Ethics statement

All animal protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC). All methods were carried out in accordance with the approved guidelines and protocols.

Animals, diets and treatment

The study timeline is shown in Fig. 6. A total of 36 male Tg APPSwDI (human APP with Swedish, Dutch and Iowa mutations on a C57BL/6 background) were raised under equal dietary conditions for 2 months. At 10 weeks of age, the animals were divided into three groups of 12 animals each and received a diet consisting of 60% of kcal in carbohydrates with equal amounts of maltodextrin and sucrose plus either waxy maize starch (high-GI diet groups) or AIN-93G purified diet (controls). For detailed diet composition, see the supplementary material. During the first week of dietary acclimatization, all animals received a 45-mg sucrose pellet daily. After that, the group fed the high-GI diet plus ghrelin agonist received a 45-mg sucrose pellet containing 1.66% ghrelin agonist39 (LY444711; Eli Lilly, Indianapolis, IN) every day (30mg/kg/day, parallel to our previous study12, dose determined according to previous work by Giddings et al. 2008, abstract added to supplement); the other groups continued to be treated with sucrose pellets as placebos. Treatment took place daily at the same time between 2:00 and 4:00 pm during the animals’ light cycle and continued until the animals were sacrificed (treatment period: week 11 until week 30). Staff watched all animals take and eat the pellets and noted the days when the pellet was not consumed. This was only the case for few animals during dietary acclimatization. During the treatment period all animals ate the pellets. The amount of food consumed by all groups was measured every 2 weeks and the threshold of food restriction for the ghrelin-agonist-treated group was set at the average level of food consumption of the group fed the high-GI diet alone.

Figure 6 Timeline of the study. Full size image

Behavioral and cognitive assessments

All behavioral and cognitive assessments took place between weeks 22 and 24 (see Fig. 6). All tests took place during the light cycle. Feeding times were not changed throughout the assessments.

Open field test

The maze consisted of a 42 by 42 cm2 arena with clear sides (20 cm high). The animal was placed in the arena and observed for 4 minutes with a camera-driven tracker system (Ethovision 9.5, Noldus, The Netherlands). The arena was subdivided into the open center area and the sides. The system recorded the position of the animal at 5 frames/s.

Water maze

The water maze apparatus and procedure were described in detail before40. Briefly, we used a blue plastic pool, 120 cm in diameter and a see-through round platform, 10 cm in diameter, located 0.5 cm below the water surface. During days 1 through 5 of the testing period, the mice were trained to find the hidden platform, which was kept in a constant position throughout these 5 days. Three trials were run per day; all starting positions were used equally in a pseudo-random order. The mice were given 60 s to find the platform and 10 s to stay on the platform. If the mouse did not find the platform in the assigned time, it was manually put onto the platform. The inter-trial interval during which the mouse was placed in a towel-bedded drying cage lasted 1 minute. Learning of the task was evaluated by recording the latency time to find the platform. At the end of the four trials on day 5 of the testing period, the mice were tested in a 60-s probe trial with no escape platform present. Mice that had learned the platform position predominantly searched in the “correct” quadrant of the pool during the probe trial or entered the correct quadrant faster. Trials were recorded by using a camera-driven tracker system (Ethovision 9.5, Noldus, The Netherlands).

Zero maze

For the zero maze test, we used a round maze with a diameter of 61 cm designed for mice (SD Instruments, San Diego, CA). At the beginning of the trial, all mice were placed on the same open part facing in the same direction. Velocity, distances moved and time spent in the open and closed parts were recorded for 4 minutes by using a camera-driven tracker system (Ethovision 9.5, Noldus, The Netherlands).

Light-dark-box

We used a custom-built plastic light-dark box (46.5 cm length, 22 cm width, 28 × 22 cm light part, 18.5 × 22 cm dark part). Time spent in the light and dark parts as well as the number of entries into the dark part were recorded for 5 minutes by using a camera-driven tracker system (Ethovision 9.5, Noldus, The Netherlands). Mice were placed in the light part of the box facing away from the entrance to the dark part.

Immunohistochemistry

Animals were sacrificed at week 30 for immunohistochemical, Western blot and ELISA analyses. Mice were anesthetized with ketamine/xylazine (100/10 mg/kg) and perfused with cold saline. The brains were removed and cut in half sagittally and the right hemisphere of the brain was placed in 4% paraformaldehyde overnight. The left hemisphere was dissected into four pieces (rostral cortex, caudal cortex, hippocampus and midbrain/brainstem) and stored frozen at −80 oC for protein analysis (ELISA, Western blot). The right half and the intact whole brains from 12 animals, 4 per group, were put in 30% sucrose for cryoprotection and 30-μm thick coronal sections were cut on a freezing-sliding microtome.

Sections from 29 brains were stained for Aβ with the W0-2 antibody (human Aβ 4-10 ; 1:2000; The Genetics Company, Schlieren, Switzerland). Another series of sections from the same 29 brains was stained for Iba-1 (1:1000; Wako, Richmond, VA) as a marker for activated microglia. For Aβ staining, sections were pretreated for 30 minutes in 85 °C sodium citrate solution (pH = 6.5). Following incubation with the primary antibody in TBS-T overnight at room temperature, tissues were rinsed three times and incubated with the appropriate biotinylated secondary antibody for 2 hours at room temperature. Sections were again rinsed three times and put for 2 hours with the tertiary antibody, extra Avidin-peroxidase. After another three rinses, metal-enhanced DAB staining was used for visualization. For each antibody, all sections were processed in one staining tray. All slides were air-dried, cleared in xylene and coverslipped with DPX.

ImageJ software (NIH open source; http://imagej.nih.gov/ij/) was used to analyze the area occupied by Aβ and glial reactivity in stratum oriens of the dorsal hippocampus and in the dorsal dentate gyrus. Images of the appropriate brain areas were acquired with an Olympus DP70 digital camera. All images were acquired in one session to avoid changes in light levels. ImageJ measurements were performed by a scientist who was blind to the study design. Few images had to be excluded due to staining/tissue preparation problems (see Fig. 2).

Oral glucose tolerance test

In order to avoid a priori differences in baseline blood glucose levels, mice had no access to food for a period of six hours before the glucose tolerance test. For the oral glucose tolerance test, 300 μl of a solution of 16.7 g glucose in 100 ml of purified water was administered directly into the mice’s stomach via gavage needles. Blood samples were taken from tail veins and immediately measured with the TRUE2Go blood measurement system41 for one baseline time point and then after 17, 34, 60 and 90 min. The mice were placed in a plastic retainer system during the procedure. One mouse was excluded from the analysis because it did not tolerate the gavage process.

Protein extraction and Western blotting

For ELISA and Western blots, brain tissue was homogenized in RIPA (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 50 mM Tris, pH 8, 20 mM NaF, 2 mM EGTA, 0.5% levamisole, 1 mM NaVO4) plus protease inhibitor cocktail (p2714 Sigma-Aldrich, St Louis, MO) by use of the fast homogenization process Minilys® (Precellys, Bertin, France). After protein estimation with the Bradford method42, samples were diluted to an appropriate concentration.

For Western blotting, p-IRS Ser636 antibody (Santa Cruz Biotechnology, Dallas, TX), synaptophysin antibody clone SVP-38 (Sigma-Aldrich, St Louis, MO), pSAPK/JNK Thr183/Tyr185 (Cell Signaling Technologies, Danvers, MA) and PSD-95 antibody (Upstate/Millipore, Billerica, MA) were used. After electrophoresis and transfer to nitrocellulose, samples were incubated with the primary antibody overnight and were then incubated with the suitable secondary antibody for 90 minutes. For measuring TNF-alpha, a commercial ELISA kit was used (EMTNFA, ThermoScientific, Rockford, IL).

Blood samples

Blood samples were taken after a 6-hour fasting period via intracardial puncture from the left ventricle shortly before the animals were perfused. Samples of 250 μl of blood were collected in chilled EDTA tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) that were prefilled with 5 μl of 200 mM AEBSF stock yielding a final concentration of 4 mM AEBSF. Samples were centrifuged for 20 minutes at 17000 rpm and 4 °C and the plasma collected was immediately acidified with 200 μl of 1 M HCl per 1 ml of plasma. pH was adjusted accordingly before ELISA measurements for insulin and ghrelin. Acyl ghrelin and des-acyl ghrelin was measured with a custom-built 2-site sandwich ELISA43. For the measurement of insulin a commercially available ELISA kit was used (EZRMI-13K, EMD-Millipore, Billerica, MA).

Quantitative magnetic resonance imaging

In vivo body composition (total body fat and lean tissue) of mice was determined by using an EchoMRI™ 3-in-1 quantitative magnetic resonance (QMR) machine (Echo Medical Systems, Houston, TX). A system test was performed by using a known fat standard before the measurements were taken. Mice were weighed and then placed into a clear holding tube capped with a stopper that restricted vertical movement but allowed constant airflow. The tube was inserted into the machine and the mouse was scanned by using Normal Precision mode.

Metabolic cages

Twenty-four-hour patterns of food intake, energy expenditure (indirect calorimetry) and physical activity were measured by using CLAMS (Columbus Instruments Inc., Columbus, OH). This instrument also enforced the feeding regimens in an automated, computer-controlled manner. Body weight was monitored weekly.

Activity measurements

Additional activity measurements over a period of five consecutive light and dark cycles were performed at week 21 by using a custom-built infrared-based beam-breaking system that recorded horizontal and vertical movements. Mice were placed in the system in their home cages with reduced bedding in order to not disrupt the continuous infrared measurements. Only data recorded on days 2 to 4 were included in the analysis.

Statistical methods

All datasets were tested for Gaussian distribution using a D’Agostino & Pearson omnibus normality test. Whenever a normal (Gaussian) distribution could be validly assumed, a one-way ANOVA, then a post-hoc Tukey’s test for multiple comparisons was used to test for significant differences between groups (referred to as “ANOVA/Tukey’s”). Nonparametric samples were tested using the Kruskal-Wallis test and Dunn’s test for multiple comparisons as a post-hoc test (referred to as “Kruskal-Wallis/Dunn’s”). Whenever only two groups were involved in the measurements, differences were tested using a t-test for paired/unpaired samples in parametric distributions or a Kolmogorov-Smirnov test for nonparametric distributions. Being aware of the nested data problem44, we only compared values on the same level of analysis to decrease the likelihood of type-1 errors. All analyses were performed with GraphPad Prism software version 6.05 (GraphPad Software, Inc., La Jolla, CA).