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. 2020 Dec 18;130(3):605–616. doi: 10.1152/japplphysiol.00641.2020

Exercise during pregnancy mitigates negative effects of parental obesity on metabolic function in adult mouse offspring

Rhianna C Laker 1,2, Ali Altıntaş 2, Travis S Lillard 1, Mei Zhang 1,3, Jessica J Connelly 1,4, Olivia L Sabik 5,6, Suna Onengut 6, Stephen S Rich 6, Charles R Farber 5,6,7, Romain Barrès 2, Zhen Yan 1,3,4,8,✉
PMCID: PMC8091938  PMID: 33332990

Abstract

Parental health influences embryonic development and susceptibility to disease in the offspring. We investigated whether maternal voluntary running during gestation could protect the offspring from the adverse effects of maternal or paternal high-fat diet (HF) in mice. We performed transcriptomic and whole-genome DNA methylation analyses in female offspring skeletal muscle and targeted DNA methylation analysis of the peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc-1α) promoter in both male and female adult offspring. Maternal HF resulted in impaired metabolic homeostasis in male offspring at 9 mo of age, whereas both male and female offspring were negatively impacted by paternal HF. Maternal exercise during gestation completely mitigated these metabolic impairments. Female adult offspring from obese male or female parent had skeletal muscle transcriptional profiles enriched in genes regulating inflammation and immune responses, whereas maternal exercise resulted in a transcriptional profile similar to offspring from normal chow (NC)-fed parents. Maternal HF, but not paternal HF, resulted in hypermethylation of the Pgc-1α promoter at CpG-260, which was abolished by maternal exercise. These findings demonstrate the negative consequences of maternal and paternal HF for the offspring’s metabolic outcomes later in life possibly through different epigenetic mechanisms, and maternal exercise during gestation mitigates the negative consequences.

NEW & NOTEWORTHY Maternal or paternal obesity causes metabolic impairment in adult offspring in mice. Maternal exercise during gestation can completely mitigate metabolic impairment. Maternal obesity, but not paternal obesity, results in hypermethylation of the Pgc-1α promoter at CpG-260, which can be abolished by maternal exercise.

Keywords: exercise, glucose intolerance, obesity, offspring, pregnancy

INTRODUCTION

Overweightness and obesity are growing epidemics and are predicted to affect 58% of adults globally by 2030 (1). Obesity during pregnancy increases susceptibility of the offspring to metabolic dysfunction and contributes to a vicious cycle of transgenerational disease transmission (2). Maternal exercise improves pregnancy outcomes in humans (3) and confers lifelong benefits to offspring health in animal models (4, 5).

An underlying mechanism for parent–offspring transmission of disease propensity in humans and rodents is epigenetic regulation, such as DNA methylation, histone modifications, and small RNAs (4, 6). Type 2 diabetes cause not only hypermethylation of the peroxisome proliferator-activated receptor-γ coactivator 1α (Pgc-1α or Ppargc1a) gene, a transcriptional coactivator for mitochondrial biogenesis and oxidative metabolism, in skeletal muscle in humans (7), but also maternal obesity induced by high-fat diet (HF) in mice results in metabolic impairment and hypermethylation of the Pgc-1α promoter at the -260 CpG site along with reduced Pgc-1α and downstream target gene expression in offspring skeletal muscle (4). Importantly, maternal exercise before and during gestation prevents all these abnormalities, providing the first evidence of the positive impact of maternal exercise on epigenetic regulation of an important metabolic regulator in mitigating parent-offspring transmission of a noncommunicable disease (4).

An outstanding question is whether exercise in obese mothers after conception has beneficial metabolic and epigenetic outcomes in the offspring. This question is a clinically relevant one for exercise prescription to obese women following their first clinic visit post conception with huge implications for stemming the transgenerational transmission of metabolic diseases. Previously, paternal obesity has been shown to be detrimental to the offspring pancreatic β-cell, glucose homeostasis, and kidney function in animal models (8, 9). Therefore, another important question is whether obesity in the father also transmits poor health outcomes through epigenetic changes to the offspring, which could be mitigated by maternal exercise. To address these questions, we utilized a mouse model of HF and voluntary wheel running during pregnancy, measured metabolic outcomes in male and female adult offspring, and performed transcriptomic and DNA methylation analyses in the female offspring skeletal muscle to identify genes and pathways that may be altered.

METHODS

Animals

All procedures were approved by the Animal Care and Use Committee at the University of Virginia. Mice were provided food and water ad libitum and housed in 12/12-h light/dark cycle at 22–24°C. In maternal HF study, female mice (C57BL/6, 8-wk-old) were subjected to either normal chow (NC; 5.8% fat, Envigo, WI) or HF (60% fat, Research Diets, NJ) for 6 wk before mating and during gestation. For mating, a lean male mouse (14-wk-old) was placed in the cage with 2 female mice overnight. When confirmed pregnant (by vaginal plug), the female mice were housed individually in cages equipped with running wheels (Ex), which were locked for the sedentary groups (Sed). Hence, there were three experiment groups denoted by NC-Sed, M-HF-Sed, and M-HF-Ex groups (M, maternal), with n = 6 mating female mice per group. In paternal HF study, male mice (8-wk-old) were subjected to either NC (n = 4) or HF diet (n = 4) for 6 wk. These male mice were then mated with lean female mice (14-wk-old), and the female mice were then housed individually with running wheels as described previously. The three experimental groups were denoted by NC-Sed, P-HF-Sed, and P-HF-Ex groups (P, paternal), with n = 6 mating female mice per group. From the day of birth onward, all dams and offspring were provided normal chow and remained sedentary. Adult offspring were humanely euthanized with CO2 at 10 mo of age, and fasted blood and quadricep muscle were collected. Maternal HF study and paternal HF study were conducted in parallel, allowing the NC-Sed group to serve as the control group for both studies.

Glucose and Insulin Tolerance Test

Glucose tolerance test (GTT) was conducted in male and female 9-mo-old offspring after 6 h of fasting following glucose injection (3.0 g/kg body wt, i.p.). Blood glucose was assessed before and 15, 30, 60, and 90 min after injection (10). Insulin tolerance test (ITT) was performed 8 h into light cycle (∼2 PM) following injection (1 U/kg body wt, i.p.) and mice did not have access to food during the test. Blood glucose was assessed before and at 15, 30, and 60 min after injection.

Fasting Serum Glucose, Insulin, and Homeostatic Model Assessment of Insulin Resistance

Plasma glucose was measured using a glucose colorimetric assay kit (Cayman Chemical), and insulin was measured using an ultrasensitive mouse insulin ELISA kit (Crystal Chem). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as (glucose × insulin)/22.5, with glucose units as mmol/L and insulin as μIU/mL.

RNA Sequencing

RNA from three female offspring/group was subject to the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol (Illumina) followed by 100-bp single-end sequencing (HiSeq 2500, Illumina). Reads were mapped to mm10 with STAR (11) v2.5.2b, and gene read coverages were computed with featureCounts (12) v1.5.2 using GENCODE (13) vM13 annotation. Approximately 23 million reads/sample were assigned to genes, and differentially expressed genes were identified using DESeq2 (14) v1.11.8 with independent filtering while adjusting P values using Benjamini and Hochberg’s method (15). Differentially expressed genes were identified by applying an adjusted P value cutoff of 0.1 (Padj ≤ 0.1). Pathway enrichment analysis was performed using Fisher’s exact test via the Gene Ontology (GO) database (domain: biological processes) for differentially expressed genes. The terms from the GO database with the same adjusted P value (15) cutoff (Padj ≤ 0.1) were considered as significantly enriched.

Reduced Representation Bisulfite Sequencing

Reduced representation bisulfite sequencing (RRBS) libraries were generated from female offspring skeletal muscle DNA as described earlier (16) and subjected to 75-bp single-end sequencing (NextSeq 2500, Illumina). Reads were trimmed using Trim Galore! v0.4.3 with the –rrbs flag. Approximately 20 million trimmed reads/samples were aligned to mm10, GENCODE (13) (vM13), and methylation levels estimated using Bismark (17) v0.18.1 and Bowtie2 (18) v2.2.5 on default settings. The SNPs for dbSNP (19) (build 142) with major allele frequency 0.95 at most in autosomal chromosomes and CpGs with coverage >10 times the 95th percentile of coverage in each sample were filtered out. Differential methylation analysis was performed by edgeR v3.24.0 using “(sequencing) pool” (20) as a covariate for maternal and paternal lines separately. CpGs with minimum eight counts were considered for differential methylation analysis, and CpGs (or CpGs in promoters or gene bodies) with an adjusted P value of ≤0.1 was considered as differentially methylated. Reactome (21) pathway enrichment analysis using Fisher’s exact test was performed by using genes in close proximity to differentially methylated CpG sites and promoters for maternal and paternal studies, respectively. The terms from Reactome database with the same adjusted P value (15) cutoff (Padj ≤ 0.1) were considered as significantly enriched. A relevant subset of the enrichment results is reported in the figures. All sequencing data are available using GEO Accession Number GSE144112.

Targeted DNA Methylation Analysis

Pyrosequencing was performed on male and female skeletal muscle DNA exactly as described previously (4).

Statistical Analysis

Data are presented as means ± SE and separated by sex. Comparisons for fasting glucose, insulin, HOMA-IR, area under the GTT curve, and pyrosequencing were done by one-way ANOVA followed by the Student–Newman–Kuels post hoc test with P < 0.05 as statistically significant. For GTT analyses, two-way ANOVA with repeated measures was conducted.

RESULTS

Maternal Exercise during Gestation Protects Offspring from Maternal Obesity

Maternal body weight was moderately but significantly higher (8%) in HF-fed compared with NC-fed mice (22.1 ± 0.3 vs. 20.3 ± 0.5 g, respectively). However, during pregnancy, sedentary HF-fed dams (M-HF-Sed) had much greater body weight (∼19% higher) than HF-fed dams with voluntary running (M-HF-Ex) and control mice (41.9 ± 2.8, 35.1 ± 1.7, 35.3 ± 1.0 g at term, respectively). Running distance averaged 11.3 and 7.6 km/day in weeks 1 and 2, respectively, and declined to 0.9 km/day in week 3. Offspring birth weight was similar between groups (not shown) with no differences in growth trajectory (Fig. 1, A and B).

Figure 1.

Figure 1.

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Gestational environment impacts adult male and female offspring metabolic homeostasis differently. Adult offspring (9-mo-old) from NC-fed sires mated with females on NC (NC-Sed), on HF with sedentary activity during gestation (M-HF-Sed) or voluntary running exercise (M-HF-Ex) were assessed. Male (A) and female (B) offspring growth profile; male fasting insulin (C), glucose (D), and HOMA-IR (E). Female fasting insulin (F), glucose (G), and HOMA-IR (H). *P < 0.05, ***P < 0.001, ****P < 0.0001 (n = 7–11 mice). HF, high-fat diet; NC, normal chow.

We detected sex differences at 9 mo of age, such that male offspring from obese dams showed significantly higher fasting insulin, glucose, and HOMA-IR than NC-Sed offspring (Fig. 1, C–E), whereas female offspring showed no differences between M-HF-Sed and NC-Sed groups (Fig. 1, F–H). Interestingly, maternal exercise provided significant metabolic benefits to both male and female offspring. Male offspring in M-HF-Ex group were protected from the detrimental effects of maternal HF, showing significantly lower fasting insulin, glucose, and HOMA-IR than M-HF-Sed offspring, whereas female offspring in M-HF-Ex group showed a significant reduction in fasting glucose with trends toward lower insulin (P = 0.09) and HOMA-IR (P = 0.06) compared with NC-Sed offspring (Fig. 1, F–H). GTT revealed significantly higher glucose excursion in male M-HF-Sed offspring at 30 min than NC-Sed offspring, with a trend (P = 0.07) toward higher area under the glucose curve (AUC; Fig. 2, A–C). Maternal exercise improved male offspring glucose tolerance with lower glucose excursion at 15 and 30 min compared with M-HF-Sed offspring and significantly reduced AUC (Fig. 2, A and B). There were no differences between groups during ITT (Fig. 2C). These parameters were not significantly different in female offspring (Fig. 2, D–F).

Figure 2.

Figure 2.

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Exercise during gestation prevents maternal HF-induced glucose intolerance in the male offspring. Adult offspring (9-mos-old) from NC-fed sires mated with females on NC (NC-Sed), on HF with sedentary activity during gestation (M-HF-Sed) or voluntary running exercise (M-HF-Ex) were assessed. Male offspring blood glucose during GTT (A), blood glucose AUC (B), and blood glucose during ITT (C). Female offspring blood glucose during GTT (D), blood glucose AUC (E), and blood glucose during ITT (F). *P < 0.05, **P < 0.01 M-HF-Sed vs. NC-Sed; #P < 0.05, ##P < 0.01 M-HF-Ex vs. M-HF-Sed. (n = 7–11 mice). GTT, glucose tolerance test; HF, high-fat diet; ITT, insulin tolerance test; NC, normal chow.

To understand the role of transcriptional changes in the impact of maternal obesity and exercise on the offspring, we performed RNAseq analysis in adult female offspring skeletal muscle. We observed 17 (M-HF-Sed vs. NC-Sed), 11 (M-HF-Ex vs. NC-Sed), and 10 (M-HF-Ex vs. M-HF-Sed) differentially expressed genes between groups as indicated. Three genes that were upregulated in the offspring of obese dams, which was mitigated by maternal exercise (Fig. 3, A–C), were aspartate dehydrogenase domain containing (Aspdh), which plays a role in NAD metabolism (Fig. 3A), bile acid-coenzyme A:amino acid N-acyltransferase (Baat) in long- and very-long-chain fatty acid metabolism (Fig. 3B), and the histocompatibility 2, K1, K region (H2-K1) in immune regulation (Fig. 3C). Conversely, the arestin domain containing 3 (Arrdc3) gene was upregulated by maternal exercise compared with that in M-HF-Sed (Fig. 3D), which has been reported to play a role in liver (22) and adipose (23) metabolism as well as respond to changes in anabolic and catabolic stimuli in skeletal muscle (24). Gene ontology enrichment analyses revealed distinct transcriptional profiles between M-HF-Sed and both NC-Sed and M-HF-Ex offspring (Fig. 3E). M-HF-Sed offspring showed gene ontologies related to immune and inflammation pathways compared with NC-Sed offspring (Fig. 3E). In contrast, an upregulation of pathways related to “heat generation and adipose tissue development” was observed in M-HF-Ex offspring (Fig. 3E) and were predominantly driven by changes in Arrdc3. Importantly, the transcriptional profile of M-HF-Ex offspring was similar to NC-Sed, highlighting the protective effects of exercise during gestation against the deleterious impact of maternal HF.

Figure 3.

Figure 3.

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Maternal exercise prevents maternal obesity-induced transcriptional and epigenetic changes in female adult offspring skeletal muscle. RNAseq (n = 3 mice) and RRBS (n = 4–7 mice) were performed for adult female offspring (9-mo-old) skeletal muscle. A–D: genes identified from RNAseq analysis for Aspdh, Baat, Cpn1, and Arrdc3; gene ontology enrichment analysis for pathways that are over- or under-represented in RNAseq data set (E); Venn diagram representing the number of differentially methylated promoter regions (F); pathway enrichment analysis of CpG methylation showing gene pathways that are up- or downregulated in response to the change in methylation status (G). RRBS, reduced representation bisulfite sequencing.

Genome-wide DNA methylation analysis in the female offspring skeletal muscle revealed 29 differentially methylated promoter regions (DMRs) between M-HF-Sed and NC-Sed offspring and 15 DMRs between M-HF-Sed and M-HF-Ex, with 7 DMRs in common (Fig. 3F). Between M-HF-Ex and NC-Sed offspring, there were 13 DMRs with two of these also present between M-HF-Sed and M-HF-Ex. Pathway enrichment analysis of the CpG methylation data set revealed that the “metabolism of carbohydrates” pathway was downregulated in M-HF-Ex versus NC-sed offspring with a parallel upregulation of “fatty acid metabolism” (Fig. 3G). We did not observe any relevant pathways that changed between M-HF-Sed and M-HF-Ex offspring that could explain the metabolic benefits of maternal exercise. Pyrosequencing for Pgc-1α promoter CpG methylation at -260 site revealed that M-HF-Sed offspring had hypermethylation, which was abolished by exercise during gestation (Fig. 4A), as we reported previously (4). This effect was not statistically significant when separated by sex in either males (Fig. 4C) or females (Fig. 4E), but showed a similar trend, probably due to insufficient sample size. Quantitative RT-PCR for Pgc-1α, Cox4, and Glut4 did not reveal any differences in gene expression between the groups (Fig. 4, B, D, and F). Therefore, despite similar epigenetic influences of maternal obesity, male offspring are more vulnerable in developing age-related metabolic impairment, and exercise during pregnancy alone exerts positive epigenetic and metabolic impacts to both male and female offspring.

Figure 4.

Figure 4.

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Maternal exercise prevents maternal obesity-induced Pgc-1a -260 CpG hypermethylation in adult offspring skeletal muscle. Pyrosequencing (n = 9–19 mice) of Pgc-1a -260 CpG methylation was performed for adult offspring (9-mo-old) skeletal muscle. Data are presented combined (A) as well as separated by male (n = 5–11) (C) and female (n = 4–8) (E) offspring. Real-time quantitative PCR was performed for Pgc-1α, Cox4, and Glut4 and presented combined (B) as well as separated by male (D) and female (F) offspring. *P < 0.05; **P < 0.01.

Maternal Exercise during Gestation Protects Offspring from Paternal Obesity

In male mice, 6 wk of HF induced ∼30% increase in body weight (37.8 ± 1.0 vs. 29.6 ± 1.4 g in NC). Paternal obesity did not affect maternal weight gain during gestation (data not shown). Running distances in pregnant dams averaged 8.3 and 8.9 km/day in weeks 1 and 2, respectively, and declined to 1.7 km/day during week 3. Birth weight was significantly lower in offspring sired by obese male mice regardless of maternal exercise (data not shown); however, growth trajectory was unaffected (Fig. 5, A and B).

Figure 5.

Figure 5.

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Neither paternal HF nor maternal exercise impacts adult offspring fasting metabolic parameters. Adult offspring (9-mo-old) from sires on NC mated with female dams on NC (NC-Sed) or from sires on HF mated with female mice on NC with sedentary activity during gestation (P-HF-Sed) or with voluntary running exercise (P-HF-Ex) were assessed. Male (A) and female (B) offspring growth profile. Male offspring fasting insulin (C), blood glucose (D), and HOMA-IR (E). Female offspring fasting insulin (F), blood glucose (G), and HOMA-IR (H). (n = 6–11 mice). HF, high-fat diet; NC, normal chow.

Interestingly, neither paternal obesity nor maternal exercise had significant impacts on offspring fasting insulin, glucose, or HOMA-IR regardless of sex (Fig. 5, C–H). However, paternal obesity resulted in glucose intolerance in male offspring at 9 mo of age, with significantly higher glucose excursion during GTT at 15 and 30 min (Fig. 6A) as well as higher AUC (Fig. 6B) compared with NC-Sed. Female offspring showed a similar pattern with significantly higher glucose excursion at 15 min during GTT (Fig. 6D), whereas AUC did not reach statistical significance (Fig. 6E) compared with NC-Sed. Maternal exercise protected offspring from paternal HF-induced glucose intolerance in male offspring (Fig. 6, A and B). Female offspring in P-HF-Ex group showed a similar blood glucose level compared with NC-Sed with significantly lower glucose level during GTT at 90 min and lower glucose level at the start of ITT compared with M-HF-Sed offspring (Fig. 6, D and F). No other differences in ITT were detected between any groups regardless of sex (Fig. 6, C and F).

Figure 6.

Figure 6.

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Exercise during gestation prevents paternal HF-induced glucose intolerance in male and female offspring. Adult offspring (9-mo-old) from sires on NC mated with female dams on NC (NC-Sed) or from sires on HF mated with female mice on NC with sedentary activity during gestation (P-HF-Sed) or with voluntary running exercise (P-HF-Ex) were assessed. Male offspring blood glucose during GTT (A), blood glucose AUC (B), and blood glucose during ITT (C). Female offspring blood glucose during GTT (D), blood glucose AUC (E), and blood glucose during ITT (F). *P < 0.05, **P < 0.01 P-HF-Sed vs. NC-Sed; #P < 0.05, ##P < 0.01 P-HF-Ex vs. P-HF-Sed. (n = 7–11 mice). GTT, glucose tolerance test; HF, high-fat diet; ITT, insulin tolerance test; NC, normal chow.

To understand the role of transcriptional changes in the impact of paternal obesity and maternal exercise on the offspring, we performed RNAseq analysis in adult female offspring skeletal muscle. Only one gene was differentially expressed between P-HF-Sed versus NC-Sed and P-HF-Sed versus P-HF-Ex. However, there were 268 differentially expressed genes between P-HF-Ex and NC-Sed groups. In searching for differentially expressed genes, which may explain the positive impact of maternal exercise on the metabolic parameters in the offspring, we found increased expression of insulin receptor (Insr; Fig. 7A); lipin 1 (Lpin1; Fig. 7B) with a role in autophagy in muscle, caseinolytic mitochondrial matrix peptidase chaperone subunit X (Clpx; Fig. 7C), a regulator of mitochondrial proteostasis, and patatin-like phospholipase domain containing 8 (Pnpla8; Fig. 7D), a mitochondrial calcium-independent phospholipase A2γ critical for mitochondrial function, in P-HF-Ex offspring skeletal muscle that was higher than in NC-Sed offspring. Gene ontology analysis revealed that P-HF-Ex-reduced gene sets involved in muscle development and function compared with NC-Sed with terms such as “regulation of myotube formation,” “regulation of striated muscle cell differentiation,” and “positive regulation of myotube differentiation” (Fig. 7E). Intriguingly, the gene ontology enriched and upregulated in P-HF-Ex and P-HF-Sed (Fig. 7E) was almost identical to that identified between M-HF-Ex and M-HF-Sed (Fig. 3E), whereas P-HF-Sed offspring showed gene ontologies related to immune and inflammation pathways compared with NC-Sed offspring (Fig. 7E).

Figure 7.

Figure 7.

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Maternal exercise prevents paternal HF-induced transcriptional changes with minimal impact to DNA methylation on adult female offspring skeletal muscle. RNAseq (n = 3 mice) and RRBS (n = 6–8 mice) were performed for adult female offspring (9-mo-old) skeletal muscle. A–D: genes identified from RNAseq analysis for Insr, Lpin1, Clpx, and Pnpla8. Gene ontology enrichment analysis for pathways that are over- or under-represented in the RNAseq data set (E); Venn diagram representing the number of differentially methylated promoter regions in offspring skeletal muscle (F); pathway enrichment analysis of promoter methylation showing gene pathways that are expected to be up- or downregulated in response to the changes in promoter methylation (G). HF, high-fat diet; RRBS, reduced representation bisulfite sequencing.

Genome-wide DNA methylation analysis in the offspring skeletal muscle revealed 16 DMRs in P-HF-Sed versus NC-Sed and 29 DMRs in P-HF-Sed versus P-HF-Ex, with only one DMR in common (Fig. 7F). In addition, there were 13 DMRs between P-HF-Ex and NC-Sed offspring with 5 also present between P-HF-Sed and P-HF-Ex. Pathway enrichment analysis for promoter methylation revealed a downregulation of pathways involved in “glycogen synthesis” and “glycogen breakdown” in P-HF-Ex versus P-HF-Sed (Fig. 7G). These may suggest a transition toward reduced glycolytic metabolism in the offspring by maternal exercise. Unlike findings in maternal HF study, neither paternal obesity nor maternal exercise impacted methylation of the Pgc-1α promoter at CpG -260, regardless of sex (Fig. 8, A, C, and E). Pgc-1α gene expression was not altered by paternal HF compared with NC-Sed; however, maternal exercise significantly increased expression when sexes were combined (P-HF-Sed versus P-HF-Ex; Fig. 8B). This effect held true in male offspring, and a similar, but not statistically significant pattern, was observed in female offspring (Fig. 8, D and F). There were no significant differences in Cox4 or Glut4 mRNA expression between groups regardless of sex (Fig. 8, B, D, and F). The findings suggest that although male offspring are more vulnerable, paternal obesity is less potent in causing age-related metabolic impairment in adult mouse offspring. A different epigenetic mechanism(s) from that of maternal obesity may be involved.

Figure 8.

Figure 8.

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No effects of paternal HF or maternal exercise on Pgc-1a -260 CpG methylation in adult offspring skeletal muscle. Pyrosequencing (n = 9–19 mice) of Pgc-1a -260 CpG methylation was performed for adult offspring (9-mo-old) skeletal muscle. Data are presented combined (A) as well as separated by male (n = 10–11) (C) and female (n = 7–9) (E) offspring. Real-time quantitative PCR was performed for Pgc-1α, Cox4, and Glut4 and presented combined (B) as well as separated by male (D) and female (F) offspring. #P < 0.05 M-HF-Ex vs. M-HF.Sed. HF, high-fat diet.

DISCUSSION

It is widely accepted that parental health plays a large role in the metabolic health of the next generation. This parent-offspring influence may amplify over generations (25). Encouragingly, lifestyle intervention, including parental exercise, before and during pregnancy, confers health benefits to the parent and offspring, and there is now a large body of evidence to support this outcome in rodent models and humans (3–5) [and reviewed in (26)]. In this study, we show that maternal HF is more potent than paternal HF in predisposing the offspring, particularly the male offspring, to adulthood metabolic impairment, and maternal exercise during gestation had only positive impacts on both male and female offspring.

It is well known that women who exercised during various times of gestation have benefits for pregnancy outcomes in lowering the risk for gestational diabetes, hypertension, preeclampsia, gestational weight gain, and prematurity (27–30); however, there have been no long-term studies investigating the outcomes for the children’s later health. We and others have demonstrated the benefits of maternal exercise in mice before and during gestation in the protection against maternal HF on offspring glucose handling (4, 31), adiposity, and hepatic glucose production (31). Carter et al. (32) reported that maternal exercise before and during gestation and nursing in mice even in the absence of HF enhances skeletal muscle glucose homeostasis in both male and female adult offspring. Therefore, multiple-organ systems appear to be responsible for the beneficial effects of maternal exercise. Stanford et al. also investigated the effects of maternal exercise timing on male offspring metabolic outcomes, but in the absence of maternal HF, and showed the greatest benefits in terms of glucose tolerance, fasting insulin, fat mass, and body weight when maternal exercise was performed both before and during gestation. Furthermore, exercise only during gestation improved offspring glucose tolerance at 8 and 12 wk of age and reduced body weight at 52 wk (33). We addressed the question of maternal exercise impact after conception in the protection from maternal or paternal HF induced 6 wk before gestation (and during gestation in maternal HF study) and showed that maternal exercise during gestation alone is sufficient to protect offspring from glucose intolerance caused by maternal or paternal HF feeding. These findings provide a strong rationale for translation to humans in implementing exercise intervention during pregnancy for high-risk mothers.

We also investigated transcriptional and epigenetic changes in skeletal muscle and found similarly increased DNA methylation of Pgc-1α promoter at CpG -260 in offspring from obese dams as we previously reported (4). Here, skeletal muscle Pgc-1α gene expression was not different between groups, which appear to diverge with the hypothesized impact of promoter methylation. One difference is that the methylation change is small, with great variability possibly due to the short duration of exercise only during gestation. Pgc-1α transcription may also need to be induced long enough to unmask the effect of promoter methylation. Perhaps a large impact would become apparent with aging. Previous studies have shown the importance of the Pgc-1α promoter methylation in mice and humans (4, 7).

RNAseq gene ontology analysis revealed the upregulation of genes in inflammation and immune responses in offspring from HF-fed dams. These findings suggest that chronic inflammation in skeletal muscle may in part explain the impaired metabolic phenotype. Whole-genome DNA methylation analysis revealed maternal exercise-induced changes in promoters associated with fatty acid and carbohydrate metabolism but failed to show a direct link of DNA methylation to the transcriptional changes. It is possible that the improvement in metabolic regulation by maternal exercise is mediated by other epigenetic mechanisms. Indeed, Son et al. (34) reported that maternal exercise induces demethylation of the Prdm16 promoter with increased gene expression in offspring brown adipose tissue (BAT) along with increased BAT activation and browning of white adipose tissue at weaning. The authors attributed these findings to elevated levels of Apelin in the maternal and fetal circulation. Apelin has been shown to increase α-ketoglutarate and ten-eleven translocase (TET), a DNA demethylase in BAT (34). Future investigations should focus on the metabolic and transcriptional phenotype of adipose and other tissues. Finally, the similarity of transcriptional profiles between NC-Sed and M-HF-Ex offspring suggests that maternal exercise maintains the status quo of the gestational milieu in the face of deleterious effect of HF and confers long-lasting benefits to the offspring.

We also addressed the question of whether obesity in the male parent transmits poor health outcomes through epigenetic changes to the offspring and whether exercise in the dams during pregnancy could be protective. Here, male and female offspring of obese male mice demonstrated glucose intolerance at 9 mo of age. Intriguingly, paternal HF activated transcriptional programs in inflammation and immune responses similar to that caused by maternal HF, although differing in ontology. These transcriptional changes are particularly interesting considering that the genes in the embryos were never directly exposed to HF milieu during gestation. This study did not find a clear connection between DNA methylation and transcriptional regulation in the offspring skeletal muscle as the underlying the mechanism of transmission of metabolic impairment.

We have shown previously that maternal exercise in NC conditions had no effect on Pgc-1α promoter methylation (4). We therefore hypothesize that maternal exercise suppresses the HF-induced hypermethylation rather than initiates an independent process of demethylation. In this study, paternal HF did not induce increased methylation at the Pgc-1α promoter in the offspring skeletal muscle, and there was no effect of maternal exercise. Unexpectedly, maternal exercise increased skeletal muscle Pgc-1α gene expression compared with P-HF-Sed offspring in the absence of epigenetic changes to the promoter. Although others have reported altered DNA methylation as a potential mechanism for the transmission of phenotype through the paternal line (6, 35, 36), diet-induced changes in small noncoding RNAs can also occur within sperm and may be transferred to the oocyte (37). There is a growing body of evidence to support this hypothesis (6, 36, 38), suggesting that sncRNA may contribute to the long-term changes that we observed. Indeed, paternal HF in male mice has been shown to alter the sperm small RNA payload and to be associated with poor metabolic outcomes in adult male and female offspring (39).

Most importantly, offspring, particularly male ones, were protected from the deleterious metabolic outcomes of the paternal HF by maternal exercise during gestation. P-HF-Ex offspring showed similar metabolic phenotype to NC-Sed offspring, even though there were 268 differentially expressed genes in the female skeletal muscle. Many of these genes were related to muscle cell and myotube differentiation and suggest that other skeletal muscle functional outcomes should be investigated, such as muscle contractile strength, fiber-type distribution, and injury regeneration. Falcão-Tebas et al. reported the benefits of maternal treadmill exercise before and during gestation in protection from paternal obesity in rats. Female adult offspring had attenuated skeletal muscle insulin resistance and improved insulin secretion by the protection of β-cell mass (40). Others have shown that the deleterious impact of paternal HF on offspring metabolism can be reversed by paternal exercise (39). Importantly, Zheng et al. (5) showed that exercise in both parents before and during gestation confers the greatest metabolic benefits for the offspring. It would be of great value to understand whether differing epigenetic mechanisms between paternal and maternal exercise are responsible to the additive effects. Transcriptional analysis uncovered almost the exact same ontology terms between P-HF-Sed and P-HF-Ex as that uncovered between M-HF-Sed and M-HF-Ex. These findings suggest that regardless of parental obesity, exercise during gestation imposes positive influence on metabolic function in the offspring, although the underlying epigenetic mechanisms may differ.

The current studies are limited by sample size owing to resources and the number of successful pregnancies in HF-fed mothers that remained sedentary. In addition, we only performed RNAseq and RRBS in female offspring to maintain consistency with our previous studies. However, it became clear that male offspring exhibited a stronger metabolic phenotype than females, and having RNAseq and RRBS data from male offspring would provide more meaningful mechanistic insights. Future epigenetic studies of the originating gametes will also improve understanding transgenerational transmission of metabolic diseases and the benefits of maternal exercise during pregnancy.

GRANTS

This work was supported by AHA post-doctoral fellowship (14POST20450061) to R.C.L.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.C.L., J.J.C., S.S.R., C.R.F., R.B., and Z.Y. conceived and designed research; R.C.L., A.A., T.S.L., M.Z., O.L.S., and S.O. performed experiments; R.C.L., A.A., T.S.L., M.Z., O.L.S., S.O., S.S.R., C.R.F., R.B., and Z.Y. analyzed data; R.C.L., A.A., J.J.C., O.L.S., S.O., S.S.R., C.R.F., R.B., and Z.Y. interpreted results of experiments; R.C.L., A.A., and Z.Y. prepared figures; R.C.L. and Z.Y. drafted manuscript; R.C.L., A.A., J.J.C., O.L.S., S.S.R., C.R.F., R.B., and Z.Y. edited and revised manuscript; R.C.L., A.A., T.S.L., M.Z., J.J.C., O.L.S., S.O., S.S.R., C.R.F., R.B., and Z.Y. approved final version of manuscript.

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