Impacts of maternal diet and exercise on offspring behavior and body weights

Accepted Manuscript Impacts of maternal diet and exercise on offspring behavior and body weights

Virginia C. Moser, Katherine L. McDaniel, Emily A. Woolard, Pamela M. Phillips, Jason N. Franklin, Christopher J. Gordon PII: DOI: Reference:

S0892-0362(17)30047-8 doi: 10.1016/j.ntt.2017.07.002 NTT 6704

To appear in:

Neurotoxicology and Teratology

Received date: Revised date: Accepted date:

1 March 2017 25 July 2017 26 July 2017

Please cite this article as: Virginia C. Moser, Katherine L. McDaniel, Emily A. Woolard, Pamela M. Phillips, Jason N. Franklin, Christopher J. Gordon , Impacts of maternal diet and exercise on offspring behavior and body weights, Neurotoxicology and Teratology (2017), doi: 10.1016/j.ntt.2017.07.002

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ACCEPTED MANUSCRIPT BRIEF COMMUNICATION

IMPACTS OF MATERNAL DIET AND EXERCISE ON OFFSPRING BEHAVIOR AND BODY WEIGHTS

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Virginia C Moser1, Katherine L. McDaniel1, Emily A. Woolard2, Pamela M Phillips1, Jason N. Franklin1, Christopher J Gordon1

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Meredith College, Raleigh, NC

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Toxicity Assessment Division, National Health Effects and Environmental Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC

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Dr. Chris Gordon MD B105-04 US EPA RTP, NC 27711 [email protected]

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Corresponding author:

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This manuscript has been reviewed following the policy of the National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, and was approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency.

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ACCEPTED MANUSCRIPT ABSTRACT Human and animal studies indicate that maternal obesity can negatively impact aspects of metabolism and neurodevelopment in the offspring. Not known, however, is whether maternal exercise can alter these adverse outcomes. In this study, Long-Evans female rats were provided a high fat (60%; HFD) or control diet (CD) 44 days before mating and throughout gestation and

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lactation. Running wheels were available to half of each diet group during the gestational period

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only, resulting in four conditions: CD diet with (CDRW) or without (sedentary; CDSED) exercise, and HFD with (HFRW) or without (HFSED) exercise. Only male offspring (one per

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litter) were available for this study: they were put on control diet two weeks after weaning and examined using behavioral evaluations up to four months of age. Before weaning, offspring of

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CDRW dams weighed less than offspring from CDSED or HFD dams. After weaning, the lower weight in CDRW offspring generally persisted. Adult offspring from HFSED dams performed

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worse than the HFRW group in a Morris water maze during initial spatial training as well as reversal learning; memory was not impacted. No differences between groups were seen in tests

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of novel object recognition, social approach, or chocolate milk preference. Thus, maternal diet and exercise produced differential effects on body weights and cognitive behaviors in the

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offspring, and the data demonstrate a positive impact of maternal exercise on the offspring in that

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it ameliorated some deleterious behavioral effects of a maternal high fat diet.

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ACCEPTED MANUSCRIPT 1. INTRODUCTION The National Institutes of Health reports that more than two-thirds of American adults are overweight or obese, due to an energy imbalance from high-calorie intake and/or sedentary lifestyle (NIH, 2016). Overweight or obese adults have a greater risk of numerous health conditions (e.g., type 2 diabetes, metabolic syndrome), and in addition, mothers who were overweight or obese before becoming pregnant increase their chances of having pregnancy

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complications (e.g., gestational diabetes, preeclampsia) as well as offspring with physical or

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neurological defects (March of Dimes, 2016). Some studies have shown that maternal obesity

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and metabolic disorders are associated with greater risk of neurological syndromes such as autism spectrum disorders, developmental delays, and attention deficit disorders (e.g.,

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Krakowiak et al., 2012; Van Lieshout et al., 2011).

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In rodent models, there is a considerable literature showing that maternal obesity induced with high-fat or unbalanced diets produces metabolic disorders in offspring (Li et al., 2011). In

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addition, there is growing evidence of neurodevelopment changes in the offspring including spatial cognitive deficits, anxiety, and abnormal social behaviors, some of which have been

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associated with neural inflammation and altered development of neural pathways (e.g., Bilbo et al., 2010; Can et al., 2012; Page et al., 2014; Sullivan et al., 2014; Tozuka et al., 2010; White et

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al., 2009). On the other hand, maternal exercise improves metabolic and cognitive function in offspring (e.g., Akhavan et al., 2008; Carter et al., 2013; Pampiansil et al., 2003; Robinson and

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Bucci, 2014; Stanford et al., 2015). Studies combining high fat diet and exercise with

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neurodevelopmental endpoints were not found.

In this study, female rats were provided a high fat (60%) or control diet before mating and throughout gestation and lactation. Running wheels were available to half of each diet group during the gestational period only. This paper presents exploratory data from male offspring that were placed on control diet after weaning and examined using several behavioral evaluations from 1 to 4 months after weaning.

2. METHODS 3

ACCEPTED MANUSCRIPT 2.1 Animals 2.1.1 Housing The US EPA animal facility was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and studies were approved by the US EPA NHEERL Institutional Animal Care and Use Committee. All rats were housed in standard acrylic cages with dimensions of (h, l, w: 20, 42, 20 cm), with

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hardwood chip bedding (Beta-Chip®, Northeastern Products, Warrensburg, NY) and shredded

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paper (Enviro-Dri®, Shepherd Specialty Papers, Watertown, TN). Pregnant rats were singly

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housed, and after weaning, pups were housed two/cage until two months of age, when they were

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moved to single cages.

2.1.2 Maternal treatment

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Initial dietary treatments and breeding were performed by Charles River Laboratories in their Raleigh NC facilities. Thirty day old female Long-Evans rats were started on either a control

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(TD.08806) or high fat (TD.06414) diet from Harlan Tecklad Diets (Madison, WI). The control diet (CD) had a caloric composition of 10.4% fat, 69.1% carbohydrate, and 20.5% protein,

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whereas the high fat diet (HFD) had a composition of 60.3% fat, 21.8% carbohydrate, and 18.4% protein. After 44 days on these diets, rats were bred over 3 consecutive days. Successfully

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mated rats (presence of sperm plug) were shipped to the US EPA animal facility on the same day

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of verification, which was designated as gestational day (GD) 1.

Upon receipt, the pregnant dams were assigned to either a sedentary (normal housing; SED) or

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active (placed in cages with running wheels; RW) group. Diet treatments were maintained through pregnancy, birth and lactation. Thus, the four maternal treatment groups were: control diet, sedentary (CDSED, n=13); control diet, active (CDRW, n= 14); high fat diet, sedentary (HFSED, n= 18); and, high fat diet, active (HFRW, n=18). The running wheels were removed on the day after birth to protect the pups from injury. For a more detailed explanation of maternal treatments, see Gordon et al. (2017).

2.1.3 Offspring

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ACCEPTED MANUSCRIPT On postnatal day (PND) 6, litters were culled to 8 (4 males and 4 females where possible), and pups were weaned on PND21. Pups remained on the same diets as the dams until PND33 to allow time for planned physiological tests (reported elsewhere). At this age, the offspring used for behavioral testing were placed on Purina 5001 rat chow with a composition by calories of 13.4% fat, 56.7% carbohydrate, and 29.8% protein. One male per litter was randomly selected and assigned to the behavioral tests described here (n=11-14/treatment). All other offspring

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were used for other studies. Dams and offspring were weighed approximately weekly.

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2.2 Experimental procedures 2.2.1 Running wheels

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The running wheel system was a stainless steel wire wheel (33 cm diameter; 1.02 m circumference; Starr Life Sciences, Oakmont, PA) placed in the home cage, and wheel

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revolutions were detected with a magnetic switch.

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2.2.2 Novel object recognition

Rats were tested on PND54-57 for novel object recognition (Ennaceur and Delacour (1988).

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Red acrylic test boxes were 60x50x36 cm, and the objects were travel mugs (filled with water) of

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similar size that differed in color, shape, and surface.

On the first day, rats were habituated to the box for 10 minutes. The next day, they were returned

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to the box with two different objects in adjacent corners, and allowed 5 minutes to interact with the objects. One hour later, one of the objects was replaced with a different, novel object, and

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the rat was allowed 2 minutes to explore these objects (counted when the rat’s nose appeared to be less than about 2 cm from object). Between each rat, the box was cleaned with disinfectant.

The number of visits as well as time spent exploring each object were recorded in the training and test sessions by an observer who was unaware of the rat’s treatment. Overall activity was the total time exploring and total number of visits to both objects. For the test session, preference for the novel object was calculated as the ratio of time spent or number of visits to the novel object compared to the total time or visits. Discrimination was measured by comparing each treatment group to a value of 0.5, which indicates equal exploration of both objects. 5

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2.2.3 Social approach Social approach testing (Varlinskaya and Spear, 2008) took place on PND63-64, using the novel object recognition box with clear acrylic dividers creating three compartments. The test rat was placed in the center compartment. A stimulus (stranger) rat, of the same age and gender as the test rat, was placed in one of the side compartments. Each rat was tested for 10 minutes, and the

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number of visits and time spent in close proximity (nose appearing to be less than 2 cm away) of

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either divider wall was recorded by an observer (unaware of the rat’s treatment). The box was

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cleaned between each trial. Overall activity was calculated, and preference and discrimination

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were measured comparing exploration of the side with the stranger rat to the empty side.

2.2.4 Morris water maze

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Rats were tested in the Morris water maze at age PND83-94. Test procedures have been previously described (Moser et al., 2001; Vorhees et al., 2006). Swimming was monitored by a

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video system (HVS Image, Hampton, UK), allowing analysis of latency, distance, location, and spatial patterns throughout the tank. For initial place training, rats were given two trials per day

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(approximately 5 min between trials) for 9 days, with a hidden platform in a fixed position. Starting positions varied in a pseudorandom order, and each rat was given 60 seconds to find the

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platform where it remained for 15 seconds. On the tenth day, a reference memory probe (spatial bias for the target quadrant) was conducted with no platform (60 s free swim). This was

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followed by a visual probe, in which a raised platform with a black band at the water’s surface was located in a quadrant opposite that of the original target. For the next three days, using two

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trials per day, a reversal procedure was conducted with the submerged platform located in the opposite quadrant: all other aspects of training were the same as before.

Learning was evaluated as a change across days in latency, path length, and path ratio (ratio of path taken to the most direct path) to find the platform. On the no-platform probe, the time spent in the target quadrant was measured. For all procedures, percent time floating, active swim speed, and time in the thigmotaxis zone (within 7 cm of tank wall) and the middle annulus were recorded.

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ACCEPTED MANUSCRIPT 2.2.5 Chocolate milk preference Rats were tested for chocolate milk preference on PND104-106. On the first day, rats were given a 1-hour exposure to chocolate milk (1:3 dilution of 1% fat chocolate milk). The next day, water bottles were removed for 4 hours, and then a bottle with tap water and one with chocolate milk were placed on each cage (milk bottle alternated sides across each group). After two hours, bottles were removed and weighed to determine consumption. Preference was measured as the

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consumption of milk compared to total fluid intake. For additional details, see Slotkin et al.

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(1999) and Roegge et al. (2008).

2.3 Statistics

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All data were analyzed using ANOVA (SAS, Cary, NC), using repeated measures where appropriate (e.g., body weight over days, water maze path length over daily blocks). In cases of

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overall significance, treatment groups were compared using Tukey’s post-hoc test. Level of

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significance in all tests was p<0.05.

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3. RESULTS 3.1 Maternal body weight and exercise

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Upon receipt (GD1), rats fed the HFD weighed significantly more than the CD rats (HFD, 266±4 g, CD, 212±3 g, mean ± SEM) (Figure 1A). Wheel running activity increased from GD1 to

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GD12, followed by a gradual lowering of activity until the day of parturition. There was no significant effect of diet on wheel running: cumulative revolutions were 98,839 ± 10,376 and

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104,688 ± 11,270 (mean ± SEM, in m) in the CD and HFD groups, respectively (Gordon et al.,

There was a significant effect of diet and exercise on dam body weight (Figure 1A) during gestation (day*treatment F(9, 49)=17.97, p<0.0001) and lactation (day*treatment F(6, 49)=12.54, p<0.0001). Body weights of the HFSED dams remained significantly higher than other groups from GD7 to lactation day (LD) 13, and on LD21 the HFSED group was not different from HFRW. During gestation, the CDRW group weighed the least, and was significantly lower than the CDSED in late gestation (GD14, 21) and lower than HFRW during most of gestation and 7

ACCEPTED MANUSCRIPT lactation (GD7, 21, LD6-21). Interestingly, the HFRW and CDSED groups were not different during RW access and through LD6. With no RW access during lactation, the CD groups were not different from each other but were lower than both HFD groups (LD13, 21).

3.2 Offspring body weight Before PND33, weights represented litter averages of males and females, and thereafter weights

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were recorded for the individual rats used in this study. During the time that dams and offspring

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were provided the two different diets (up to PND33), body weight showed significant change

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over days (day*treatment F(9, 147)=26.75, p<0.0001) (Figure 1B). There were no significant interactions between sex and treatment, thus males and females were combined for each litter.

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Before weaning (PND6, 13), pups in the CDRW group had significantly lower body weights than all other groups, and the CDSED group weighed less than the HFSED group on PND13. As

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the pups grew (PND21, 27), there was a significant difference due to diet (HFD groups heavier

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than CD) but within each diet group, the impact of maternal exercise was no longer significant.

On PND33, rats were placed on standard control rat chow for the rest of the study. The

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treatment factor did not reach statistical significance (F(3, 45)=2.75, p=0.0536), but average

3.3 Behavior

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3.3.1 Morris water maze

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weights appeared highest in HFSED and lowest in CDRW groups (Figure 1C).

All rats learned the platform location over two weeks of training with no treatment-related

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differences in latency or path length (Figure 2A). However, during this time the path ratio (overall treatment F(3, 45)=2.84, p=0.0487, no interaction with day) was significantly higher in the HFSED group compared to the HFRW group, suggesting that a less direct, and less efficient, path was taken even though they did reach the target (Figure 2B). There were no other treatment-related differences during spatial training or in the probes.

When the platform was moved for reversal training, the HFSED group did not learn the new position as quickly (Figure 2C), with a significant interaction of treatment across days (day*treatment F(6, 45)=2.38, p=0.0348). The significant difference was between days 1 and 2, 8

ACCEPTED MANUSCRIPT when all but the HFSED group decreased in path length as well as time to find the platform. There was essentially no change from day 1 to 2 in the HFSED rats (Figure 2C), and this was statistically different from the HFRW and CDSED groups (comparison to CDRW did not reach significance). By the third day all groups were similar. This suggests that the HFSED group was slower to “unlearn” the previous platform position but did learn the new position after

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another day.

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3.3.2 Other behaviors

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There were no significant differences across any groups in novel object memory (group preferences ≥ 0.63), time spent investigating a stranger rat (group preferences ≥ 0.81), or

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chocolate milk preference (group preferences 92-93%).

4. DISCUSSION

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This study examined the effects of maternal diet with and without access to running wheel exercise on the behavioral development in the offspring. An appropriate litter allocation (one per

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litter) was possible with males but not females. Because of this, comparable data for females were not obtained, and it is unknown whether there would be sex differences in the behaviors

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studied here.

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The influence of exercise during gestation was evaluated in offspring from CD dams, generally showing a lower body weight gain in the male CDRW offspring. This could reflect altered

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metabolic parameters, e.g., glucose homeostasis, as has been shown previously (e.g., Carter et al., 2013). Given that the sedentary dams would be considered most like “controls” in typical toxicological studies, the lower body weight could be interpreted as an adverse outcome. On the other hand, it could represent an improved and healthier metabolic status, especially since studies show that long-term health is better in rats whose weight gain is lowered with caloric restriction (Sohal and Forster, 2014). Unlike other reports using similar test methods (water maze, novel object recognition; Akhavan et al., 2008; Dayi et al., 2012; Pampiansil et al., 2003), this study did not detect improved cognitive function in offspring of exercised dams.

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ACCEPTED MANUSCRIPT A maternal HFD has been shown to produce offspring predisposed to obesity and metabolic syndrome in animal models (Li et al., 2011). The current study demonstrated a similar effect of a maternal HFD on body weight of the offspring up to PND27, when offspring were continuing on the HFD. However, once rats were placed on CD, an effect of the maternal HFD on body weight of adult offspring was less apparent. We conclude that a persistent effect of maternal HFD and exercise on body weight of adult offspring on a control diet may be subtle and more

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difficult to detect.

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Studies of the impacts of maternal obesity on cognition and other behaviors of offspring have been less than consistent (e.g., Bilbo et al., 2010; Page et al., 2014; Sullivan et al., 2014; White et

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al., 2009). For example, impaired learning and memory were reported in offspring of maternal HFD regardless of post-weaning diet (Page et al., 2014), but White et al. (2009) observed

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memory deficits only in offspring maintained on HFD as adults. In the present study, spatial learning, but not memory, was impaired in offspring of sedentary HFD dams compared to the

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HFD dams with running wheels. This was evidenced by less effective path during training, and slower reversal learning. HFRW offspring were not different from offspring of dams on control

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diets, with or without running wheel access.

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It is possible that running wheel access during gestation impacted maternal behavior, and this could account for the differences between the HFD exercised and sedentary groups on behavioral

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CD group.

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measures. However, in this study differences between RW and SED groups were not seen in the

In summary, maternal high fat diet and exercise appears to have elicited differential effects on growth and selective behaviors of adult offspring. These data suggest a positive impact of maternal exercise in that it 1) lowered body weight in offspring in the CD group, and 2) ameliorated the deleterious behavioral effects of the HFD on water maze performance. However, compared to some published studies, our lack of effects on behavior in the CD group, and relatively subtle differences in the HFRW and HFSED groups, may reflect differences in exercise regimen, testing protocols, or other experimental variables. The current data are

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ACCEPTED MANUSCRIPT preliminary and suggestive, and may contribute to a growing understanding of the impact of

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maternal exercise and diet on growth and behavior of offspring.

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ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS The authors thank Drs. A. Johnstone and S. Snow for their review of this manuscript.

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FUNDING

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This research was funded via the intramural research program of the Office of Research and

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Development, US EPA.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS

Figure 1. Effects of diet and exercise on body weight; see text for specific treatment differences across groups. A: Weight of dams throughout gestation (GD) and lactation (LD). B: Weight of litters (males and females together/litter) until control diet was introduced for all rats. C: Weight

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of male rats throughout behavioral testing. All data plotted as mean ± SEM.

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Figure 2. Effects of diet and exercise on Morris water maze learning. A: Path length to find the platform over 9 days of training, followed by 3 days of reversal training. B: Path ratio averaged

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across initial 9 days of spatial training. HFSED group significantly higher than HFRW. C: Change in path length from first to second day of reversal training. HFSED group significantly

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different from HFRW and CDSED groups. All data plotted as mean ± SEM.

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Fig. 1

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