Metabolic dysfunction following obesity induction

Female Sprague-Dawley rats underwent a 10 week obesity induction protocol using a high-fat/sucrose diet. By week 2 of obesity induction, high-fat/sucrose-fed females were significantly heavier than the age-matched lean reference group and remained so until mating (Supplementary Fig. S1). Diet-induced obese dams also had higher blood glucose and plasma insulin concentrations compared to lean dams during an oral glucose tolerance test (OGTT) that was performed following obesity induction (Supplementary Fig. S1). Obese dams displayed higher fasting plasma leptin, which is produced in proportion to fat mass and lower peptide-YY (PYY), an appetite-reducing satiety hormone whose blood concentration is decreased in obesity in rats13 and humans12, compared to the lean reference group (leptin: obese 1162.7 ± 67.9 pmol L−1, lean: 749.5 ± 92.8 pmol L−1, P = 0.002; PYY: obese 13.7 ± 0.8 pmol L−1, lean 17.3 ± 1.1 pmol L−1, P = 0.028). Consistent with previous work14, diet-induced obesity altered gut microbiota composition, where obese dams had lower relative abundance of fecal Bifidobacterium spp. and higher relative abundance of Clostridium clusters XI and I as measured by real-time qPCR (Supplementary Table S1). Finally, whole-body metabolism of obese and lean dams was compared using 1H NMR serum metabolomics. Differences between metabolic profiles of obese and lean dams included variations in fatty acid oxidation (carnitine, o-acetylcarnitine, 2-hydroxybutyrate, ketone bodies), amino acid metabolism, gut microbial metabolites (propionate, formate, acetate, methanol) and phospholipid metabolism (cytidine) (Supplementary Fig. S2).

Prebiotic normalizes gestational weight gain

To examine the effect of supplementing a maternal high-fat/sucrose diet with the prebiotic oligofructose, diet-induced obese dams were fed either a high-fat/sucrose diet ad libitum (obese control HFS group), the high-fat/sucrose diet supplemented with 10% wt/wt oligofructose ad libitum (OFS group), or a restricted amount of the high-fat/sucrose diet in order to match body weight to the OFS group (weight matched group, WM). The WM group was included so that the effect of oligofructose could be discerned independently from its known ability to lower body weight in the context of high-fat/sucrose diet-induced obesity14. Pre-pregnancy body weight did not differ between the maternally obese groups (HFS: 447.4 ± 7.2 g; OFS: 446.0 ± 7.2 g, WM: 439.3 ± 7.3 g, P = 0.738). Of the 42 diet-induced obese female rats, 32 mated successfully. Maternal body weight over gestation and lactation was dependent on maternal diet (time × diet interaction P = 0.001). HFS dams were heavier by their due date than both OFS and WM dams (Fig. 1a) and remained heavier than OFS dams until weaning, but were heavier than WM dams only on lactation days 7 and 14. As per experimental design, OFS and WM body weight did not differ throughout the study. To assess whether the disparity in maternal body weight between dietary groups was associated with differences in energy intake, food intake data was analyzed. Accordingly, HFS dams had the highest energy intake throughout pregnancy and lactation, while OFS and WM dams had similar energy intake (Fig. 1b). Body composition was measured using dual energy x-ray absorptiometry (DXA) at weaning, where HFS dams had higher fat mass and percent body fat compared to OFS and WM dams (Fig. 1c), indicating that the obese phenotype was maintained to a greater extent in HFS dams throughout pregnancy and lactation.

Figure 1 Effect of oligofructose supplementation on maternal body weight, energy intake, body composition, glycemia and insulinemia. During gestation and lactation, diet-induced obese dams were fed either a high-fat/sucrose diet (HFS obese control group), the high-fat/sucrose diet supplemented with 10% wt/wt oligofructose (OFS group), or a restricted amount of the high-fat/sucrose diet in order to match body weight to the OFS dams (WM). A lean reference group was maintained on control AIN-93 G diet through pregnancy and lactation for body weight and body composition measurements but was not included in statistical analysis. Maternal glycemia and insulinemia was determined from blood collected during oral glucose tolerance tests (OGTTs) performed on gestation day 14 and lactation day 19. Dams were fasted overnight (12 h) and an OGTT was performed after gavage with glucose (2 g/kg body weight). Blood samples were collected at 0, 15, 30, 60, 90 and 120 minutes during the OGTT. (a) Maternal body weight during gestation and lactation (HFS, n = 11; OFS, n = 12; WM, n = 9). (b) Maternal energy intake during gestation and lactation (HFS, n = 7; OFS, n = 12; WM, n = 9; Independent-Samples Kruskall-Wallis Test with adjusted significance where *P < 0.05 compared to all other groups). (c) Maternal fat mass and percent body fat at weaning (HFS, n = 10; OFS, n = 9; WM, n = 8). (d,e) Maternal glycemia and insulinemia (HFS, n = 7; OFS, n = 12; WM, n = 9). (f) Area under the curve (AUC) for glucose and insulin concentrations during the OGTTs (HFS, n = 7; OFS, n = 12; WM, n = 9; Student’s t-test). Graphs represent mean +/− SEM. Mean values without a common letter are significantly different using one-way ANOVA (P < 0.05). For body weight, glycemia and insulinemia measurements, the data were analyzed using repeated measures one-way ANOVA, where the between subjects factor was maternal diet and the within-subjects factor was time. If a statistically significant interaction was observed, a one-way ANOVA between groups was performed. Full size image

The effect of oligofructose on maternal glucose and insulin response was investigated on gestation day 14 and lactation day 19 via OGTTs. During gestation, WM dams had the highest fasting glucose, HFS dams had the highest peak glucose and OFS dams had higher blood glucose than HFS dams at 120 minutes (Fig. 1d). There was no main effect of diet on maternal blood glucose during either gestation (P = 0.842) or lactation (P = 0.076). There were no differences in plasma insulin concentration during either gestation or lactation (Fig. 1e). However, maternal glucose area under the curve (AUC) increased in all groups from gestation to lactation, while insulin AUC decreased (Fig. 1f).

Maternal prebiotic reduces offspring fat mass and glycemia

To determine the potential impact of a maternal high-fat/sucrose diet and oligofructose supplementation on offspring body weight and composition in early life, litters were weighed on postnatal days 1, 7, 14 and 21 (weaning) and body composition of, whenever possible, two male and two female offspring from each dam was measured via DXA scan at weaning. There was a significant interaction of offspring sex and body weight only at birth (P < 0.001), where male pups weighed more than female pups (males: 6.6 ± 0.1 g; females 6.2 ± 0.1 g, P = 0.001). HFS offspring weighed more than OFS and WM offspring by postnatal day 14 and remained heavier until weaning (Fig. 2a). HFS offspring had higher fat mass and percent body fat at weaning compared to OFS and WM offspring, which did not differ (Fig. 2b). These results were consistent with the increased plasma concentration of leptin in the HFS offspring (251.4 ± 17.8 pmol L−1) compared to both OFS offspring (113.3 ± 15.5 pmol L−1, P < 0.001) and WM offspring (133.3 ± 12.3 pmol L−1, P < 0.001). Fasting plasma glucose levels in HFS offspring (8.9 ± 0.3 mmol L−1) were significantly higher than both OFS (8.0 ± 0.2 mmol L−1, P = 0.013) and WM offspring (7.5 ± 0.1 mmol L−1, P < 0.001), which were not different. There were no differences in plasma insulin concentration among offspring (HFS offspring: 52.7 ± 5.3 pmol L−1; OFS offspring: 45.5 ± 4.6 pmol L−1; WM offspring: 58.8 ± 4.8 pmol L−1; P = 0.176). Finally, there was no difference in litter size between maternal groups throughout the study (HFS: 8.8 ± 0.4 pups; OFS: 8.8 ± 0.3 pups; WM: 8.6 ± 0.4 pups, P = 0.674), thus controlling for the potential influence of small versus large litter size on the programming of obesity in offspring15.

Figure 2 Effect of maternal oligofructose supplementation on offspring body weight and body composition. Pregnant and lactating diet-induced obese dams were fed either a high-fat/sucrose diet (HFS obese control group), the high-fat/sucrose diet supplemented with 10% wt/wt oligofructose (OFS group), or a restricted amount of the high-fat/sucrose diet in order to match body weight to the OFS dams (WM). Offspring body weight was measured weekly and body composition analyzed at weaning using DXA scan. Offspring body weight was calculated using litter averages as individual values. (a) Offspring body weight throughout lactation (HFS, n = 12; OFS, n = 12; WM, n = 9). (b) Offspring fat mass and percent body fat (HFS offspring, n = 31, 15 males and 16 females; OFS offspring, n = 29, 13 males and 16 females; WM offspring, n = 30, 13 males and 16 females). Values are mean ± SEM. Mean values without a common letter are significantly different using one-Way ANOVA (P < 0.05). Full size image

Rapid changes in maternal gut microbiota

To assess the impact of pregnancy and lactation and maternal diet on the composition of the maternal gut microbiota, we performed gut microbial profiling of maternal fecal samples collected on gestation days 1, 14 and 21 and lactation days 1 and 19. Relative abundance of Bacteroides/Prevotella spp., Clostridium cluster XI, C. leptum, Enterobacteriaceae and Methanobrevibacter spp., changed significantly over gestation and lactation (main effect of time P < 0.05, Fig. 3) and approached significance in Clostridium cluster I (P = 0.052). Relative abundance of Bacteroides/Prevotella spp., C. coccoides, C. leptum, Clostridium cluster XI, Methanobrevibacter spp. and Roseburia spp. was dependent on maternal diet (time × diet interaction P < 0.05, Fig. 3). Across gestation and lactation, OFS dams had higher relative abundance of Bifidobacterium spp. and Bacteroides/Prevotella spp. (main effect of diet P < 0.05, Supplementary Table S2) compared to both HFS and WM dams. Notably, the increase in Bifidobacterium spp. in OFS dams was evident after 24 hours on the oligofructose supplemented diet (Fig. 3). Relative abundance of Clostridium cluster I differed only between HFS and OFS dams, while relative abundance of Roseburia spp. differed only between OFS and WM dams (Supplementary Table S2). The relative abundance of C. leptum, Clostridium cluster XI and Methanobrevibacter spp. across gestation and lactation differed between all three maternal groups, although OFS dams had the lowest relative abundance. Finally, consistent with the fermentation of prebiotics by the gut microbiota, cecum weight was highest in OFS dams (OFS: 1.7 ± 0.2 g; HFS: 0.58 ± 0.04 g; WM: 0.67 ± 0.04 g; P < 0.001 versus both HFS and WM dams).

Figure 3 Relative microbial abundance of fecal microbiota changes throughout pregnancy and lactation and is affected by diet. Abundance of fecal microbiota from pregnant and lactating dams fed either a high-fat/sucrose diet (HFS obese control group), the high-fat/sucrose diet supplemented with 10% wt/wt oligofructose (OFS group), or a restricted amount of the high-fat/sucrose diet in order to match body weight to the OFS dams (WM) was determined using real-time qPCR. Microbial abundance was measured as 16 S rRNA gene copies per 20 ng DNA and reported here as the relative abundance (%) of bacterial taxa per total bacteria. Longitudinal analysis of maternal fecal relative abundance on gestation days 1, 14 and 21 (G1, G14, G21) and lactation days 1 and 19 (L1, L19) was performed using repeated measures one-way ANOVA, where the between subjects factor was maternal diet and the within-subjects factor was timepoint (gestation days 1, 14 and 21 and lactation days 1 and 19). Values are mean ± SEM. Mean values without a common letter are significantly different using one-way ANOVA (P < 0.05) and indicate there was a significant interaction between time and maternal diet (P < 0.05). HFS, n = 11 except for A. muciniphila and C. coccoides where n = 10; OFS, n = 12; WM, n = 9. Full size image

Prebiotic offspring have distinct microbiota from HFS and WM

Maternal gut microbiota composition has been demonstrated to affect the colonization of offspring gut microbiota in early life, potentially affecting lifelong metabolic health7. Therefore, microbial profiling was also performed on cecal matter collected from offspring at weaning. Offspring sex affected the relative abundance of Roseburia spp. (P = 0.02) with males having higher relative abundance than females (males: 0.8 ± 0.3%; females: 0.15 ± 0.04%; P = 0.033). While maternal diet affected the relative abundance of 9 out of 11 microbial groups, in 4 out of 11 microbial groups (Bifidobacterium spp., C. coccoides, C. leptum, Enterobacteriaceae), HFS and WM offspring abundance were the same while OFS offspring differed (Table 1). WM offspring differed from both HFS and OFS offspring, which were the same, within Clostridium clusters I and XI, while HFS offspring differed from both OFS and WM offspring only within Bacteroidetes/Prevotella spp. As such, overall microbial profiles of OFS offspring were more distinct from HFS and WM offspring, which were more similar.

Table 1 Relative abundance of cecal gut microbiota from offspring of maternally obese dams fed different diets during pregnancy and lactation. Full size table

Finally, correlation analysis was used to assess the relationship between each measured maternal gut microbial group during the perinatal (gestation day 21 and lactation day 1) and weaning (lactation day 19) period with the corresponding offspring microbial abundance at weaning. Of the ten strongest correlations, seven were from the perinatal period (Table 2). Notably, the second strongest association was with Bifidobacterium spp. abundance from dams on lactation day 19. Overall, these data provide supporting evidence that maternal gut microbiota plays a role in the colonization of offspring gut microbial profiles in early life.

Table 2 Top ten correlations between microbial abundance of offspring cecal microbiota at weaning and maternal fecal microbiota during the perinatal and weaning period. Full size table

Prebiotic increases maternal and offspring gut hormones

The reduction in energy intake in the OFS dams led us to examine whether circulating levels of the satiety hormones peptide-YY (PYY) and glucagon-like peptide-1 (GLP-1), both of which are associated with a reduction in food intake16,17 and increase upon prebiotic feeding18, were elevated in OFS dams. Relative to their pre-pregnancy levels, plasma PYY AUC increased in OFS dams to a greater extent compared to HFS dams by gestation day 14 and both WM and HFS dams by lactation day 19 (Fig. 4a). Plasma GLP-1 AUC also increased most in OFS dams (Fig. 4b). The gut trophic hormone glucagon-like peptide-2 (GLP-2) is co-secreted with GLP-1 by intestinal L-cells, is associated with intestinal health and its secretion is also increased in response to prebiotic intake19. Accordingly, at weaning, portal plasma GLP-1 and GLP-2 were highest in OFS dams (Fig. 4c,d), confirming the influence of oligofructose fermentation on the secretion of glucagon-like peptides by the host. Finally, to test whether these satiety hormones were also increased in the offspring of OFS dams, we measured the concentration of PYY, GLP-1 and GLP-2 in cardiac plasma of offspring at weaning. Notably, plasma PYY, GLP-1 and GLP-2 were highest in OFS offspring compared to offspring of both HFS and WM dams (Table 3).

Table 3 Offspring gut and satiety hormone levels at weaning. Full size table

Figure 4 Oligofructose feeding increases circulating levels of peptide YY (PYY), glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2). Blood samples for PYY and GLP-1 area under the curve were collected during the glucose tolerance tests performed pre-pregnancy, on gestation day 14 and lactation day 19 from diet-induced obese dams fed either a high-fat/sucrose diet (HFS obese control group), the high-fat/sucrose diet supplemented with 10% wt/wt oligofructose (OFS group), or a restricted amount of the high-fat/sucrose diet in order to match body weight to the OFS dams (WM) during pregnancy and lactation. Portal blood samples for GLP-1 and GLP-2 measurement were collected at euthanasia of the dams. (a,b) Relative increase of PYY and GLP-1 area under the curve (AUC) on gestation day 14 and lactation day 19 compared to pre-pregnancy AUC (PYY: HFS, n = 11; OFS, n = 12; WM, n = 9; GLP-1: HFS, n = 11; OFS, n = 10; WM, n = 9). (c,d) Portal plasma GLP-1 and GLP-2 concentration (GLP-1: HFS, n = 10; OFS, n = 9; WM, n = 7, GLP-2: HFS, n = 10; OFS, n = 9; WM, n = 8). Means without a common letter are significantly different using one-way ANOVA (P < 0.05). For relative AUC measurements, the data were analyzed using repeated measures one-way ANOVA, where the between subjects factor was maternal diet and the within-subjects factor was timepoint (pre-pregnancy, gestation and lactation). If a statistically significant interaction was observed, one-way ANOVAs between groups was performed. *P < 0.05 Independent-Samples Kruskall-Wallis Test with adjusted significance. Full size image

Maternal body fat correlates with offspring body fat

Following gut microbial and satiety hormone profiles in offspring, we sought to determine what factors might be contributing to the reduced percent body fat in offspring of OFS and WM dams. While nine offspring variables were significantly correlated with offspring percent body fat, the variable that explained the largest amount of variance in offspring adiposity was maternal adiposity (R = 0.542, P < 0.01, Supplementary Table S3).

Distinct maternal metabolomics signatures

Finally, we tested whether we could detect differences in whole-body metabolism between maternal groups during gestation and lactation and thus identify potential maternal mechanisms involved in nutritional programming of offspring obesity risk. We therefore examined the differences in metabolite profiles between maternal groups using 1H NMR serum metabolomics analysis. Analysis was performed on serum collected on gestation day 14 and lactation day 19. In order to maximize differences between groups, multivariate analysis was performed using orthogonal partial least-squares discriminant analysis (OPLS-DA), where samples were clustered according to maternal diet. The explained variance (R2Y), predictability (Q2Y) and details of each model are described in Supplementary Table S4.

Metabolomics analysis was able to clearly separate the maternal dietary groups during gestation (Fig. 5). Coinciding with the start point of the separation of body weight from HFS dams, metabolic profiles of OFS and WM dams were most similar, as indicated by the lowest R2Y and Q2Y (Supplementary Table S4). The strongest separation between maternal groups occurred between the HFS and the WM dams. HFS dams had increased levels of and precursors to ketone bodies (3-hydroxybutyrate, acetone and acetoacetate, 2-oxoisocaproate) and metabolites involved in lipid metabolism (o-phosphocholine, cytidine). HFS dams also had elevated branched chain amino acids (isoleucine, leucine, valine) and decreased glucogenic amino acids (alanine, proline). OFS dams were characterized by altered levels of metabolites associated with changes in gut microbial composition, including short chain fatty acids (SCFAs) (isobutyrate, propionate, formate, butyrate, acetate, ethanol) and higher myo-inositol levels, a marker of increased insulin sensitivity20. WM dams displayed mainly increased glutamine and gluconeogenic substrates, including propylene glycol21. While there were general changes in amino acid metabolism across all maternal groups, OFS dams in particular exhibited increased levels of metabolites involved in arginine metabolism (arginine, ornithine, citrulline, proline). Interestingly, differences in choline and methionine occurred only during gestation, both of which are methyl donors and implicated in prenatal epigenetic histone methylation patterns in animal models22, potentially affecting the transgenerational transmission of obesity23.

Figure 5 Metabolomic analysis of maternal serum collected during pregnancy. Comparison of maternal serum metabolites from dams fed a high-fat/sucrose diet (HFS obese control group), the high-fat/sucrose diet supplemented with 10% oligofructose (OFS group), or a restricted amount of the high-fat/sucrose diet to weight-match to the OFS group (WM) during gestation and lactation. (a–c) Pairwise OPLS-DA loadings plots comparing maternal serum metabolites from samples collected on gestation day 14. Metabolites included in the OPLS-DA modeling were selected following preliminary pairwise univariate analysis of the 57 metabolites identified and quantified by 1H NMR spectroscopy. Bold metabolites represent the variables that contributed most to the discrimination of dietary groups, which were identified using a combination of VIP > 1 and p(corr) > 0.4, with those contributing most to the separation located furthest from the center. The directions of the arrows indicate the maternal group corresponding to increased levels of those metabolites. HFS, n = 7; OFS, n = 12; WM n = 9. Full size image

Figure 6 shows the metabolic profiles of lactating dams. Similar to gestation profiles, OFS and WM metabolic profiles in lactation were most similar, while HFS and WM dams were most distinct, indicated by their respective R2Y and Q2Y values (Supplementary Table S4). The modeled predictability (Q2Y) increased from gestation to lactation in all analyses, suggesting a strong effect of pregnancy itself on maternal metabolism.

Figure 6 Metabolomic analysis of maternal serum collected during lactation. Comparison of maternal serum metabolites from dams fed a high-fat/sucrose diet (HFS obese control group), the high-fat/sucrose diet supplemented with 10% oligofructose (OFS group), or a restricted amount of the high-fat/sucrose diet to weight-match to the OFS group (WM) during gestation and lactation. (a–c) Pairwise OPLS-DA loadings plots comparing maternal serum metabolites from samples collected on lactation day 19. Metabolites included in the OPLS-DA modeling were selected following preliminary pairwise univariate analysis of the 57 metabolites identified and quantified by 1H NMR spectroscopy. Bold metabolites represent the variables that contributed most to the discrimination of dietary groups, which were identified using a combination of VIP > 1 and p(corr) > 0.4, with those contributing most to the separation located furthest from the center. The directions of the arrows indicate the maternal group corresponding to increased levels of those metabolites. HFS, n = 10; OFS, n = 9; WM, n = 8. Full size image

In lactation, HFS dams continued to exhibit markers of increased ketone body synthesis, altered lipid metabolism and a marker of impaired fasting glucose (3-methyl-2-oxovalerate)24. Finally, HFS dams had higher levels of threonine, an amino acid that may be elevated when the tricarboxylic acid (TCA) cycle slows in the presence of excess hepatic fatty acids25. OFS dams continued to be characterized by altered levels of SCFAs and increased myo-inositol, while increased levels of o-acetylcarnitine suggests decreased transport of fatty acids into the mitochondria. Finally, WM metabolic profiles were dominated by gluconeogenic substrates (glycine, alanine, aspartate, arginine, asparagine, proline, glycerol), markers of increased protein catabolism (urocanate), oxidative stress (2-aminobutyrate, pyroglutamate, taurine), de novo lipogenesis (malonate) and undernourishment (2-hydroxyisobutyrate).

Taken together, these data indicate that despite similar food intake, gestational weight gain and percent body fat in OFS and WM dams, their serum metabolomics profiles diverged considerably and represented unique metabolite signatures. The reduced adiposity observed in offspring of both OFS and WM dams therefore is likely the consequence of distinct maternal metabolic processes, the long-term impact of which remains to be elucidated.