Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring

<div><p>Offspring of murine dams chronically fed a protein-restricted diet have an increased risk for metabolic and neurobehavioral disorders. Previously we showed that adult offspring, developmentally exposed to a chronic maternal low-protein (MLP) diet, had lower body and hind-leg muscle weights and decreased liver enzyme serum levels. We conducted energy expenditure, neurobehavioral and circadian rhythm assays in male offspring to examine mechanisms for the body-weight phenotype and assess neurodevelopmental implications of MLP exposure. C57BL/6J dams were fed a protein restricted (8%protein, MLP) or a control protein (20% protein, C) diet from four weeks before mating until weaning of offspring. Male offspring were weaned to standard rodent diet (20% protein) and single-housed until 8–12 weeks of age. We examined body composition, food intake, energy expenditure, spontaneous rearing activity and sleep patterns and performed behavioral assays for anxiety (open field activity, elevated plus maze [EPM], light/dark exploration), depression (tail suspension and forced swim test), sociability (three-chamber), repetitive (marble burying), learning and memory (fear conditioning), and circadian behavior (wheel-running activity during light-dark and constant dark cycles). We also measured circadian gene expression in hypothalamus and liver at different Zeitgeber times (ZT). Male offspring from separate MLP exposed dams had significantly greater body fat (P = 0.03), less energy expenditure (P = 0.004), less rearing activity (P = 0.04) and a greater number of night-time rest/sleep bouts (P = 0.03) compared to control. MLP offspring displayed greater anxiety-like behavior in the EPM (P<0.01) but had no learning and memory deficit in fear-conditioning assay (P = 0.02). There was an effect of time on <i>Per1</i>, <i>Per 2</i> and <i>Clock</i> circadian gene expression in the hypothalamus but not on circadian behavior. Thus, transplacental and early developmental exposure of dams to chronic MLP reduces food intake and energy expenditure, increases anxiety like behavior and disturbs sleep patterns but not circadian rhythm in adult male offspring.</p></div

MLP male offspring mice have no sociability phenotype and display decreased repetitive behavior.

<p>(A) Three-chamber test (MLP and Control, n = 9 each), (B) Marble burying (MLP and Control, n = 9 each). Data is presented as mean ± SEM. P<0.05 was considered statistically significant by student t-test for (A) and (B).</p

Food intake and energy expenditure in 12-wk-old MLP and Control offspring showing values over 24 hours, and the distribution between light and dark phases.

<p>Food intake and energy expenditure in 12-wk-old MLP and Control offspring showing values over 24 hours, and the distribution between light and dark phases.</p

Body composition.

<p>(A): Body weight; (B) Body fat as percent of total body mass in male offspring (8–12 weeks age) from dams exposed to either control or MLP diet (n = 7 each). Bars are mean ± SEM and P<0.05 statistically significant by student t-test.</p

MLP male offspring mice display anxiety-like but not depression-like behavior.

<p>Elevated plus maze test (MLP and Control, n = 9 each); (A) Time spent in open arm, center and closed arm, (B) Latency to enter closed arm. (C) Tail suspension test (MLP, n = 31 and Control, n = 13) and (D) Forced swim test (MLP and Control, n = 20 each). Data is presented as mean ± SEM. P<0.05 was considered statistically significant by student t-test.</p

Circadian physical activity and gene expression is not affected in MLP male offspring mice.

<p>(A) Representative double-plotted actogram for control (left panel) and MLP (right panel) offspring. Zeitgeber times (ZT) (0, 6, 12, 18) are represented on top. Black and white blocks below ZT’s indicate dark and light conditions, respectively. Mice were individually housed first in 12 h light:12 h dark for acclimation (L:D) (white background, top half) then in constant darkness (D:D) (gray background, bottom half). Black notches represent activity. The total wheel revolutions during final 10 days of the (B) acclimation (Control, n = 9; MLP, n = 15), (C) constant darkness (Control, n = 8; MLP, n = 15), and (D) Periodicity (Tau) (Control, n = 8; MLP, n = 15) is shown. (E) Total RNA (mRNA) from hypothalamus of MLP and control offspring (n = 3/time point/group) after two weeks housing under L:D cycle were extracted at ZT 0, 6, 12, or 18. Data shown is relative gene expression for indicated circadian genes (numerator) over the housekeeping genes <i>18s</i> and <i>Actin (denominator)</i>. All data are presented as mean ± SEM. P<0.05 was considered statistically significant by Mann-Whitney U test for (A-D) and by 2-way ANOVA with Bonferroni correction for (E). * indicates statistically significant effect of Zeitgeber time points between control and MLP offspring groups.</p

MLP male offspring mice show anxiety to an unfamiliar environment but no associative learning and memory deficit.

<p>Fear conditioning test in MLP and Control male offspring mice. (A) Training phase (MLP and Control, n = 20 each), (B) Context phase, (C) pre cue phase, and (D) cue phase. MLP, n = 18; Control, n = 19 for (B-D). Data is presented as mean ± SEM. P<0.05 was considered statistically significant by Mann Whitney test.</p

Spontaneous cage activity, measured as infra-red beam breaks in 12-wk-old MLP and Control offspring; values over 24 hours, and the distribution between light and dark phases are shown.

<p>Spontaneous cage activity, measured as infra-red beam breaks in 12-wk-old MLP and Control offspring; values over 24 hours, and the distribution between light and dark phases are shown.</p

MLP male offspring mice display dysfunctional night time resting/sleeping patterns.

<p>(A): Total resting/sleeping time/day; (B): Sleep bouts/day; (C): Sleep bout duration; (D): Maximum sleep bout duration (n = 7/group, 12 weeks age). Data shown is mean ± SEM with P<0.05 considered statistically significant by student t-test.</p