March 2016
Volume 57, Issue 3
Open Access
Lens  |   March 2016
Hyperglycemia Enhances the Production of Amyloid β1–42 in the Lenses of Otsuka Long-Evans Tokushima Fatty Rats, a Model of Human Type 2 Diabetes
Author Affiliations & Notes
  • Noriaki Nagai
    Faculty of Pharmacy Kinki University, Higashi-Osaka, Osaka, Japan
  • Yoshimasa Ito
    Faculty of Pharmacy Kinki University, Higashi-Osaka, Osaka, Japan
  • Hiroshi Sasaki
    Department of Ophthalmology, Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan
  • Correspondence: Yoshimasa Ito, Faculty of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan; itoyoshi@phar.kindai.ac.jp 
Investigative Ophthalmology & Visual Science March 2016, Vol.57, 1408-1417. doi:10.1167/iovs.15-19026
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      Noriaki Nagai, Yoshimasa Ito, Hiroshi Sasaki; Hyperglycemia Enhances the Production of Amyloid β1–42 in the Lenses of Otsuka Long-Evans Tokushima Fatty Rats, a Model of Human Type 2 Diabetes. Invest. Ophthalmol. Vis. Sci. 2016;57(3):1408-1417. doi: 10.1167/iovs.15-19026.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: It has been reported that the accumulation of amyloid β1–42 (Aβ1–42) in human lenses can cause some forms of lens opacification. However, the factors leading to changes in the accumulation of Aβ in the lens remain obscure. In this study, we investigate the effect of hyperglycemia on Aβ1–42 accumulation in lenses.

Methods: Otsuka Long-Evans Tokushima Fatty (OLETF) rats and the human lens epithelial cell line SRA 01/04 (HLE cells) were used. The expression of mRNA was determined using a quantitative real-time RT-PCR method; Aβ1–42 levels were analyzed by an ELISA method.

Results: Otsuka Long-Evans Tokushima Fatty rats at more than 20 weeks of age develop diabetes mellitus with hyperglycemia. Additionally, the levels of the mRNAs for Aβ1–42, amyloid precursor proteins (APP), β-(BACE1), and·γ-secretase (PS) rise in the lenses of OLETF rats with age; high Aβ1–42 levels are observed in the lens capsule–epithelium and cortex. The enhanced expression of the genes for APP, BACE1, and PS in the lenses of OLETF rats is prevented by food restriction (25 g/d/rat). When the effect of glucose levels on the production of Aβ1-42 was investigated in the human lens epithelial cell line SRA 01/04 (HLE cells), the mRNA levels for APP, BACE1, and PS, as well as Aβ1-42 protein levels, were significantly higher under high glucose conditions (20 mM) than under normal glucose conditions (5.6 mM).

Conclusions: High glucose leads to the increased expression of genes related to Aβ production, resulting in the accumulation of Aβ in the lens.

Amyloid β (Aβ) denotes peptides of 39 to 43 amino acids formed by the cleavage of amyloid precursor protein (APP); Aβ1–40, Aβ1–42, and Aβ1–43 peptides result when APP is cleaved after residues 40, 42, and 43, respectively.15 Amyloid β accumulation in the brain is the hallmark of Alzheimer's disease. Amyloid β is produced by the sequential proteolytic processing of APP by β-secretase (β-site APP cleaving enzyme, known as BACE1)6 and γ-secretase (a presenilin complex comprising PS1 and PS2).7 Disintegrin and metalloprotease domain protease 10 (ADAM10) are the major proteases for the α-cleavage of APP.810 Amyloid precursor protein undergoes α-cleavage within the Aβ domain to generate non-amyloidogenic soluble APPα. In a previous study, Aβ accumulation was observed in the cytosol of lens fiber cells of people with Alzheimer's disease.11 In addition, Jun et al.12 suggest that cortical cataracts are coheritable with the future development of Alzheimer's disease. Frederikse et al. found the expressions of β-secretase and γ-secretase in mammalian lenses13,14 and show that oxidative stress enhances Aβ production via an increase in APP in mammalian lenses.15 Moncaster et al. also report enhanced Aβ expression in the lens of a patient with Down Syndrome, which resulted in amyloidogenic interactions with other structural proteins (e.g., αB-crystallin) within the cytoplasm of supranuclear lens fiber cells that led to increased lens protein aggregation, light scattering, and opacification.16 In contrast, Michael et al.17 and Hopkin et al.18 claim Aβ is not present in the lenses of Alzheimer's and/or Parkinson's disease patients; therefore, the question of whether or not Aβ accumulates in human cataracts is currently controversial. On the other hand, substantial epidemiologic evidence indicates that patients with diabetes mellitus are at increased risk of developing Alzheimer's disease,19 and researchers have found that a decrease in insulin-degrading enzyme activity under high glucose conditions reduces Aβ degradation and elimination from the brain.20,21 In addition, it has been reported that the Aβ and tau pathology of Alzheimer's disease is induced by the onset of diabetes in a rabbit model.22 However, it remains unclear whether diabetes mellitus also leads to Aβ accumulation in the lens and whether high glucose conditions affect the Aβ production system. Therefore, it is necessary to elucidate the relationship between high glucose conditions and Aβ accumulation in the lenses of diabetic patients. 
In previous studies, streptozotocin-induced diabetic rats and galactose-fed rats, both models for insulin-dependent diabetes mellitus, have been used to investigate diabetic cataracts2327; however, the general pathophysiology of these models differs from that in humans because most diabetes mellitus in humans is not insulin dependent. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a known model for human type 2 diabetes mellitus via a metabolic syndrome.28 Nearly 100% of male OLETF rats show hyperglycemia and hyperinsulinemia as a result of islet cell hyperplasia and peripheral insulin resistance and develop diabetic syndrome spontaneously at 20 weeks of age.2832 With continued aging, OLETF rats develop hypoinsulinemia that results from the deterioration of islet β cells,31,32 and the plasma insulin levels in OLETF rats greater than 60 weeks of age are lower than in their normal controls, Long-Evans Tokushima Otsuka (LETO) rats.33,34 In addition, lens opacification has been observed in OLETF rats greater than 60 weeks of age.35 Based on these findings, OLETF rats may provide a better model than streptozotocin-induced diabetic rats or galactose-fed rats for studies to clarify the expression of Aβ in diabetic lens. In the present study, we examined hyperglycemia and the accumulation of APP, Aβ, and the mRNAs for APP processing enzymes BACE, PS1, PS2, and ADAM10 in the lenses of OLETF and wild-type control rats. 
Methods
Animals and Materials
Male LETO (normal rats) and OLETF rats provided by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan) and the human lens epithelial cell line SRA 01/04 (HLE cells)36 were used in this study. All procedures were performed in accordance with the Kinki University School of Pharmacy Committee for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. The Glucose Assay Kit and ELISA Insulin Kit were provided by BioVision, Inc. (Milpitas, CA, USA) and Morinaga Institute of Biological Science, Inc. (Kanagawa, Japan), respectively. Accutrend GCT was obtained from Roche Diagnostics (Mannheim, Germany), and the Cholesterol E-Test Kit and Rat β Amyloid (42) ELISA Kit were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of the highest purity commercially available. 
Food Restriction
The diet groups were allowed free access to a commercial diet (CE-2; Clea Japan, Inc., Tokyo, Japan) and water until 40 weeks of age; food restriction was carried out in OLETF rats from 40 to 80 weeks of age (diet group). The diet group was fed a commercial diet (25 g/d/rat) and water (free access). The amount of food intake (25 g/d/rat) was determined based on the amount eaten by 10-week-old OLETF rats fed freely. 
Blood Test for Diabetes Mellitus
Blood parameters for diabetes mellitus were assessed according to a previous study.35 Rats were fasted for 15 hours, and blood was drawn from a tail vein at 9 AM without anesthesia. Plasma glucose (Glu), triglycerides (TG), total cholesterol (Cho), and insulin were measured by a Glucose Assay Kit, Accutrend GCT, Cholesterol E-Test Kit, and ELISA Insulin Kit, respectively, according to the manufacturers' instructions. 
RNA Preparation
Rats were euthanized by injection of a lethal dose of sodium pentobarbital, the eyes were removed, and the lenses were isolated. Total RNA was obtained from the lenses using the acid guanidium thiocyanate-phenol-chloroform extraction method.37 For cultured HLC cells, total RNA was extracted and purified using an RNeasy Mini Kit and RNase-Free DNase Set (Qiagen, Tokyo, Japan), respectively. 
Quantitative Real-Time RT-PCR
The RT reaction was performed using an RNA PCR Kit (AMV Ver 3.0; Takara Bio, Inc., Shiga, Japan) according to the manufacturer's instructions. The RT reaction was performed at 42°C for 15 minutes, followed by 5 minutes at 95°C. The PCR reactions were performed using LightCycler FastStart DNA Master SYBR Green I according to the manufacturer's instructions (Roche Diagnostics Applied Science, Mannheim, Germany). The specific primers used for PCR (10 pM) are shown in Table 1. The conditions for PCR were as follows: 95°C for 10 minutes (hot start), 60 cycles of 95°C for 10 seconds (denaturing), 61°C (samples from rat lenses), or 63°C (samples from cultured human cells) for 10 seconds (annealing), and 72°C for 5 seconds (extension). The quantities of the PCR products were measured fluorometrically in a real-time manner using a LightCycler DX 400 (Roche Diagnostics Applied Science). The differences in the threshold cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other groups (APP, ADAM10, BACE1, PS1, and PS2) were used to calculate the levels of mRNA expression in OLETF rats. 
Table 1
 
Sequences of Primers Used for Quantitative RT-PCR Analysis
Table 1
 
Sequences of Primers Used for Quantitative RT-PCR Analysis
Measurement of Aβ1–42 Levels
Rats were euthanized by injection of a lethal dose of sodium pentobarbital, and the lenses were removed (total lens). The epithelium-containing capsules (capsule–epithelium) were carefully removed and separated from the nucleus and cortical portions. The cortex was also separated, and the remaining lens other than the capsule–epithelium and cortex was used as the nucleus. Cultured HLE cells were collected with a cell scraper (Asahi Glass Co., Ltd., Tokyo, Japan). The total lens, separated lens (parts of capsule–epithelium, cortex, nucleus), and cell samples were homogenized in 100–500 μL diethylamine solution (0.2% diethylamine and 50 mM NaCl) and centrifuged at 100,000g for 1 hour at 4°C. The supernatants were used for the measurement of soluble Aβ1–42. The pellets were dissolved in 200 μL formic acid solution and neutralized with 1 M Tris buffer. These solutions were for the measurement of insoluble Aβ142.38,39142 levels were measured using a rat β amyloid (42) ELISA Kit (dynamic range 0.1–20 pM) according to the manufacturer's instructions (Wako).39 Amyloid β1–42 levels reflect total Aβ1–42 levels (both soluble and insoluble Aβ1-42) for each lens and are expressed as fmol/g or pmol/g protein. The protein levels in samples used to determine Aβ1–42 levels were assessed according to the method of Bradford40 using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). 
Oral Glucose Tolerance Test
Normal and OLETF rats, 40 or 80 weeks of age, were fasted for 15 hours prior to the oral administration of glucose (3 g/kg). Blood samples were taken from the tail veins at intervals between 0 (just before glucose administration) and 180 minutes, and Glu and insulin levels were determined by a Glucose Assay Kit and ELISA Insulin Kit, respectively, according to the manufacturers' instructions. The rats were euthanized under deep ether anesthesia 180 minutes after glucose administration, and the eyes were removed. The aqueous humor was collected from the eyes through a 29-gauge injection needle, and the lenses were isolated for the measurement of glucose, insulin, mRNA, and Aβ1–42 levels. 
Image Analysis of Cataract Development in OLETF Rats
The experiment was performed as described previously.35 The rat pupils were dilated by the instillation of 0.1% pivalephrine (Santen Pharmaceutical Co., Osaka, Japan) without anesthesia. Changes in the transparency of the lenses were monitored using an EAS-1000 (Nidek, Gamagori, Japan). The outline of the lens image was determined by selecting four points on the image, and the opaque area within the outline and defining level were set automatically by the software. The EAS-1000 conditions were set to slit length and width 5.0 mm, flash level 100 W/s, flash power index 2300 ± 66, threshold level 30. The total area of opacity of the lenses, expressed as pixels, was calculated based on the following equation: pixels within opacity area (pixel) = pixels within outline − pixels within transparent area. 
Western Blot Analysis
The experiment was performed as described previously.39 Rats were euthanized by injection of a lethal dose of sodium pentobarbital, and the lenses were removed (total lens). The HLE cells were collected by a scraper. The total protein (10 μg) in the rat lens or HLE cell was separated in a 15% polyacrylamide SDS gel, and the proteins were then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). After transfer, the blots were probed with 0.06 mg/L rabbit anti-rat or -human amyloid β polyclonal antibody (Rockland Immunochemicals, Inc., Pottstown, PA, USA) or with 0.07 mg/L rabbit anti-rat or -human GAPDH polyclonal antibody (Imgenex, CA, USA) for 2 hours at room temperature. After that, the membranes were incubated with secondary alkaline phosphatase–conjugated anti-rabbit IgG (1:7000 dilution; Promega, Madison, WI, USA) for 2 hours at room temperature and incubated with a stabilized substrate for alkaline phosphatase (Promega). The anti-rat and anti-human amyloid β polyclonal antibodies detect a higher-molecular-weight precursor form of amyloid β protein, which appears as a band at 37–50 kDa (APP-Aβ intermediate protein). 
Cell Culture and Treatments
Human lens epithelial cells were cultured according to our previous report.36 The percentages of cells in culture flasks were determined under a light microscope; treatments were carried out when the cells were approximately 80% confluent, usually on the third day after seeding (4.0 × 103 cells/cm2). The culture medium was changed every other day, as well as 1 hour before each experiment. For experiments, culture medium without fetal bovine serum was used, and the cells were incubated in the presence of 5.6 (control) or 20 mM glucose for 24 hours. 
Statistical Analysis
Unpaired Student's t-test, Aspin-Welch's t-test, or 1-way ANOVA followed by Dunnett's multiple comparison was used to evaluate statistical difference. P < 0.05 was considered significant. All data are expressed as the mean ± SE of the mean. 
Results
Changes in Aβ1–42 Levels in the Lenses of OLETF Rats During Diabetes Mellitus Development
Figure 1 shows body weight (A), food intake (B) and Glu (C), TG (D), Cho (E), and insulin (F) levels in blood from 10- to 80-week-old normal and OLETF rats. Food intake was significantly greater in OLETF rats compared with normal rats, with 80-week-old OLETF rats consuming 35.4 ± 1.9 g/d/rat. The Glu, TG, Cho, and insulin levels in 10-week-old OLETF rats were similar to those in 10-week-old normal rats. These values did not change with age in normal rats, but increased in OLETF rats. By 20 to 40 weeks of age, type 2 diabetes mellitus had developed in the OLETF rats with metabolic syndrome, and body weights and Glu, PG, TG, Cho, and insulin levels were all significantly higher than in normal rats. By 60–80 weeks of age, the OLETF rats had developed hypoinsulinemia, and the body weights and plasma insulin levels were lower than those of normal rats. Figure 2 shows the levels of opacity and Aβ1–42 in the lenses of 10- to 80-week-old normal and OLETF rats as determined by EAS-1000 and ELISA methods. Opacity levels in the anterior subcapsular area increased slightly after 40 weeks in the OLETF rats, and the accumulation of Aβ1–42 was observed in 40- to 80-week-old OLETF rats. In addition, the Aβ1–42 levels in the capsule–epithelium, cortex and nucleus of lenses of 80-week-old OLETF rats were 16.1, 13.25, and 1.15 fmol/g protein, respectively. Figures 3A through 3E show the expressions of the mRNAs for APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) in the lenses of 10- to 80-week-old normal and OLETF rats as determined by quantitative real-time PCR methods. Although, the gene expression levels of proteins related to Aβ production (APP, ADAM10, BACE1, PS1, and PS2) and Aβ1–42 levels did not change with age in normal rats, they all rose in the lenses of OLETF rats. Figure 3F shows the expressions of full-length and broken APP in the lenses of 80-week-old normal and OLETF rats as determined by Western blot. The antibody detected the full-length APP at just above 100 kDa; however, the amount of full-length APP was slightly. The amount was similar between normal and OLETF rats, and no band around 4 kDa (Aβ) was detected. On the other hand, a band at 37–50 kDa (APP-Aβ intermediate protein) was clearly observed, and the amount was higher in the lenses of 80-week-old OLEF rats compared with 80-week-old normal rats. 
Figure 1
 
Changes in body weight (A), food intake (B), and Glu (C), TG (D), Cho (E), and insulin (F) levels in the blood from 10- to 80-week-old normal and OLETF rats. Open circles, normal rats; closed circles, OLETF rats. Data are presented as means ± SE of 7–10 independent rats. *P < 0.05 versus normal rats for each group. The increase in food intake corresponded with increased glucose, cholesterol, and TG in OLETF rats. The body weight increased to a maximum at 40 weeks and then decreased corresponding with the insulin levels.
Figure 1
 
Changes in body weight (A), food intake (B), and Glu (C), TG (D), Cho (E), and insulin (F) levels in the blood from 10- to 80-week-old normal and OLETF rats. Open circles, normal rats; closed circles, OLETF rats. Data are presented as means ± SE of 7–10 independent rats. *P < 0.05 versus normal rats for each group. The increase in food intake corresponded with increased glucose, cholesterol, and TG in OLETF rats. The body weight increased to a maximum at 40 weeks and then decreased corresponding with the insulin levels.
Figure 2
 
Changes in opacification and Aβ1–42 levels in 10- to 80-week-old normal and OLETF rats. (A) Scheimpflug slit images of lenses from 80-week-old normal and OLETF rats. The outline of the lens image used for analysis (dashed lines) was set by selecting four points on the software, and the opaque area within the outline was measured as pixels. In the analysis of lens opacity, the area of reflected light (472 ± 32 pixels) was removed. (B) Images of lenses from postmortem 80-week-old normal and OLETF rats. (C) Opacity and (D) Aβ1–42 levels in the total lenses of 10- to 80-week-old normal and OLETF rats. (E) Aβ1–42 levels in the separated lens (capsule–epithelium, cortex, and nucleus) of 80-week-old normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of 5–10 independent rat lenses. *P < 0.05 versus normal rats for each group. The Aβ1–42 production was enhanced in the capsule–epithelium and cortex of lenses from OLETF rats but not in the nucleus.
Figure 2
 
Changes in opacification and Aβ1–42 levels in 10- to 80-week-old normal and OLETF rats. (A) Scheimpflug slit images of lenses from 80-week-old normal and OLETF rats. The outline of the lens image used for analysis (dashed lines) was set by selecting four points on the software, and the opaque area within the outline was measured as pixels. In the analysis of lens opacity, the area of reflected light (472 ± 32 pixels) was removed. (B) Images of lenses from postmortem 80-week-old normal and OLETF rats. (C) Opacity and (D) Aβ1–42 levels in the total lenses of 10- to 80-week-old normal and OLETF rats. (E) Aβ1–42 levels in the separated lens (capsule–epithelium, cortex, and nucleus) of 80-week-old normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of 5–10 independent rat lenses. *P < 0.05 versus normal rats for each group. The Aβ1–42 production was enhanced in the capsule–epithelium and cortex of lenses from OLETF rats but not in the nucleus.
Figure 3
 
Expressions of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate protein (F) measured in total lenses of normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of five to six independent rat lenses. *P < 0.05 versus normal rats for each group. The mRNA for enzymes necessary for the proteolysis of APP to Aβ and the APP-Aβ intermediate protein (37–50 kDa) increased with age in the lenses of OLETF rats.
Figure 3
 
Expressions of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate protein (F) measured in total lenses of normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of five to six independent rat lenses. *P < 0.05 versus normal rats for each group. The mRNA for enzymes necessary for the proteolysis of APP to Aβ and the APP-Aβ intermediate protein (37–50 kDa) increased with age in the lenses of OLETF rats.
Effect of Glucose Levels on Aβ1–42 Production in the Lenses of OLETF Rats
Figures 4A and 4B show the changes in Glu levels in 40- (A) and 80-week-old (B) normal and OLETF rats subjected to the oral glucose tolerance (OGT) test. Glucose levels of 40- and 80-week-old normal and OLETF rats reached a peak 30–60 minutes after the oral administration of glucose. The Glu levels in 40- and 80-week-old normal rats then decreased gradually and reached preprandial levels 120 minutes after glucose administration. In contrast, the Glu levels in 40- and 80-week-old OLETF rats had still not returned to preprandial levels 180 minutes after glucose administration. Figure 4C shows the plasma insulin levels in 40- and 80-week-old normal and OLETF rats subjected to the OGT test. Although plasma insulin levels rose in 40-week-old OLETF rats following glucose administration, no changes in plasma insulin levels were observed in 80-week-old OLETF rats either with or without glucose administration. On the other hand, glucose levels in the aqueous humor rose following glucose administration in both 40- and 80-week-old OLETF rats (Fig. 4D). Figure 5 shows the mRNA expression levels for APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) and APP-Aβ intermediate protein (F), as well as the Aβ1-42 levels (G), in the lenses of 40- and/or 80-week-old normal and OLETF rats 180 minutes after glucose administration. Although no changes in the expression levels of genes related to Aβ production (APP, ADAM10, BACE1, PS1, and PS2) or in Aβ1–42 levels were observed in normal rats, the mRNA expression levels for APP, BACE1, PS1, and PS2 were increased in the lenses of OLETF rats at 180 minutes after glucose administration. The APP-Aβ intermediate protein expression in the lenses of OLETF rats was also increased slightly by glucose administration. On the other hand, the mRNA expression levels for ADAM10 and Aβ1-42 levels were similar in the lenses of OLETF rat with or without the administration of glucose. Table 2 shows the body weights and plasma Glu, TG, Cho, and insulin levels, and Figure 6 shows the lens gene expression levels of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), PS2 (E), APP-Aβ intermediate protein (F), and Aβ1–42 levels (G) for 80-week-old OLETF rats on a food-restricted diet (diet group). The body weights and some blood test values for diabetes mellitus (Glu, TG, Cho, and insulin), as well as the gene expression levels of proteins related to Aβ production (APP, ADAM10, BACE1, PS1, PS2), in the diet group were similar to those of normal 80-week-old rats. Moreover, the expression of the APP-Aβ intermediate protein was slightly lower in the diet groups compared with the control groups. However, Aβ1–42 levels in the diet group were significantly higher than in normal rats. Amyloid β1–42 levels in the capsule–epithelium, cortex, and nucleus of the lenses from 80-week-old food-restricted OLETF rats were 6.41 ± 0.46, 9.18 ± 0.48, and 1.32 ± 0.15 fmol/g protein, respectively (mean ± SE, n = 5). Figure 7 shows the changes in plasma Glu, Aβ1–42 levels, and lens opacity in OLETF rats with aging. The increase in opacity was observed following the accumulation of Aβ1–42 in the lenses of OLETF rats. 
Figure 4
 
Changes in glucose and insulin levels in 40- and 80-week-old normal and OLETF rats in response to an OGT test. (A, B) Changes in Glu levels in 40- (A) and 80-week-old (B) normal and OLETF rats after oral administration of glucose (3 g/kg). Open circles, normal rats; closed circles, OLETF rats. The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats without glucose administration. *2P < 0.05 versus OLETF rats without glucose administration. (C, D) Insulin levels in blood (C) and glucose levels in the aqueous humor (D) of 40- and 80-week-old normal and OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *3P < 0.05 versus normal rats for each category. *4P < 0.05 versus Control for each category. Increased glucose levels in the OLETF rats at 40 and 80 weeks of age corresponded with insulin levels in the blood and increased glucose levels in the aqueous humor of OLETF rats relative to normal rats.
Figure 4
 
Changes in glucose and insulin levels in 40- and 80-week-old normal and OLETF rats in response to an OGT test. (A, B) Changes in Glu levels in 40- (A) and 80-week-old (B) normal and OLETF rats after oral administration of glucose (3 g/kg). Open circles, normal rats; closed circles, OLETF rats. The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats without glucose administration. *2P < 0.05 versus OLETF rats without glucose administration. (C, D) Insulin levels in blood (C) and glucose levels in the aqueous humor (D) of 40- and 80-week-old normal and OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *3P < 0.05 versus normal rats for each category. *4P < 0.05 versus Control for each category. Increased glucose levels in the OLETF rats at 40 and 80 weeks of age corresponded with insulin levels in the blood and increased glucose levels in the aqueous humor of OLETF rats relative to normal rats.
Figure 5
 
Changes in APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 40- and 80-week-old normal and/or OLETF rats after oral administration of glucose (3 g/kg). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats for each category. *2P < 0.05 versus Control for each category. The increase in glucose levels in the blood correspond to the increased mRNA levels for enzymes necessary for the proteolysis of APP to Aβ.
Figure 5
 
Changes in APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 40- and 80-week-old normal and/or OLETF rats after oral administration of glucose (3 g/kg). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats for each category. *2P < 0.05 versus Control for each category. The increase in glucose levels in the blood correspond to the increased mRNA levels for enzymes necessary for the proteolysis of APP to Aβ.
Table 2
 
Body Weight and Some Blood Test Values for Diabetes Mellitus in 80-Week-Old OLETF Rats With Food Restriction
Table 2
 
Body Weight and Some Blood Test Values for Diabetes Mellitus in 80-Week-Old OLETF Rats With Food Restriction
Figure 6
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 80-week-old normal and OLETF rats with food restriction. In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Food restriction was carried out on OLETF rats from 40 to 80 weeks of age (diet group). Normal: 80-week-old normal rats allowed free access to a commercial diet and water. Control: 80-week-old OLETF rats allowed free access to a commercial diet and water. Diet: 80-week-old OLETF rats fed a commercial diet (25 g/d/rat) and water (free access). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal group for each category. *2P < 0.05 versus control group for each category. The increase in mRNA for enzymes necessary for proteolysis of APP to Aβ and the APP-Aβ intermediate protein in the lenses of OLETF rats were improved by food restriction.
Figure 6
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 80-week-old normal and OLETF rats with food restriction. In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Food restriction was carried out on OLETF rats from 40 to 80 weeks of age (diet group). Normal: 80-week-old normal rats allowed free access to a commercial diet and water. Control: 80-week-old OLETF rats allowed free access to a commercial diet and water. Diet: 80-week-old OLETF rats fed a commercial diet (25 g/d/rat) and water (free access). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal group for each category. *2P < 0.05 versus control group for each category. The increase in mRNA for enzymes necessary for proteolysis of APP to Aβ and the APP-Aβ intermediate protein in the lenses of OLETF rats were improved by food restriction.
Figure 7
 
Temporal relationships between plasma Glu and Aβ1–42 levels and lens opacity in OLETF rats. Glucose levels in the blood (circles) and Aβ1–42 levels in the total lenses (triangles) were determined by an ELISA method. Lens opacity (squares) was measured using the EAS-1000. For each parameter, the highest value attained was taken as 100%. The data are presented as means ± SE of 5–10 rats. Opacity in anterior subcapsular increased slightly after the accumulation of Aβ1–42 in the lenses of OLETF rats.
Figure 7
 
Temporal relationships between plasma Glu and Aβ1–42 levels and lens opacity in OLETF rats. Glucose levels in the blood (circles) and Aβ1–42 levels in the total lenses (triangles) were determined by an ELISA method. Lens opacity (squares) was measured using the EAS-1000. For each parameter, the highest value attained was taken as 100%. The data are presented as means ± SE of 5–10 rats. Opacity in anterior subcapsular increased slightly after the accumulation of Aβ1–42 in the lenses of OLETF rats.
Effect of Glucose Levels on Aβ1–42 Production in HLE Cells
Figure 8 shows the mRNA expression levels for APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E), APP-Aβ intermediate protein (F), and Aβ1–42 levels (F) in HLE cells cultured in the presence or absence of high glucose concentrations. The ADAM10 mRNA level was the same in HLE cells regardless of glucose treatment; however, in contrast, APP, BACE1, PS1, and PS2 mRNA levels, as well as the Aβ1–42 level, were significantly higher under high glucose conditions. Moreover, the expression of the APP-Aβ intermediate protein was also enhanced by treatment with glucose. 
Figure 8
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, APP-Aβ intermediate protein (F), and Aβ1–42 levels (G) in HLE cells cultured with or without of high glucose (20 mM). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, nontreated HLE cells (Control); closed columns, glucose-treated HLE cells (Glucose). The data are presented as means ± SE of 10 independent experiments. *P < 0.05 versus nontreated HLE cells (Control). In the presence of 20 mM glucose, HLE cells showed increased mRNA expression of enzymes required for the proteolysis of APP and APP-Aβ intermediate protein. A 4.12-fold increase in Aβ1–42 was observed after 24 hours in culture.
Figure 8
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, APP-Aβ intermediate protein (F), and Aβ1–42 levels (G) in HLE cells cultured with or without of high glucose (20 mM). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, nontreated HLE cells (Control); closed columns, glucose-treated HLE cells (Glucose). The data are presented as means ± SE of 10 independent experiments. *P < 0.05 versus nontreated HLE cells (Control). In the presence of 20 mM glucose, HLE cells showed increased mRNA expression of enzymes required for the proteolysis of APP and APP-Aβ intermediate protein. A 4.12-fold increase in Aβ1–42 was observed after 24 hours in culture.
Discussion
The body weights of 40-week-old OLETF rats were approximately 1.48-fold higher than those of 40-week-old normal rats. Plasma concentrations of Glu, TG, and Cho increased with age in OLETF rats, and plasma insulin levels in 40-week-old OLETF rats were also higher than in 40-week-old normal rats. These results indicate that 40-week-old OLETF rats developed diabetes mellitus with metabolic syndrome and insulin resistance (Fig. 1). In contrast with the results in 40-week-old OLETF rats, the body weights and plasma insulin levels of 60- to 80-week-old OLETF rats were lower than those of 60- to 80-week-old normal rats (Fig. 1). Hirashima et al.33 report atrophy or the disappearance of β cells in OLETF rats older than 60 weeks of age and found that plasma insulin levels are lower than in normal rats.34 These findings suggest that the type 2 diabetes mellitus in 60-week-old OLETF rats has reached a fairly advanced stage and that the reduced body weight and plasma insulin changes may be due to the deterioration of islet β cells with the progression of the disease. 
In this study, we investigated the changes in Aβ in the lenses of OLETF rats with diabetes mellitus. In experiments using Aβ peptides of primate, rodent, and Dutch-hemorrhagic analogues, amyloid fibrils were found to assemble from Aβ1–40, Aβ1–42, and Aβ1–43 peptides,14,41,42 and it has been reported that Aβ1–42 and Aβ1–43 aggregate more strongly than Aβ1–40 in the brains of knock-in mice containing a pathogenic PS-1 R278I.5 Moreover, the accumulation of Aβ1–42 was found to be higher than that of Aβ1–43 in the brains of the knock-in mice.5 On the other hand, Moncaster et al. reported that enhanced Aβ expression in the lens results in amyloidogenic interactions with other structural proteins such as αB-crystallin, resulting in lens opacification.16 In addition, Aβ accumulation has been observed in the cytosol of lens fiber cells of people with Alzheimer's disease,11 and we previously reported that the accumulation of Aβ1–42 is related to lens opacification in cataract model rats.39 From these findings, we believe that the measurement of Aβ1–42 levels, which are increased in the cataract model,39 may be more important than the measurement of Aβ1–40 and Aβ1–43 levels when trying to elucidate the relationship between Aβ accumulation and toxicity in the lens. Therefore, we investigated changes in Aβ1–42 levels in the lenses of OLETF rats. At first, we attempted to measure the amount of Aβ1–42 and its localization in lenses using an immunologic method; however, precise, reproducible results could not be obtained by this method for OLETF rats. We believe that Aβ1–42 levels in the lens are remarkably low and that the lenses of 60- and 80-week-old OLETF rats, which accumulate Aβ1–42, are just starting to undergo lens opacification. Based on these findings, we measured the amounts of Aβ1–42 using a highly sensitive ELISA method, and dissected the lenses into capsule–epithelium, cortex, and nucleus to clarify Aβ1–42 localization. Amyloid β1–42 levels in the lenses of OLETF rats were found to increase with aging (Fig. 2D), and high levels were observed in the lens capsule–epithelium and cortex (Fig. 2E). In addition, the gene expression levels of proteins related to Aβ production (APP, BACE1, PS1, and PS2) rose in the lenses of OLETF rats with aging (Fig. 3), and the levels of both soluble and insoluble Aβ1–42 were increased in the lenses OLETF rats (soluble, 5.01 ± 0.20 fmol/g protein; insoluble, 6.55 ± 0.31 fmol/g protein; mean ± SE, n = 6). These results suggest that the development of diabetes mellitus causes an increase in the production of Aβ1–42 in the lenses of OLETF rats. On the other hand, the expression of the gene for ADAM10, which·is related to the suppression of Aβ production, was also found to increase significantly in the lenses of OLETF rats with aging (Fig. 3B). The induction of ADAM10 gene expression may be caused by the enhanced Aβ1–42 levels in the lenses of OLETF rats. We checked the expression of the Aβ protein by Western blot. In this study, a band at 37–50 kDa (APP-Aβ intermediate protein) was clearly observed (the band for full-length APP at just above 100 kDa was faintly observed, whereas no band around 4 kDa [Aβ] was detected). We thought that this result might be due to the amount of Aβ and the sensitivity of antibody. Therefore, we compared the expression of the Aβ protein using the 37- to 50-kDa band (APP-Aβ intermediate protein) and showed that the expression of the APP-Aβ intermediate protein (37–50 kDa) is higher in the lenses of 80-week-old OLEF rat compared with 80-week-old normal rats. 
Next, we investigated the effect of hyperglycemia on Aβ accumulation in the lenses using 40-week-old OLETF rats with hyperinsulinemia and 80-week-old OLETF rats with hypoinsulinemia. The glucose levels in the aqueous humor were similar to the plasma Glu levels in 40- and 80-week-old OLETF rats, and the oral administration of glucose enhanced the glucose levels in the aqueous humor (Fig. 4D). The expressions of the mRNAs for APP, BACE1, and PS were found to have increased in the lenses of OLETF rat 180 minutes after glucose administration (Fig. 5). Moreover, the increases in the gene expression levels of proteins related to Aβ production (APP, ADAM10, BACE1, PS1, PS2) in the lenses of OLETF rats were prevented by food restriction. However, the Aβ1–42 levels in the diet groups were still significantly higher than in normal rats (Table 2 and Fig. 6), and the accumulation of Aβ1–42 in the lens cortex was higher than in the capsule–epithelium. It is known that epithelial cells differentiate to become fiber cells during the growth process4345 and that this differentiation into lens fiber cells is accompanied by the degradation of cellular organelles.4648 Therefore, Aβ1–42 in lens fiber cells may be difficult to cleave because the cells contain no nuclei or other organelles. We also investigated the effects of glucose levels on the production of Aβ1–42 in cultured HLE cells. The glucose level in these in vitro experiments (5.6 and 20 mM) was selected based on the glucose levels found in the aqueous humor of normal and OLETF rats. The APP, BACE1, PS1, and Aβ1–42 levels in HLE cells rose significantly under high glucose conditions (Fig. 8). These results show that high glucose levels cause an increase in the expression of genes related to Aβ production in the lenses of rats and humans. Moreover, Aβ that has accumulated in the lens may remain even after diabetes management has been achieved. 
It is important to understand the connection between lens opacification and Aβ1–42. It is known that the accumulation of Aβ in the brain of Alzheimer's patients is related to the damage of neuronal cells. On the other hand, enhanced Aβ expression in the lens causes amyloidogenic interactions with other structural proteins within the cytoplasm of supranuclear lens fiber cells and leads to increased lens protein aggregation, light scattering, and opacification.16 Moreover, it has been reported that aldose reductase (AR) activity increases in the lenses of OLETF rats greater than 40 weeks of age, causing an elevation in sorbitol levels; thus, the polyol pathway via AR may be related to the development of cataracts.49 We also reported that oxidative stress in the lenses of OLETF rats may be related to lens epithelial cell apoptosis and lens opacification.35 Under in vivo physiologic conditions, low concentrations of Aβ5058 cause mitochondrial damage.5962 Cytochrome c oxidase activity in the lenses of 80-week-old OLETF rats is significantly lower than in 80-week-old normal rats (normal, 2179 ± 303 U/g protein; OLETF, 1001 ± 238, U/g protein; means ± SE, n = 5), and our previous report showed that mitochondrial damage results in lens opacification.39,63 We observed lens opacification in 80-week-old OLETF rats; however, no lens opacification was observed in 40-week-old OLETF rats with hyperglycemia and enhanced sorbitol levels (Figs. 2C and 7).49 Also, lens opacification in OLETF rat was observed following the rise in the level of Aβ1–42 (Fig. 7). Together, the data suggest that hyperglycemia increases glucose levels in the aqueous humor of OLETF rats and an increase in Aβ production. In lenses, the mRNAs for APP, BACE, PS1, and PS2 were elevated, consistent with the observed increase in lens Aβ. Aβ accumulation can cause mitochondrial damage and oxidative stress. Further studies are needed to elucidate the precise mechanism for the accumulation and toxicity of Aβ in the lenses of OLETF rats. In addition, it is important to clarify the effect of Aβ accumulation on cataract formation in human lens. Therefore, we are now investigating Aβ accumulation in the lens epithelium of human diabetic patients. Our preliminary data suggest that total Aβ (Aβ1–40,42,43) accumulation in the lens epithelium of human diabetic patients (age, 70.4 ± 3.2 years; total Aβ levels, 16.3 ± 6.3 pmol/g protein; n = 7) is higher than that in normal patients (clear lenses) without diabetes mellitus (age, 62.2 ± 4.3 years; total Aβ levels, 9.2 ± 2.0 pmol/g protein; n = 12). 
In conclusion, we investigated changes in Aβ1–42 levels in the lenses of OLETF rats during the development of diabetes mellitus. Based on the results obtained in the present study, we hypothesize that hyperglycemia increases glucose levels in the aqueous humor of OLETF rats, resulting in an enhancement in Aβ production and elevated Aβ levels in lenses that correlate with accelerated lens opacification. These findings are significant for the design of future experimental studies aimed at reducing lens opacification in patients with diabetes mellitus. 
Acknowledgments
The authors alone are responsible for the content and writing of the paper. 
Disclosure: N. Nagai, None; Y. Ito, None; H. Sasaki, None 
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Figure 1
 
Changes in body weight (A), food intake (B), and Glu (C), TG (D), Cho (E), and insulin (F) levels in the blood from 10- to 80-week-old normal and OLETF rats. Open circles, normal rats; closed circles, OLETF rats. Data are presented as means ± SE of 7–10 independent rats. *P < 0.05 versus normal rats for each group. The increase in food intake corresponded with increased glucose, cholesterol, and TG in OLETF rats. The body weight increased to a maximum at 40 weeks and then decreased corresponding with the insulin levels.
Figure 1
 
Changes in body weight (A), food intake (B), and Glu (C), TG (D), Cho (E), and insulin (F) levels in the blood from 10- to 80-week-old normal and OLETF rats. Open circles, normal rats; closed circles, OLETF rats. Data are presented as means ± SE of 7–10 independent rats. *P < 0.05 versus normal rats for each group. The increase in food intake corresponded with increased glucose, cholesterol, and TG in OLETF rats. The body weight increased to a maximum at 40 weeks and then decreased corresponding with the insulin levels.
Figure 2
 
Changes in opacification and Aβ1–42 levels in 10- to 80-week-old normal and OLETF rats. (A) Scheimpflug slit images of lenses from 80-week-old normal and OLETF rats. The outline of the lens image used for analysis (dashed lines) was set by selecting four points on the software, and the opaque area within the outline was measured as pixels. In the analysis of lens opacity, the area of reflected light (472 ± 32 pixels) was removed. (B) Images of lenses from postmortem 80-week-old normal and OLETF rats. (C) Opacity and (D) Aβ1–42 levels in the total lenses of 10- to 80-week-old normal and OLETF rats. (E) Aβ1–42 levels in the separated lens (capsule–epithelium, cortex, and nucleus) of 80-week-old normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of 5–10 independent rat lenses. *P < 0.05 versus normal rats for each group. The Aβ1–42 production was enhanced in the capsule–epithelium and cortex of lenses from OLETF rats but not in the nucleus.
Figure 2
 
Changes in opacification and Aβ1–42 levels in 10- to 80-week-old normal and OLETF rats. (A) Scheimpflug slit images of lenses from 80-week-old normal and OLETF rats. The outline of the lens image used for analysis (dashed lines) was set by selecting four points on the software, and the opaque area within the outline was measured as pixels. In the analysis of lens opacity, the area of reflected light (472 ± 32 pixels) was removed. (B) Images of lenses from postmortem 80-week-old normal and OLETF rats. (C) Opacity and (D) Aβ1–42 levels in the total lenses of 10- to 80-week-old normal and OLETF rats. (E) Aβ1–42 levels in the separated lens (capsule–epithelium, cortex, and nucleus) of 80-week-old normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of 5–10 independent rat lenses. *P < 0.05 versus normal rats for each group. The Aβ1–42 production was enhanced in the capsule–epithelium and cortex of lenses from OLETF rats but not in the nucleus.
Figure 3
 
Expressions of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate protein (F) measured in total lenses of normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of five to six independent rat lenses. *P < 0.05 versus normal rats for each group. The mRNA for enzymes necessary for the proteolysis of APP to Aβ and the APP-Aβ intermediate protein (37–50 kDa) increased with age in the lenses of OLETF rats.
Figure 3
 
Expressions of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate protein (F) measured in total lenses of normal and OLETF rats. Open columns, normal rats; closed columns, OLETF rats. The data are presented as means ± SE of five to six independent rat lenses. *P < 0.05 versus normal rats for each group. The mRNA for enzymes necessary for the proteolysis of APP to Aβ and the APP-Aβ intermediate protein (37–50 kDa) increased with age in the lenses of OLETF rats.
Figure 4
 
Changes in glucose and insulin levels in 40- and 80-week-old normal and OLETF rats in response to an OGT test. (A, B) Changes in Glu levels in 40- (A) and 80-week-old (B) normal and OLETF rats after oral administration of glucose (3 g/kg). Open circles, normal rats; closed circles, OLETF rats. The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats without glucose administration. *2P < 0.05 versus OLETF rats without glucose administration. (C, D) Insulin levels in blood (C) and glucose levels in the aqueous humor (D) of 40- and 80-week-old normal and OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *3P < 0.05 versus normal rats for each category. *4P < 0.05 versus Control for each category. Increased glucose levels in the OLETF rats at 40 and 80 weeks of age corresponded with insulin levels in the blood and increased glucose levels in the aqueous humor of OLETF rats relative to normal rats.
Figure 4
 
Changes in glucose and insulin levels in 40- and 80-week-old normal and OLETF rats in response to an OGT test. (A, B) Changes in Glu levels in 40- (A) and 80-week-old (B) normal and OLETF rats after oral administration of glucose (3 g/kg). Open circles, normal rats; closed circles, OLETF rats. The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats without glucose administration. *2P < 0.05 versus OLETF rats without glucose administration. (C, D) Insulin levels in blood (C) and glucose levels in the aqueous humor (D) of 40- and 80-week-old normal and OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *3P < 0.05 versus normal rats for each category. *4P < 0.05 versus Control for each category. Increased glucose levels in the OLETF rats at 40 and 80 weeks of age corresponded with insulin levels in the blood and increased glucose levels in the aqueous humor of OLETF rats relative to normal rats.
Figure 5
 
Changes in APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 40- and 80-week-old normal and/or OLETF rats after oral administration of glucose (3 g/kg). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats for each category. *2P < 0.05 versus Control for each category. The increase in glucose levels in the blood correspond to the increased mRNA levels for enzymes necessary for the proteolysis of APP to Aβ.
Figure 5
 
Changes in APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 40- and 80-week-old normal and/or OLETF rats after oral administration of glucose (3 g/kg). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Control: normal or OLETF rats 180 minutes after the oral administration of purified water. Glucose: normal or OLETF rats 180 minutes after the oral administration of glucose (3 g/kg). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal rats for each category. *2P < 0.05 versus Control for each category. The increase in glucose levels in the blood correspond to the increased mRNA levels for enzymes necessary for the proteolysis of APP to Aβ.
Figure 6
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 80-week-old normal and OLETF rats with food restriction. In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Food restriction was carried out on OLETF rats from 40 to 80 weeks of age (diet group). Normal: 80-week-old normal rats allowed free access to a commercial diet and water. Control: 80-week-old OLETF rats allowed free access to a commercial diet and water. Diet: 80-week-old OLETF rats fed a commercial diet (25 g/d/rat) and water (free access). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal group for each category. *2P < 0.05 versus control group for each category. The increase in mRNA for enzymes necessary for proteolysis of APP to Aβ and the APP-Aβ intermediate protein in the lenses of OLETF rats were improved by food restriction.
Figure 6
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, and APP-Aβ intermediate (F) and Aβ1–42 protein levels (G) in the lenses of 80-week-old normal and OLETF rats with food restriction. In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, normal rats; closed columns, OLETF rats. Food restriction was carried out on OLETF rats from 40 to 80 weeks of age (diet group). Normal: 80-week-old normal rats allowed free access to a commercial diet and water. Control: 80-week-old OLETF rats allowed free access to a commercial diet and water. Diet: 80-week-old OLETF rats fed a commercial diet (25 g/d/rat) and water (free access). The data are presented as means ± SE of six independent rats. *1P < 0.05 versus normal group for each category. *2P < 0.05 versus control group for each category. The increase in mRNA for enzymes necessary for proteolysis of APP to Aβ and the APP-Aβ intermediate protein in the lenses of OLETF rats were improved by food restriction.
Figure 7
 
Temporal relationships between plasma Glu and Aβ1–42 levels and lens opacity in OLETF rats. Glucose levels in the blood (circles) and Aβ1–42 levels in the total lenses (triangles) were determined by an ELISA method. Lens opacity (squares) was measured using the EAS-1000. For each parameter, the highest value attained was taken as 100%. The data are presented as means ± SE of 5–10 rats. Opacity in anterior subcapsular increased slightly after the accumulation of Aβ1–42 in the lenses of OLETF rats.
Figure 7
 
Temporal relationships between plasma Glu and Aβ1–42 levels and lens opacity in OLETF rats. Glucose levels in the blood (circles) and Aβ1–42 levels in the total lenses (triangles) were determined by an ELISA method. Lens opacity (squares) was measured using the EAS-1000. For each parameter, the highest value attained was taken as 100%. The data are presented as means ± SE of 5–10 rats. Opacity in anterior subcapsular increased slightly after the accumulation of Aβ1–42 in the lenses of OLETF rats.
Figure 8
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, APP-Aβ intermediate protein (F), and Aβ1–42 levels (G) in HLE cells cultured with or without of high glucose (20 mM). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, nontreated HLE cells (Control); closed columns, glucose-treated HLE cells (Glucose). The data are presented as means ± SE of 10 independent experiments. *P < 0.05 versus nontreated HLE cells (Control). In the presence of 20 mM glucose, HLE cells showed increased mRNA expression of enzymes required for the proteolysis of APP and APP-Aβ intermediate protein. A 4.12-fold increase in Aβ1–42 was observed after 24 hours in culture.
Figure 8
 
Expression of APP (A), ADAM10 (B), BACE1 (C), PS1 (D), and PS2 (E) mRNAs, APP-Aβ intermediate protein (F), and Aβ1–42 levels (G) in HLE cells cultured with or without of high glucose (20 mM). In contrast with the Western blot data shown in Figure 3F, only the area between 37 and 50 kDa is shown in F, because the bands for the full-length APP at just above 100 kDa were similar for normal and OLETF rats, and no band around 4 kDa (Aβ) was detected by the Western blot method. Open columns, nontreated HLE cells (Control); closed columns, glucose-treated HLE cells (Glucose). The data are presented as means ± SE of 10 independent experiments. *P < 0.05 versus nontreated HLE cells (Control). In the presence of 20 mM glucose, HLE cells showed increased mRNA expression of enzymes required for the proteolysis of APP and APP-Aβ intermediate protein. A 4.12-fold increase in Aβ1–42 was observed after 24 hours in culture.
Table 1
 
Sequences of Primers Used for Quantitative RT-PCR Analysis
Table 1
 
Sequences of Primers Used for Quantitative RT-PCR Analysis
Table 2
 
Body Weight and Some Blood Test Values for Diabetes Mellitus in 80-Week-Old OLETF Rats With Food Restriction
Table 2
 
Body Weight and Some Blood Test Values for Diabetes Mellitus in 80-Week-Old OLETF Rats With Food Restriction
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