Early-life physical activity reverses metabolic and Foxo1 epigenetic misregulation induced by gestational sleep disturbance

Abstract

Sleep disorders are highly prevalent during late pregnancy and can impose adverse effects, such as preeclampsia and diabetes. However, the consequences of sleep fragmentation (SF) on offspring metabolism and epigenomic signatures are unclear. We report that physical activity during early life, but not later, reversed the increased body weight, altered glucose and lipid homeostasis, and increased visceral adipose tissue in offspring of mice subjected to gestational SF (SFo). The reversibility of this phenotype may reflect epigenetic mechanisms induced by SF during gestation. Accordingly, we found that the metabolic master switch Foxo1 was epigenetically misregulated in SFo livers in a temporally regulated fashion. Temporal Foxo1 analysis and its gluconeogenetic targets revealed that the epigenetic abnormalities of Foxo1 precede the metabolic syndrome phenotype. Importantly, regular physical activity early, but not later in life, reversed Foxo1 epigenetic misregulation and altered the metabolic phenotype in gestationally SF-exposed offspring. Thus, we have identified a restricted postnatal period during which lifestyle interventions may reverse the Foxo1 epigenetically mediated risk for metabolic dysfunction later in the life, as induced by gestational sleep disorders.

Keywords: Foxo1 gene; epigenetics; offspring metabolism effect; physical activity; pregnancy sleep disruption; reverse epigenetic effects.

Fig. 1.

Experimental design. Pregnant mice were exposed to fragmented sleep (SF) or sleep control (SC) conditions. After lactation and weaning (4 wk after birth), SCo and SFo were subdivided into three groups (n = 8 per group): 1) undergoing early physical activity (PA; 4–8 wk), 2) undergoing late PA (16–20 wk), or 3) not undergoing any PA during 4–24 wk. For the indicated time points, blood and tissue samples were collected from each group of mice, and biochemical and epigenetic analyses were performed.

Fig. 2.

Late-pregnancy SF induces metabolic dysfunction of the offspring. A: SFo (n = 8, ■) accrued higher body weight than SCo (n = 8; □) measured from 0 to 24 wk after birth. Glucose tolerance test (GTT; B) and insulin (ITT; C) tolerance tests in SCo (n = 8, □) and SFo (n = 8, ■) showing altered glucose tolerance and reduced insulin sensitivity in gestational SF-exposed offspring measured at age 24 wk. SFo (n = 8; solid bars) had higher serum triglycerides (D) and cholesterol (E) than the SCo (n = 8, open bars) at 24 wk after birth. Results are expressed as means ± SE; *P < 0.05.

Fig. 3.

No significant differences in energy expenditure in SFo and SCo. Oxygen consumption (V̇o₂) did not significantly differ between offspring of SFo (n = 8; solid columns) and SCo (n = 8; striped columns) mothers during a typical 24-h cycle.

Fig. 4.

PA during early life reversed the metabolic phenotypic alterations in SFo. Measurements of body weight (A), food intake (B), and glucose tolerance tests (C) of SFo_early (n = 8, ○), SFo-PA_early (n = 8, ■), SFo_late (n = 8, open △), and SFo-PA_late (n = 8, ▲) livers was performed as described in Fig. 1. D: PA from week 4 to week 8 of life led to a normalization of visceral fat mass in offspring of SF mothers at 24 wk of age. SCo_early (n = 8; open column), SCo-PA_early (n = 8, open column), and SFo_early (n = 8, open column), and SFo-PA_early (n = 8, open column). *P < 0.05.

Fig. 5.

Validation of the epigenetic analysis in SCo and SFo livers, measured at age 20 wk. Methylated DNA immunoprecipitation (MeDIP) of 5-methyl-CpG and chromatin immunoprecipitation (ChIP) of H3K4m3 at the active Gapdh and transcriptionally silenced Tsh2b genes were performed in SCo (n = 4–6; open columns) and SFo (n = 4–6; solid columns) livers, as described in Fig. 6, B and D, but the Gapdh for ChIP and Tsh2b for MeDIP values of SCo and SFo levels were arbitrarily set to 1. The relative gene abundance sequence was calculated as the ratio of its concentration in the immunoprecipitated (IP) fraction to that in the input. Results are shown as means ± SD; *P < 0.05.

Fig. 6.

Late-gestation SF induces offspring misregulation of the Foxo1 in the liver by epigenetic mechanisms. ChIP of histone H3 acetylation (A), histone H3K4 trimethylation (B), MeDIP of DNA 5-hydroxymethyl-CpG (C), and 5-methyl-CpG (D) over the Foxo1 Putative Enhancer (P.Enhancer) and Promoter regions and control genes in SCo (n = 4–6; open columns) and SFo (n = 4–6; solid columns) livers, measured at age 20 wk. The relative gene abundance sequence was calculated as the ratio of its concentration in the IP fraction to that in the input DNA. These fold-difference values were corrected by subtraction of the nonspecific signal derived from the nonimmune rabbit IgG ChIP. It was then normalized to similar data obtained for Tbp (for ChIP and 5-hmC MeDIP) and Tsh2b (for 5-mC MeDIP). The values of SCo levels were arbitrarily set to 1, and results are shown as means ± SD; *P ≤ 0.05. E: Ratio of histones H3K4 tri-methylation/H3K4 monomethylation ChIPs relative abundance at the Foxo1 P.Enhancer was calculated for the SCo (n = 4–6, open column) and SFo (n = 4–6, solid column) livers. ChIP was performed as described in A and B, measured at age 20 wk. F: ratio of histones H3K27 dimethylation/H3K4 monomethylation ChIPs relative abundance at the Foxo1 P.Enhancer was calculated for the SCo (n = 4–6; open column) and SFo (n = 4–6, solid column) liver. ChIP was performed as described in A and B, measured at age 20 wk. *P < 0.05. G: ChIP of histone H3K27 acetylation at Foxo1 P.Enhancer and control gene. H: UCSC Genome Browser presentation of mouse Foxo1 region and histone H3K4m3 and histone H3K4m1 (ENCODE Project).The position of the two ChIP and MeDIP primer sets, spanning the regions of the Foxo1 promoter and putative enhancer, is shown.

Fig. 7.

SF during late gestation induces upregulation in the offspring of the Foxo1 in liver. Expression analyses in SCo (n = 4–6; open columns) and SFo (n = 4–6; solid columns) livers of Foxo1, control genes, and gluconeogenesis genes were measured at age 20 wk. Each bar represents the abundance of mRNA relative to Tbp mRNA. The values of SCo levels were arbitrarily set as 1, and results are shown as means ± SD; *P ≤ 0.05.

Fig. 8.

In early life, the SFo Foxo1 epigenetic abnormalities affect the putative regulatory element. ChIP of histone H3 acetylation over the Foxo1 P.Enhancer (A) and promoter region (B). ChIP of histone H3K4 trimethylation over the Foxo1 PEnhancer region (C) and promoter region (D) in SCo (n = 4–6; open column) and SFo (n = 4–6; solid column) liver were performed as described in Fig. 6, A and B at three different time points (3, 8, and 20 wk). Expression analyses of Foxo1 (E) and its gluconeogenesis target gene Pdk4 (F) in SCo (n = 4–6; open columns) and SFo (n = 4–6, solid columns) livers were performed, as described in Fig. 4 at three different time points (3, 8, and 20 wk of age). Results are shown as means ± SD; *P < 0.05.

Fig. 9.

Late-gestation SF induces gene expression misregulation of the epigenetic chromatin modification enzymes in the offspring. A: epigenetic chromatin modification enzymes RT2 Profiler PCR Array of SCo and SFo mouse liver at age 20 wk. B: list of the most differentially expressed epigenetic chromatin modification enzymes in SFo using a Log2 scale with a cut-off of twofold changes.

Fig. 10.

Epigenetic abnormalities in Foxo1 among SFo mice are reversible by instituting PA during early life. MeDIP of 5-hydroxymethyl-CpG (A), 5-methyl-CpG (B), ChIP of histone H3K4 tri-methylation (C), and histone H3K27 trimethylation (D) over the Foxo1 PEnhancer and promoter (P) regions and expression analyses (E) were performed in SCo_early (SC) (n = 4–6; open columns), SCo-PA_early (SCPA) (n = 4–6; dotted columns), and SFo_early (SF) (n = 4–6; solid columns); SFo-PA_early (SFPA) (n = 4–6; striped columns) liver, as described in Fig. 6 after PA or no PA from 4 to 8 wk. MeDIP of 5-hydroxymethyl-CpG over Foxo1 (F) and gene expression analyses (G) were performed in SCo_late (SC) (n = 4–6; open columns), SCo-PA_late (SCPA) (n = 4–6; dotted columns) and SFo_late (SF) (n = 4–6; solid columns); SFo-PA_late (SFPA) (n = 4–6; striped columns) livers, as described in Fig. 6 after PA or no PA from 16 to 20 wk. Results are expressed as means ± SE; *P < 0.05.

Fig. 11.

The epigenetic effect of the PA on the Foxo1 is SFo-specific and was not seen at a control gene Actb. MeDIP of 5-methyl-CpG (A) and 5-hydroxymethyl-CpG (B) over the control Actb gene promoter and expression analyses (C) were performed in SCo_early (n = 4–6; open columns); SCo-PA_early (n = 4–6; dotted columns) and SFo_early (n = 4–6; solid columns); SFo-PA_early (n = 4–6; striped columns) liver as described in Fig. 6, after PA or no PA from 4 to 8 wk. Results are expressed as means ± SE. *P ≤ 0.05.

Fig. 12.

Schematic design of metabolic and Foxo1 epigenetic abnormalities reverses induced by gestational sleep disturbance. Late-pregnancy sleep perturbations induce metabolic phenotype in the SF offspring at ∼20 to 24 wk after birth. Gestational sleep disturbance causes epigenetic misregulation of Foxo1 in the SF offspring. The SFo Foxo1 epigenetic abnormalities and the metabolic phenotype in the offspring were reversed by physical activity, but only when restricted to a defined window of opportunity during postnatal life: from week 4 to week 8 after birth.