All experimental procedures involving animals were conducted in accordance to the regulations and guidelines set forth by the United States Department of Agriculture (USDA) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All the experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois.

Animals

Male C57BL/6J mice (N = 24) at 22 days of age were obtained from The Jackson Laboratory (Bar Harbor, ME). Upon arrival, mice were group-housed with four animals per polycarbonate shoebox-style cage (29 × 19 × 13 cm) and acclimatized to a reversed 12 h photoperiod (lights on at 1000 and off at 2000) in a temperature-controlled (21 ± 1°C) room for one week. During this acclimation period, animals were provided ad libitum access to AIN-93G purified rodent diet (Teklad 94045; Harlan Laboratories, Indianapolis, IN) and fresh water. Cages were furnished with 1/4″ corncob bedding (Teklad 7097; Harlan Laboratories), cotton squares and cardboard tubes for nesting and environmental enrichment.

Experimental Design

Two isocaloric treatment diets containing fructose or glucose at 18% of total metabolizable energy were formulated based on the AIN-93G purified rodent diet37 and manufactured by Custom Animal Diets (Bangor, PA). Both diets replaced all sucrose and a fraction of cornstarch with either glucose or fructose. Analysis of the two diets for proximate composition was performed to validate that both diets were indeed equivalent in metabolizable energy content as based on their respective crude nutrient profiles (Table 1).

Table 1 Composition of the experimental diets Full size table

One week after arrival, animals were singly housed and randomly assigned to receive either fructose or glucose treatment diets (n = 12/group) for a total of 11 weeks (77 days) in order to assess the long-term effects of the interventional diets. Food intake and BW were measured daily at the end of the light cycle for 77 days. On days 31–40, animals received daily i.p. injections of 50 mg/kg bromodeoxyuridine (BrdU) to label dividing cells. On days 61–77, mice were evaluated for behavioral performance in a battery of tests. First, animals were placed into custom-made home cages for continuous video tracking of home cage activity over 5 days. The animals were then returned to their standard cages for 2 days where they remained undisturbed except for daily weighing of food and BW. Next, animals were evaluated for novel object recognition (3 days), then rotarod (3 days). The following 2 days animals remained undisturbed. Finally, animals were tested for contextual fear conditioning (2 days). Each of these tasks is described in more detail below. On day 78, mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in phosphate buffer solution (PBS; 0.287% sodium phosphate monobasic anhydrous, 1.102% sodium phosphate dibasic anhydrous, 0.9% sodium chloride in water). The liver, both right and left inguinal and subscapular fat pads and spleen were immediately dissected and weighed. The brain was also immediately dissected, post-fixed in 4% paraformaldehyde overnight at 4°C then transferred to 30% sucrose (w/v) in PBS at 4°C until sectioning.

Home cage activity testing

Animals were placed into clear acrylic arenas (68 × 36 × 16 cm) that were divided into four individual cages (34 × 18 × 16 cm) that allowed for physical contact and social interaction through wire grating, as previously described38. Cages were furnished with corncob bedding. Food and water were delivered from the side so as not to interfere with video tracking. Ceiling-mounted video cameras interfaced to computers running TopScan video tracking software (Clever Sys Inc., Reston, VA) were used to continuously measure distance traveled in the cage over 5 days during both the light and dark cycle. Red light not visible to the mice was used for video tracking during the dark cycle. As demonstrated in our previous experiments38, it takes a minimum of 3 days for animals to habituate and acclimatize to the cages. As such, activity in days 4 and 5 (in a 5 day protocol) were found to be reliable and reproducible measures of locomotor activity. Days 4 and 5 were chosen a priori to data collection in the present experiment as reliable measures of locomotor activity.

Energetic cost of home cage activity

The energetic cost of voluntary home cage physical activity in our study was estimated following Koteja et al.39. First, we estimated the macronutrient concentrations available to the animal for generation of usable energy (i.e., metabolizable energy), taking into consideration: a) food disappearance, b) the energy excreted in the form of feces (estimated at 22% of total food intake) and urine (estimated at 3% of total food intake) as previously shown39 and also c) food wastage due to spillage in the home cage (estimated at 10% of total food intake), as there is considerable evidence that food wasting is a significant source of error when estimating food intake40,41. Finally, a multiple regression was conducted predicting metabolizable energy intake over the 5 day home cage activity test (measured in grams of food) as a function of BW and total distance traveled (in km).

Based on the energy expenditure values, it was also possible to make an educated guess of the consequence of the differential energy expenditure on BW between the groups at the end of the 11 week intervention using an empirical energy balance technique as shown previously42 [Energy intake – Energy expenditure = Energy storage (fat mass+ fat free mass) ]. In the energy balance, we take into consideration the energy density (ρ FM ; ρ FFM ), metabolic rates (γ FM ; γ FFM ) and deposition cost (γ FM ; η FFM ) for both fat mass (FM) and fat free mass (FFM)43.

Learning, memory and motor performance

Novel Object Recognition

On day 1, mice were placed in the testing arena (50 × 38 × 20 cm) for 10 min to habituate to the environment. The following day, two identical objects were placed into the arena. Mice were allowed to explore the objects for thirty seconds and then immediately returned to their home cage. On day 3 animals were again placed into the arena, this time with one familiar object (explored the previous day) along with a novel object that the animal had never explored. The mice were allowed to explore the arena and the objects for a total of 5 min during which time TopScan measured the duration spent sniffing each object (using the sniffing module in Topscan). The automated measure obtained with Topscan sniffing module was validated against manual recording in previous pilot studies, it specifically demonstrated that the sniffing module reproduces accurately manual recording, providing a validated objective measurement. Two sets of objects were used and animals were counterbalanced with respect to which object was familiar and novel between treatment groups. A discrimination index was calculated as the percent time sniffing the novel object divided by the percent time spent sniffing both objects on the test day using (day 3).

Rotarod

Following Clark et al.44, mice were placed on a stationary rotarod (AccuRotor Rota Rod Tall Unit, 63-cm fall height, 30 mm diameter rotating dowel; Accuscan, Columbus, OH, USA) which was then accelerated at 60 rpm/min. Latency to fall was recorded. Each animal underwent four consecutive trials a day for three days.

Contextual Fear Conditioning

Following Clark et al.44, two animals from each treatment group were randomly selected to serve as controls to not receive any shocks, while the remaining animals were assigned to receive shocks. On day one, mice were placed into the fear conditioning chamber for 180 seconds. Animals in the shock group received 2 foot-shocks (0.5 mA, duration 2 s) at 120 and 150 s. Animals in the control group received no shocks. The following day, all mice were placed into the same context for 180 seconds without any shocks. Percent time spent freezing was automatically recorded by TopScan.

Immunohistochemistry

Tissue Preparation

Brains were sectioned (40 μm) by cryostat and stored in 24-well plates containing cryoprotectant (30% ethylene glycol, 25% glycerin, 45% PBS; v/v) at −20°C. A one-in-six series of sections from each animal (a series of rostral to caudal sections separated by 240 μm increments) was stained in the following way. Free-floating tissue sections were washed in tissue-buffering solution (TBS; 1.3% Trizma hydrochloride, 0.19% Trizma base, 0.9% sodium chloride) and incubated for 30 min in 0.6% hydrogen peroxide in TBS. Sections were then treated with 50% deionized formamide and 2× saline-sodium citrate (SSC) buffer for 120 minutes to denature DNA. Following DNA denaturation, sections were washed twice for 15 minutes each in SSC buffer prior to 37°C incubation in 2 M hydrochloric acid for 30 minutes. Washed sections were treated at room temperature with 0.1 M boric acid in TBS (pH 8.5), then blocked with TBS-X plus (0.3% Triton-X and 3% goat serum in TBS). Primary rat anti BrdU antibody (Cat. No.OBT0030; AbD Serotec, Raleigh, NC) was added to TBS-X plus at a dilution of 1:100 and incubated for 72 hours at 4°C. Following incubation, sections were washed with TBS and blocked with TBS-X plus for 30 minutes prior to addition of biotinylated secondary goat anti rat antibody (Cat. No. BA-9400; Vector, Burlingame, CA) in TBS-X at a dilution of 1:250 for 100 minutes at room temperature. Finally, tissue was processed using the ACB system (Cat. No. PK-6100; Vector) and stained using a diaminobenzidine (DAB) kit (Cat. No. D4418; Sigma, St. Louis, MO).

Image Analysis

The entire granule layer (bilateral) of the dentate gyrus was systemically photographed with a Zeiss brightfield microscope, using an Axiocam-to-computer interface with a total magnification of 100× to estimate the density of BrdU-positive nuclei (or cells). Photographs were analyzed using ImageJ software by tracing the granule layer and setting a threshold to remove the entire background excluding BrdU-positive nuclei. These automated counts were compared to hand counted values by simple linear regression. The relationship had an R2 value of 0.9688, represented by the equation: hand count = 0.9564*(automated count) − 0.4776. Because the automated counts slightly underestimated the value of hand-counted cells, the automated counts were corrected using the linear equation. The total number of BrdU+ cells was estimated by multiplying the linear transformed value by 0.85 to account for 15% probability that an individual BrdU+ cell was located in the top plane of the section (and therefore would be represented twice in adjacent sections) and by a factor of 6 because only 1-in-6 series was measured44.

Statistical Analysis

All statistical analyses were completed using SAS version 9.1 (SAS Institute, Cary, NC). A P-value less than 0.05 was considered statistically significant. Weekly averages for food intake and BW were analyzed using a repeated measures 2-way ANOVA with week as the within-subjects factor and dietary treatment as the between-subjects factor. Body composition measures (i.e., mass of fat pads, liver and spleen) were analyzed as raw values and expressed as a percentage of BW using two-sample unpaired t-tests assuming equal variances between groups. Distances traveled in the home cage (km/day), the discrimination index for the novel object test and number of BrdU positive cells in the granule layer were analyzed using unpaired t-tests. The average discrimination index across all animals was compared to zero using a one-sample t-test to determine whether the animals learned the task. Latency to fall from the rotarod was analyzed using a repeated measures 2-way ANOVA with day as the within-subjects factor and diet treatment as the between-subjects factor. Percent time freezing in the contextual fear conditioning apparatus was analyzed by 2-way ANOVA with shock group (shock or no shock) and dietary treatment as the two factors.