metabolism that has been identified using positron emission tomography (2). The reduction of glucose utilization was not attributed to an insufficient supply of glucose to the brain, but rather the decrease of glucose breakdown in brain tissue (3,4). Furthermore, previous studies reported that the activities of the number of enzymes and the expression of the glucose transporter involved in glucose metabolism decreased in the brain tissue of AD (5–7).

The existence of both insulin and insulin receptors in the CNS is well established (8–9). Insulin was first localized in the CNS of rats by immunohistochemical staining (10). Subsequently, the insulin mRNA was demonstrated to exist in various brain areas, suggesting that this peptide is synthesized in the brain (11). In human brain, insulin regulates enzymes associated with cerebral glucose metabolism via specific high-affinity insulin receptors, which are different from peripheral insulin receptors only in the amount of glycosylation (12,13). Additionally, insulin binds to insulin-like growth factor I (IGF-I) receptors and, via these receptors, potentially exerting more generalized trophic effects on neural cell and interacting with cholinergic neurotransmission (14,15). The binding of insulin to its receptor is followed by an autophosphorylation of tyrosine residues at the β-chain of the insulin receptor resulting in a subsequent activation of the intrinsic tyrosine kinase, which phosphorylates the first known endogenous substrate insulin-receptor substrate-1 (IRS-1) (16). Phosphorylated IRS-1 transfers its signal to a wide spectrum of cellular signal transduction pathways (17). In particular, AD patients exhibit alterations in insulin and IGF-1 levels and their receptors, leading to defective response to insulin (18).

Furthermore, the study with transgenic (Tg) 2576 mice overexpressing the Swedish mutant human amyloid precursor protein revealed an age-related cortical and hippocampal deposition of β-amyloid plaques, as well as a decreased phosphofructokinase-C (PFK-C) protein and mRNA level in cerebral cortical tissue. Additionally, 24-month-old Tg2576 mice showed reduced enzyme activity of PFK without affecting the mRNA levels of the other PFK isoforms and fructose 1,6-bisphosphatase (FBPase) in comparison to non-transgenic littermates (5). However, no studies have been conducted thus far to investigate whether the proteins involved in glucose metabolism are significantly altered in neuron-specific enolase (NSE)/hPS2m Tg mice that demonstrate AD-like pathology.

Therefore, the aim of this study was to investigate whether AD pathology induced by overexpression of human mutant PS2 protein induces changes in glucose metabolism. The findings showed that the enhancement of Aβ-42 peptides and γ-secretase activity in NSE/hPS2m Tg mice significantly induced the defect of glucose metabolism, including the decrease of insulin, the increase of glucose, as well as the alteration of their related receptors and the signal pathway. Furthermore, these results show that the insulin treatment may decrease the level of Aβ-42 peptides in the brain of NSE/hPS2m Tg mice.

Materials and methods

Care and use of NSE/hPS2m Tg mice

NSE/hPS2m Tg mice overexpressing human mutant PS2 (hPS2m, N141I), under the control of an NSE promoter were used in this study (19,20) and were obtained from the Department of Laboratory Animal Resources in Korea FAD. These Tg mice showed behavioral dysfunction, Aβ-42 deposition and the induction of caspase-3 and Cox-2 activities at 12 months of age. To analyze the protein level and insulin concentration, a total of 10 mice were used; five NSE/hPS2m Tg mice and five non-Tg littermates at 12 months of age. The mice were handled in a Pusan National University-Laboratory Animal Resources Center accredited by the Korea FDA in accordance with the USA NIH guidelines (accredited unit no. 231). The mice were maintained in a specified pathogen-free environment and were housed in cages under a strict light cycle (light period for 12 h and dark period for 12 h) and were given a standard irradiated chow diet (Purina Mills Inc., Milford, IN, USA) ad libitum.

Aβ-42 western blot analysis

For detection of the Aβ-42 level, the frozen brain of mice was sectioned with scissors and homogenized in Pro-Prep™ Protein Extraction Solution [Intron Biotechechnology Co. Ltd., Seongnam, Korea, (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM DTT, 0.5% NP-40, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, proteinase inhibitor)] with a glass homogenizer. The homogenate mixture was centrifuged at 22,250 × g for 10 min at 4°C to eliminate the nuclei and unbroken cells. The protein prepared from the brain was separated by electrophoresis in a 4–20% SDS-PAGE gel for 3 h and transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was incubated separately with the primary anti-Aβ-42 antibody (MAB1560, 6E10; Chemicon International, Temecula, CA, USA) overnight at 4°C. The membranes were washed with the washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 and 0.05% Tween-20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000 dilution; Zymed, South San Francisco, CA, USA) at room temperature for 2 h. Blots were developed using a Chemiluminescence Reagent Plus kit (ECL; Amersham Pharmacia, Piscataway, NJ, USA).

γ-secretase activity analysis

Detection of γ-secretase activity was performed according to the manufacturer’s instructions (FP003; R&D System Inc., Wiesbaden, Germany). Frozen brain tissues were sectioned with scissors and homogenized in lysis solution and 1X cell extraction buffer with a glass homogenizer into 0.5–2.0 mg/ml final concentrations. The homogenate was separated by centrifugation at 22,250 × g for 15 min at 4°C and then supernatant was collected for the protein and enzyme assays. Protein was assayed by the BCA method (Pierce, Rockford, IL, USA) using an ELISA reader. For the determination of γ-secretase activity, the 50 μl of tissue lysate (25–200 μg) prepared from the brain extract was added to each well in a 96-well microplate in triplicate and then 2X reaction buffer (50 μl) was added. The substrate (5 μl) was added to each well and the plate was incubated in the dark at 37°C for 1–2 h. Fluorescence was measured at a wavelength of 335–355 nm using a Fluorescent Microplate Reader FL600 (Bio-Tek Instrument, Inc., Winooski, VT, USA).

Radioimmunoassay (RIA) and serum biochemical analyses

Blood was collected from the abdominal vein of NSE/hPS2m Tg mice and non-Tg littermates and then incubated at room temperature for 30 min. The serum was separated by centrifugation at 890 × g for 15 min at 4°C. Serum insulin concentration was carried out as per the manufacturer’s instructions [Coat-A-Count Insulin kit; Diagnostic Products Corp., LA, CA, USA) using a gamma counter (Cobra 5010 Quantum, Cobra 5010 II; Packard Instrument Co., Inc., Meriden, CT, USA)]. The glucose concentration was assayed according to the manufacturer’s instructions by Glucose Kit using an Automatic Biochemical Analyzer (Hitachi 747, Tokyo, Japan).

Preparation of membrane protein

The brain was harvested from NSE/hPS2m and non-Tg mice. Frozen brain was sectioned with scissors and homogenized in buffer A [10 mM Tris (pH 7.4), 1 mM EDTA, 250 mM Sucrose, proteinase inhibitor (pH 7.4)] with a glass homogenizer. The homogenate mixture was centrifuged at 900 × g for 10 min at 4°C to eliminate the unbroken cells. The supernatant was transferred to a new tube and centrifuged at 110,000 × g for 75 min at 4°C to collect the microsomal fraction. The pellet containing the microsomal fraction was resuspended in lysis buffer A containing 1% Triton X-100 for use in the western blot analysis.

Western blot analysis

The protein prepared from the brain tissues was separated by electrophoresis in a 4–20% SDS-PAGE gel for 3 h and transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was incubated separately with the primary, anti-insulin receptor α (sc-710; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-insulin receptor β (sc-711; Santa Cruz Biotechnology, Inc.), anti-IGF receptor (I7151; Sigma-Aldrich, St. Louis, MO, USA), anti-Glut-1 (sc-7903; Santa Cruz Biotechnology, Inc.), anti-Glut-3 (sc7582; Santa Cruz Biotechnology, Inc.), anti-Glut-4 (400064; Calbiochem, Darmstadt, Hesse, Germany), anti-AKT (Ab3130; Chemicon International), anti-p-AKT (Ab3132; Chemicon International), anti-GSK (9332; Cell Signaling Technology, Inc., Boston, MA, USA), anti-p-GSK (9331; Cell Signaling Technology, Inc.), anti-Tau (T7194; Sigma-Aldrich) and anti-p-Tau (T8069; Sigma-Aldrich) antibodies overnight at 4°C. The membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.05% Tween-20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000 dilution; Zymed) at room temperature for 2 h. Blots were developed using a Chemiluminescence Reagent Plus kit (ECL; Amersham Pharmacia).

Insulin treatments

Initially, the 12-month-old NSE/hPS2m Tg mice were divided into three subgroups: No-treatment group [1X phosphate-buffered saline (PBS)], low-dose group (0.2 units of insulin), high-dose group (0.4 units of insulin). Insulin (Novolin N, 100 IU/ml; GreenCross, Yongin, Korea) was subcutaneously injected into NSE/hPS2m Tg mice in a volume of 0.2 ml dilution in 1X PBS solution for 4 weeks.

Statistical analysis

Tests for significance between non-Tg and Tg mice were performed using a one-way ANOVA test of variance (SPSS for Windows, Release 10.10, standard version; SPSS, Chicago, IL, USA). Post-hoc tests of variance (SPSS for Windows, Release 10.10, standard version) were used to determine significance between insulin treatment and non-treatment Tg groups. Values were reported as the mean ± standard deviation (SD). P<0.05 was considered to indicate a statistically significant difference.

Results

Identification of AD phenotypes in NSE/hPS2m Tg mice

To detect the phenotypes of AD resulting from the overexpression of the hPS2m transgene, the Aβ-42 peptide and activity of γ-secretase were quantified in total brain tissue of 12-month-old NSE/hPS2m Tg mice by western blot analysis and γ-secretase activity assay, respectively. As shown in Fig. 1A, the level of Aβ-42 peptide was significantly higher in the brain of the NSE/hPS2m Tg mice compared with that of the non-Tg littermates. Also, γ-secretase activity was significantly increased in the brain of the NSE/hPS2m Tg mice (Fig. 1C). Furthermore, the level of p-Tau (Thr 231 site) aggregated into the neurofibrillary tangle in the cytoplasm of particular pyramidal neurons was significantly increased in the brain of NSE/hPS2m Tg mice (Fig. 1B). These results suggest that NSE/hPS2m Tg mice aged 12-months possessed the AD-like phenotypes involving the deposition of Aβ-42, the increase of γ-secretase activity and Tau hyperphosphorylation.

Figure 1 Characterization of AD phenotypes in the neuron-specific enolase (NSE)/hPS2m transgenic (Tg) mice. (A) Detection of Aβ-42 peptides. Lysates prepared from the brains were separated by 10–20% SDS-PAGE and transferred to nitrocellulose membranes. Aβ-42 levels were detected with antibodies raised against human Aβ-42 followed by anti-mouse-horseradish peroxidase (HRP). (B) Tau phosphorylation. The relative level of Tau phosphorylation was calculated at a rate of p-Tau level per Tau level. (C) Activity of γ-secretase. The γ-secretase activity in lysate prepared from brain extract was measured the fluorescence intensity at 335–355 nm wavelength by a Fluorescent Microplate Reader FL600. (D) Concentration of the insulin in serum. Detection of insulin concentration was performed the procedure suggested by manufacture using Coat-A-Count Insulin kit. (E) Concentration of the glucose in the plasma. Glucose concentration was assayed with Glucose kit using Automatic Biochemical Analyzer. Data are presented as the means ± standard error of the mean (SEM) from three replicates. *P<0.05, significance level compared with the non-Tg littermates.

Alteration of insulin and glucose levels in NSE/hPS2m Tg mice

To investigate whether cortical and hippocampal disorganization caused by Aβ-42 peptides affected the insulin and glucose levels, insulin and glucose concentrations were measured in the serum obtained from NSE/hPS2m Tg mice. In the Coat-A-Count Insulin kit analysis, the concentration of insulin was significantly lower in the NSE/hPS2m Tg mice (2.1 mg/dl) than that in the non-Tg littermates (6.2 mg/dl) (Fig. 1D). By contrast, the concentration of glucose was significantly higher in the NSE/hPS2m Tg mice (210 mg/dl) compared with that in the non-Tg littermates (120 mg/dl) (Fig. 1E). Therefore, changes of the insulin and glucose concentration in Tg mice indicated that the deposition of Aβ-42 peptide was directly associated with defects in the glucose metabolism.

Differential expression of insulin receptor and IFG receptor protein in NSE/hPS2m Tg mice

To determine whether the decrease in the insulin concentration caused by Aβ-42 peptide affected its related receptor expression, the expression levels of the insulin receptor and IGF receptor protein were measured using western blot analysis from the membrane protein of the brain tissues. Results of the western blot analysis revealed that the expression of insulin receptors α and β to be significantly reduced in the NSE/hPS2m Tg mice compared with the non-Tg littermates (Fig. 2A and B). However, these analyses also revealed no differences in the expression level of IGF receptors in the brain between the NSE/hPS2m Tg mice and non-Tg littermates (Fig. 2C). These results suggest that the defect in glucose regulation was capable of significantly decreasing the level of insulin receptors α and β, although the level of IGF receptor was unaffected.

Figure 2 Expression of insulin receptor and insulin-like growth factor I (IGF-I) receptor in the brain of neuron-specific enolase (NSE)/hPS2m transgenic (Tg) mice and non-Tg littermates. (A and B) Expression levels of the insulin receptor α and β. (C) Expression level of the IGF-1 receptor. The expression levels of three proteins in the membrane fraction prepared from the brain extract were detected with the primary anti-insulin receptor, and anti-IGF receptor antibody. Data are presented as the means ± standard error of the mean (SEM) from three independent experiments. *P<0.05, significance level compared with the non-Tg littermates.

Effect of glucose regulation defect on the insulin receptor signal transduction pathway

To test the hypothesis that the defect of the glucose metabolism in the AD model would alter the phosphorylation of signal protein in the insulin receptor signal pathway, the phosphorylation rate of AKT and GSK proteins in the brain of NSE/hPS2m Tg mice was detected. AKT2 is a key signaling molecule in the insulin signaling pathway that induces glucose transport (21). Glycogen synthase kinase 3 (GSK-3) involved in the Wnt signaling cascade may be inhibited following phosphorylation by AKT (22). The level of p-AKT protein involved in the downstream signal pathway of the insulin receptor was reduced in the brain of NSE/hPS2m Tg mice compare with non-Tg littermates (Fig. 3A). By contrast, the p-GSK-3β level was higher in the NSE/hPS2m Tg mice than the non-Tg littermates (Fig. 3B). Thus, this result suggests that the decrease in the insulin receptors α and β significantly induced the change of signal protein activation in the downstream signal pathway of the insulin receptor, respectively.

Figure 3 Phosphorylation of (A) AKT and (B) GSK in the neuron-specific enolase (NSE)/hPS2m transgenic (Tg) mice. The top panels demonstrate 40 μg of protein per sample, with an antibody against phosphorated protein. The bottom panels demonstrate the total protein, which was assessed using antibodies recognizing these proteins regardless of their phosphorylated states. Data are presented as the means ± standard error of the mean (SEM) from three independent experiments. *P<0.05 is the significance level compare with the non-Tg littermates.

Differential expression of glucose transporter in NSE/hPS2m Tg mice

To determine whether the decrease of the glucose level affected the expression of glucose transporter, western blot analysis was performed to detect the expression level of the glucose transporter in the membrane protein of mice brains. Fig. 4 shows the differences in the distribution of various molecular mass forms of Glut-1 and -3 in total cerebral homogenate. Two discrete bands at 45- and 55-kDa are observed in total homogenate. The density of the 45- and 55-kDa band in Glut-1 was significantly reduced in NSE/hPS2m Tg mice compared with that of non-Tg littermates (Fig. 4A). The 45-kDa band in Glut-3 was significantly reduced in the NSE/hPS2m Tg mice when this level was compared with non-Tg littermates. However, the density of the 55-kDa band was not significantly different between the NSE/hPS2m Tg and control mice (Fig. 4B). By contrast, there was no difference in the Glut-4 protein level between the NSE/hPS2m Tg mice and non-Tg littermates (Fig. 4C). These results suggest that the defect of glucose regulation may be attributed to the decrease in the expression level of Glut-1 and -3 proteins, but not for Glut-4.

Figure 4 Expression of glucose transporters in the brain of neuron-specific enolase (NSE)/hPS2m transgenic (Tg) mice and non-Tg littermates. The expression level of (A) anti-Glut-1, (B) anti-Glut-3 and (C) anti-Glut-4 in the membrane fraction prepared from the brain extract was detected using the specific primary antibody. Glut-1 and -3 proteins were detected as two different bands of 55 and 45-kDa. Data are presented as the means ± standard error of the mean (SEM) from three independent experiments. *P<0.05, significance level compared with the non-Tg littermates.

Effect of insulin administration on the deposition of the Aβ-42 peptide

Previously, Boyt et al(23) suggested that glucose ingestion and the subsequent elevation of plasma concentration of glucose and insulin lead to a decrease in the level of amyloid precursor protein in plasma. Therefore, to examine whether treatment of insulin in the NSE/hPS2m Tg mice affected the concentration of the Aβ-42 peptide, the insulin was injected into 12-month-old Tg mice. In RIA and serum biochemical analysis, the insulin concentration was significantly higher in the serum of insulin-treated mice than that of non-treated Tg mice. However, the glucose concentration in the insulin-treated mice was 3–4-fold lower than in the non-treated Tg mice, respectively (Fig. 5A). Furthermore, the western blot analysis revealed that the level of Aβ-42 peptide in the brain of the insulin-treated mice was slightly reduced compare with that of non-treated mice (Fig. 5B). Altering the level of Aβ-42 peptide from insulin-treated mice as shown by western blot analysis, suggests the possibility that insulin treatment is directly associated with a decrease in the Aβ-42 peptide (Fig. 5C).

Figure 5 Effect of insulin administration on the Aβ-42 peptide in the neuron-specific enolase (NSE)/hPS2m transgenic (Tg) mice. The NSE/hPS2m Tg mice were treated with 0.2 or 0.4 units of insulin for 4 weeks, while the control group were treated with 1X PBS solution for the same period of time. (A) The insulin level in serum was assayed with radioimmunoassay, (B) while the glucose levels were assayed with serum biochemical analysis. (C) The levels of Aβ-42 peptides were detected with anti-Aβ-42 antibody in the brain extracts from insulin-treated and non-treated Tg mice. Data are the means ± standard error of the mean (SEM) from three independent experiments. *P<0.05, significance level compared with the no-treated group. **P<0.05, significance level compared to the 0.2 units-treated group.

Discussion

One of the key functional disturbances in AD is the reduction in glucose utilization, which may be related to the increased Aβ-42 peptide deposition that occurs in the neocortical region and in walls of cerebral blood vessels. Glucose metabolism is important in brain disorders, as glucose is the energy source in the brain. In addition, the reduction in insulin concentration could induce disturbing glucose metabolism (24–26). Therefore, the present study was conducted with NSE/hPS2m Tg mice and non-Tg mice to observe the correlation between the β-amyloid peptide and glucose metabolism in the brain. A significant behavioral dysfunction in the water maze test and the levels of Aβ-42, caspase-3 and Cox-2 expression were especially observed in the brains of NSE/hPS2m Tg mice at 12-months of age (19). These Tg mice showed a 40–50% increase in the Aβ-42 peptide and γ-secretase activity from their brains at 12-months of age (Fig. 1A and B). A previous study reported that the NSE/hPS2m Tg mice developed a greater number of fibrillar Aβ deposits in the cortex and hippocampus than the non-Tg littermates (27). Furthermore, our results suggest that the NSE/hPS2m Tg mice used in this study exhibit AD-like phenotypes at 12-months of age.

Insulin is derived from a common precursor, proinsulin, from which these peptides are released in equimolar amounts by proteolytic cleavage (28). Insulin and Aβ peptide are also common substrates for insulin-degrading enzyme (29), which is activated in various tissues including brain tissues (30) and may be important in eliminating toxic amyloidogenic peptides (31,32). It has been demonstrated by cell culture and animal experiments that insulin in the brain potently effects neuronal glucose metabolism and cell differentiation (12,33). In our study, the level of insulin and its receptor between NSE/hPS2m Tg mice and non-Tg littermates were determined to examine whether the incidence of AD was able to affect the glucose metabolism pathway. RIA revealed that insulin concentration was significantly reduced in the NSE/hPS2m Tg mice compared with the non-transgenic mice. In addition, the expression of insulin receptor α and β chain decreased in the brain of the NSE/hPS2m Tg mice compared with that of the non-Tg littermates. With respect to insulin receptors in NSE/hPS2m Tg mice, we have, to the best of our knowledge, shown for the first time that the expression of insulin receptors increased in the brain of NSE/hPS2m Tg mice thereby increasing the Aβ-42 peptides by the overexpression of the mutant PS2 gene under the control neuron-specific promoter. We have also confirmed IGF-I receptor expression in the NSE/hPS2m Tg mice, as shown earlier in AD patients (18,34,35). The densities of these neurotrophic receptors were unchanged in NSE/hPS2m Tg mice in contrast to the insulin receptor. These results provide further evidence for specific involvement of brain insulin receptors in the pathogenesis of AD.

Additionally, insulin binds to IGF receptors and, via its receptor, possibly exerts more generalized trophic effects on neural cells and interaction with cholinergic neurotransmission (14,15). The binding of insulin to its receptor is followed by the autophosphorylation of tyrosine residues at the β-chain of insulin receptor resulting in subsequent activation of the intrinsic tyrosine kinase, which phosphorylates the initial endogenous substrate IRS-1 (16). Phosphorylated IRS-1 transfers signals to a wide spectrum of cellular signal transduction pathways (17). In the AD patients, the activation of AKT was decreased in the signaling pathway of insulin receptor downstream. By contrast, it was reported that the GSK-3β and Tau protein on the downstream of AKT protein were significantly activated in the brains of AD patients compared with the age-matched controls (36). Our results have shown that the phosphorylated AKT protein significantly decreased in the brains of NSE/hPS2m Tg mice compared with non-Tg littermates, while activated GSK-3 and Tau significantly increased. These observations suggest that expression of the hPS2m transgene might accelerate the pathogenic changes in glucose metabolism defect, through an AKT, a GSK-3 and a Tau phosphorylation, either directly or indirectly for underlying AD.

Findings of previous studies have shown decreased protein levels involved in glucose metabolism in AD patient brains. In senile dementia of Alzheimer type, the concentration of plasma glucose resulted in a significant increase compared with the age-matched control group (37,38). Significantly decreased cortical glucose transporter subtype (Glut-1 and Glut-3) was identified in the brain of AD patients compared with the age-matched controls (6,7,39). Glut-1 and -3 are expressed the major glucose transporter in the brain. Glut-1 can be detected as two molecular mass forms of 45- and 55-kDa, which differ in their extent of glycosylation (40). Our data have identified reduced Glut-1 and 3 proteins in the brain of NSE/hPS2m Tg mice, while no difference was observed in the Glut-4 protein level. Furthermore, the density of the 45- and 55-kDa bands in Glut-1 was significantly reduced in NSE/hPS2m Tg mice compared with controls. The 55-kDa band in Glut-3 was significantly reduced in the NSE/hPS2m Tg mice compared to the non-Tg littermates. However, the density of the 45-kDa bands was not significantly different between the NSE/hPS2m Tg mice and non-Tg littermates. These results suggest the defect of glucose metabolism in the brain of NSE/hPS2m Tg mice is significantly associated with the lower expression of the Glut-1 and -3 proteins, but not with Glut-4.

We also examined the effect of insulin on the deposition of Aβ-42 peptides in the brain. Previous studies have suggested that the subsequent elevation of plasma insulin leads to a decrease in plasma amyloid precursor protein concentration (23,41). In this study, when the NSE/hPS2m Tg mice were treated with insulin by subcutaneous injection for 4 weeks, the level of the Aβ-42 peptide was slightly decreased in the treated NSE/hPS2m Tg mice compared with the non-treated NSE/hPS2m Tg mice. This observation suggests that insulin is important in Aβ-42 peptide degradation processing, although the nature of this role and the specific mechanisms remain to be elucidated. Therefore, more studies are required to investigate the detailed mechanism for the correlation between the insulin concentration and amyloid precursor protein and the clinical significance of the physiological changes in the insulin treatment condition.

Acknowledgements

We would like to thank Dr Jun Yong Cho at the Korea National Sport University for consulting of glucose metabolism analyses. This study was supported by the 2012 Specialization Project Research Grant funded by the Pusan National University.

References