Contribution of genetic and dietary insulin resistance to Alzheimer phenotype in APP/PS1 transgenic mice

Abstract According to epidemiological studies, type-2 diabetes increases the risk of Alzheimer’s disease. Here, we induced hyperglycaemia in mice overexpressing mutant amyloid precursor protein and presenilin-1 (APdE9) either by cross-breeding them with pancreatic insulin-like growth factor 2 (IGF-2) overexpressing mice or by feeding them with high-fat diet. Glucose and insulin tolerance tests revealed significant hyperglycaemia in mice overexpressing IGF-2, which was exacerbated by high-fat diet. However, sustained hyperinsulinaemia and insulin resistance were observed only in mice co-expressing IGF-2 and APdE9 without correlation to insulin levels in brain. In behavioural tests in aged mice, APdE9 was associated with poor spatial learning and the combination of IGF-2 and high-fat diet further impaired learning. Neither high-fat diet nor IGF-2 increased β-amyloid burden in the brain. In male mice, IGF-2 increased β-amyloid 42/40 ratio, which correlated with poor spatial learning. In contrast, inhibitory phosphorylation of glycogen synthase kinase 3β, which correlated with good spatial learning, was increased in APdE9 and IGF-2 female mice on standard diet, but not on high-fat diet. Interestingly, high-fat diet altered τ isoform expression and increased phosphorylation of τ at Ser202 site in female mice regardless of genotype. These findings provide evidence for new regulatory mechanisms that link type-2 diabetes and Alzheimer pathology.

amyloid plaques in the cortex and hippocampus beginning at the age of 3 months. Amyloid pathology advances faster in female than male mice. With increasing age, these mice manifest memory impairment and various degenerative changes in the brain, similar to AD. As a model of insulin resistance, we used transgenic mice over-expressing insulin-like growth factor 2 (IGF-2) 2.
In these mice, beta-islet hyperplasia leads to hyperinsulinemia and development of insulin resistance probably through downregulation of insulin receptor number and function or postreceptor pathways, causing hyperglycemia, which may further increase insulin secretion 2. Both these mouse lines were back-crossed for more than 10 generations to the common C57B6 strain and maintained in pathogen-free conditions at the Laboratory Animal Center, University of Eastern Finland, Kulopio, Finland. The housing conditions were controlled (temperature +22°C; 12-hour light/12-hour dark cycle -lights on at 07:00 h; humidity 50-60%), and fresh food and water were freely available. After 2 months of age, all the mice were kept in individual cages. The experiments were conducted according to the Council of Europe (Directive 86/609) and Finnish guidelines, and approved by the National Animal Ethics Committee of State Provincial Office of Southern Finland.

Behavioral testing
Spontaneous exploratory activity was monitored in a transparent test cage (L 26 x W 26 x H 39 cm) with automated infrared photo detection (TruScan, Coulbourn Instruments, Allentown, PA, USA).
The apparatus was equipped with two rings of infrared photocell sensors enabling separate monitoring of horizontal (XY-movement over time) and vertical activity (rearing) and was connected to a personal computer. A session started by placing the animal in the center of the field.
Mice were allowed to explore the apparatus for 10 min in two separate sessions with a retention interval of 48 h. The test cages were cleaned with 70% ethanol and dried before the next mousewas tested to remove any odour from urine and feces of the mice.
Spatial memory was assessed with the Morris swim task. The apparatus was a black plastic pool with a diameter of 120 cm. A black escape platform (square, 14 x 14 cm) was located 1.0 cm below (hidden) the water surface. The temperature of the water was kept constant throughout the experiment (20 ± 0.5°C), and a 10-min recovery period was allowed between the training trials.
First, the mice were pre-trained to find and climb onto the platform for 2 days by using an alley (L 1 m x W 14 cm x H 25 cm) leading to the submerged platform. The experiment was performed for 5 consecutive days, with five trials (T1-T5) per day and a single trial on 6th day (D1 -D6). The maximum swim time was 60 s for the first 4 days and 40 s for the 5th and 6th day. If the mouse failed to find the escape platform within the maximum time, the animal was gently placed on the platform by the experimenter and left for 10 s. When the mouse found the platform, 10 s of orientation time was given before it was lifted. During the 5 days of testing, the mice were trained with a hidden platform. The platform location was kept constant, and the starting position varied between four constant locations at the pool rim. Mice were placed in the water with their nose pointing toward the wall. There were two probe trials, the 5th trial on day 5 and the only trial on day 6, to help the determination of the spatial search bias of the mice. During the probe trials, the platform was removed and the mice were allowed to swim for 40 s. Between the probe trials, there was a reminder trial (without any recordings) with platform to assure mice regarding the presence of platfrom in the same position. The start and end of the trial were marked by the experimenter with a remote controller, while the swim path was monitored with a video tracking system (HVS Image, Hampton, UK). Thigmotaxis (swimming along the sides of the wall) was assessed by dividing the entire pool into three concentric zones of equal surface area and calculating the time spent in the outer zone. Search bias during the probe trial was measured by calculating the time the mice spent in the immediate vicinity of previous platform location. This was defined as a target area centered on the platform with a diameter of 30 cm. This target area comprised 6.25% of the total surface area. To prevent hypothermia, following each trial mice were towel-dried and returned to a pre-warmed cage.

Insulin and glucose metabolism
Glucose tolerance test (GTT). After an overnight fasting (16 h) an i.p. injection of 2 mg/g Dglucose as a 20% solution (prepared in normal saline) was given. Blood samples for the determination of glucose and insulin levels (50 -75 µl) were collected at 0, 15, 30 and 60 min from the saphenous vein. The glucose values were determined immediately using a glucometer (One Touch, LifeScan Inc., Milpitas, CA, USA).

Insulin tolerance test (ITT).
After fasting for 4 h in the morning, an i.p. injection of insulin (Actrapid, NovoNordisk, Sweden) at 0.25 mU/g (prepared in normal saline) was given. Blood samples were collected at 0, 20, 40 and 80 min, and the glucose values were recorded using the glucometer (One Touch). At the end of the experiment, the mice received D-glucose at 0.5 mg/g i.p.
to neutralize the effects of the remaining insulin. Same mice were used in both tests (GTT & ITT) with 2 weeks of recovery between the tests. If blood glucose dropped to 2.0 mM or below, the mouse was carefully observed and an extra glucose sample was taken after 5 min. If the blood glucose levels still remained low, 100 μl of 20% glucose was injected i.p. and the mouse was excluded from the analysis of the ITT experiment.

Analysis of insulin levels
During GTT, blood samples were collected using fine glass capillary tubes (50-75 µl) lined with anti-coagulant (Na-EDTA), and the blood was drained in to Eppendorf tubes and centrifuged (10000 x g for 1 min) to obtain plasma. The plasma was frozen immediately in liquid nitrogen and kept under -70°C until analysed. Plasma insulin levels were analysed with rat insulin enzyme-linked immunosorbent assay kit (INSKR020, Crystal Chem Inc., Chicago, IL, USA), with mouse insulin (INSSM021, Crystal Chem Inc., Chicago, IL, USA) as a standard.

Perfusion technique
At the end of the experimental series, all mice were deeply anesthetized with a cocktail of 420 mg/kg of chloralhydrate and 105 mg/kg of pentobarbital (i.p.) and transcardially perfused with ice cold saline at 9 ml/min for 7 min. Before starting the perfusion the descending aorta was clamped to avoid perfusion into the peripheral organs and the right atria was cut open. All mice from the acute group and most females of the diet group were used to obtain fresh brain samples for biochemical assays. The brain was carefully removed and dissected on ice into following samples (frontal cortex, dorsal posterior cortex, ventral posterior cortex, hippocampus bilaterally and the cerebellum). The liver was removed and divided into two pieces. All these samples were snap frozen in liquid nitrogen and stored at -70°C. In addition, the pancreas was collected from the diet group and immersion-fixed in a histology cassette in 4% paraformaldehyde (PFA) and kept refrigerated until prepared for histology. The left hemi-brain from 3 mice of each genotype and treatment group among the female mice in the diet group was immersion-fixed in 4% PFA for 4 h, followed by 30% sucrose overnight, and stored in a cryoprotectant at -20 °C. All male mice in the diet group were transcardially perfused with 4% PFA, and the brains were post-fixed with the same solution for 2 h and then treated similarly to the brains with immersion fixation.

Islet isolation for insulin gene expression
A separate cohort of 4-month-old mice (n = 10 of all four genotypes; 4 males + 6 females) were euthanized with cervical dislocation; the pancreas was collected and proceeded for islet isolation.
Pancreatic islets were isolated according to the method modified from 3. Shortly, the islets were separated by digestion with 1 mg/ml collagenase XI (C7657, Sigma-Aldrich, St. Louis, MO, USA) in Hanks' balanced salt solution (HBSS, 14185, Gibco, Grand Island, NY, USA) supplemented with 20 mM Hepes solution, pH 7,4, (H0887, Sigma-Aldrich) and 0.5 % BSA (A3311, Sigma-Aldrich), by injecting the collagenase through the common bile duct. The collagenase-filled pancreas was incubated by shaking at + 37ºC for 8-10 minutes to digest the exocrine tissue and washed twice with the buffer described above (without collagenase). The islets were hand-picked under a dissection microscope, washed once with PBS, and snap-frozen in liquid nitrogen. Arbp (Mm00725448_s1) (Applied Biosystems, Foster City, CA, USA). Insulin mRNA expression data were normalized to Arbp (36B4 gene) mRNA expression. Gene expression data were pooled from both sexes and the gene expression levels were calculated as a percentage related to average wild-type gene expression in the same RT-PCR run. For liver cDNA, Taqman gene expression assays used were: G6Pc3 (Mm00440636-m1), pck1 (Mm00616234_m1) and beta-actin (Assay by design from Applied Biosystems, sequence available from the authors upon request). G6Pc3 and pck1 gene expression were normalized to β-actin expression, and calculated as a percentage related to wild-type gene expression.

ELISA analysis of liver and brain Aβ levels
Liver Aβ levels were analyzed from female mice of the acute group. The tissue was homogenized into a PBS buffer with phosphatase and EDTA-free protease inhibitor cocktail (Thermo Scientific) in 1:1 proportion to the sample weight. To analyze total Aβ levels, the mouse ventral cortex was homogenized in a guanidine buffer (5 M guanidine-HCl/50 mMTris-HCl, pH 8.0, with EDTA-free phosphatase and protease inhibitor cocktail (Thermo Scientific) in 1:1 proportion to sample weight.
The samples and Aβ peptides used as standards were prepared to contain 0.5 M guanidine-0.5% BSA-1 mM AEBSF in the final composition. The levels of transgenic human Aβ40 and Aβ42 were quantified using the Signal Select TM Beta Amyloid ELISA Kits (BioSource International Inc., CA, USA) according to the manufacturer's protocol.

Histological analysis
Pancreas: The pancreas was fixed in 4 % PFA (pH 7) for at least 24 hours, dehydrated in graded ethanol, embedded in paraffin, and processed into histological slides. For morphometric analysis, immunohistochemical detection of islet area (insulin-positive area) was performed in three sections The images were captured using a microscope (Zeiss: Stemi 2000 -C) attached to a camera (Canon powershot G9).
The sections were incubated overnight at room temperature on a shaker table. Following incubation, the sections were rinsed thoroughly with TBS-T and transferred to the solution containing the secondary antibody; Goat anti-mouse biotin 1:1500 (Sigma). After 2 hours of incubation, the sections were rinsed three times and transferred to a solution containing Streptavidin (GE Healthcare, UK) for 2 hours. Visualization of Aβ plaques was achieved by incubation with DAB-Ni solution. Stained sections were mounted on gelatin-coated slides and dehydrated in alcohol series, cleared with xylene and mounted in Depex. For CD45 staining, the sections were pretreated because of endogenous peroxidase with 0.3% H 2 O 2 in MeOH for 30 minutes. After that sections were blocked in 10% NGS in TBS-T (Tris-buffered saline with Triton X-100). The sections were put in primary, polyclonal rat anti-mouse CD45 antibody for overnight incubation at room temperature on a shaker table. Following incubation, sections were rinsed thoroughly with TBS-T and transferred to the solution containing secondary antibody, sheep anti-rat biotin 1:500 (Serotec, UK). After 2 hours of incubation, the sections were rinsed with TBS-T and transferred to a solution containing streptavidin 1:1000 (GE Healthcare, UK). Visualization of microlia was achieved by incubation with DAB-Ni solution. Stained sections were mounted on gelatin-coated slides. Aβ plaques were stained with Congo Red. For this, the slides were put overnight in 4% PFA. Next day, the slides were rinsed with dH 2 O and put in alcoholic NaCl-hydroxide solution for 20 minutes.
After that the slides were submerged in Congo Red solution (Congo Red, Sigma, USA) for 30 minutes. The slides were dehydrated shortly in alcohol series, cleared with xylene and mounted in Depex. The area of the hippocampus and the overlying cortex, and the amyloid plaques were measured using Adobe Photoshop CS3 extended (version 10). The images were captured with a microscope (Olympus C3275), attached with an Olympus camera.