Sex-Specific Lasting Metabolome Effects of Adverse Gestational Conditions and Prevention by Myo-Inositol Supplementation During Suckling

Abstract

This study examines the protective effects of myo-inositol supplementation during suckling on long-term negative outcomes caused by fetal energy restriction, using a metabolomics approach.Offspring of rats from both control and 25% gestational calorie-restricted dams received either myo-inositol or the vehicle during suckling and were exposed to a Western diet (WD) between 5 and 7 months of age. Metabolomics analysis of plasma samples at 7 months allowed the identification of 164 metabolites, revealing marked sex differences to gestational restriction. In males, maternal calorie restriction resulted in alterations in 19 metabolites, while only six metabolites showed significant variations in females, consistent with a lower impact of gestational calorie restriction (GCR) on the adult phenotype. Supplementation with myo-inositol normalized the levels of 16 metabolites in males and all six in females. Functionally, myo-inositol mostly targeted liver-associated functions in males and metabolic control functions in females. Mild/moderate GCR leads to significant changes in the metabolomic profiles of adult offspring, with males experiencing higher metabolic alterations. Early postnatal myo-inositol supplementation may be a promising strategy to alleviate the negative metabolic effect of maternal undernutrition. Sex-specific differences in the metabolomic response emphasize the necessity of considering both sexes for effective interventions.

Keywords: Fetal programming; calorie restriction; metabolomics; myo‐inositol; sex differences.

FIGURE 1

Principal component analysis (PCA) plot of plasma metabolites from male and female rats of the different groups studied (n = 52). The plots illustrate the layout of the samples colored according to sex (A), maternal diet during gestation (B), and myo‐inositol supplementation during suckling (C). C, control; F, females; GCR, gestational calorie restriction; M, males; Myo, myo‐inositol; V, vehicle.

FIGURE 2

(A) Volcano plot illustrating the effect of gestational calorie restriction versus ad libitum access on all detected metabolites in plasma of males (n = 14) and females (n = 13). (B) Partial least squares discriminant analysis (PLS‐DA) plot. (C) Variable of importance in projection (VIP) score plots for the top 15 most important metabolites identified by PLS‐DA in males and females. This analysis has been carried out only with vehicle‐treated males and females, comparing the offspring of dams exposed to gestational calorie restriction with those of dams with ad libitum feeding. C, control; GCR, gestational calorie restriction.

FIGURE 3

(A) Volcano plot depicting the effect of myo‐inositol supplementation versus vehicle treatment on all detected metabolites in males (n = 14) and females (n = 15). (B) Partial least squares discriminant analysis (PLS‐DA) plot. (C) Variable of importance in projection (VIP) score plots for the top 15 most important metabolites identified by PLS‐DA in males and females. This analysis has been carried out only with control males and females, comparing those supplemented with myo‐inositol during suckling with those treated with the vehicle. Myo, myo‐inositol; V, vehicle.

FIGURE 4

Heatmaps displaying the differential metabolites detected in the comparison of CR‐V or CR‐Myo vs. C‐V in (A) male and (B) female plasma samples (n = 5–8). C, control; GCR, gestational calorie restriction; Myo, myo‐inositol; V, vehicle.

FIGURE 5

(A, B) Metabolites with differential levels due to maternal calorie restriction during gestation and/or myo‐inositol supplementation during suckling in plasma samples of males and/or females (n = 5–8/group). Data are mean ± SEM. Circles correspond to individual data. Statistical analysis was conducted using a three‐way ANOVA to examine the influence of sex (S), maternal calorie restriction during gestation (R), myo‐inositol supplementation during suckling (M), and their interactions (SxR, RxM, SxM, SxRxM), with significance set at p < 0.05. Within each sex, a two‐way ANOVA was then performed to evaluate the effects of maternal calorie restriction during gestation (R), myo‐inositol supplementation during suckling (M), and their interaction (RxM), also at p < 0.05. Additionally, a one‐way ANOVA followed by Duncan's Multiple Range Test (DMS) was performed separately for males and females to further identify group differences. Single comparisons between groups were conducted using the Mann–Whitney U test, with asterisks (*) indicating significant differences from the respective vehicle‐treated group and hashtags (#) indicating differences from the respective control group, all at p < 0.05. P values and False Discovery Rate (FDR) for these comparisons are listed from left to right below the graph. C, control; GCR, gestational calorie restriction; Myo, myo‐inositol; V, vehicle.

FIGURE 5

(A, B) Metabolites with differential levels due to maternal calorie restriction during gestation and/or myo‐inositol supplementation during suckling in plasma samples of males and/or females (n = 5–8/group). Data are mean ± SEM. Circles correspond to individual data. Statistical analysis was conducted using a three‐way ANOVA to examine the influence of sex (S), maternal calorie restriction during gestation (R), myo‐inositol supplementation during suckling (M), and their interactions (SxR, RxM, SxM, SxRxM), with significance set at p < 0.05. Within each sex, a two‐way ANOVA was then performed to evaluate the effects of maternal calorie restriction during gestation (R), myo‐inositol supplementation during suckling (M), and their interaction (RxM), also at p < 0.05. Additionally, a one‐way ANOVA followed by Duncan's Multiple Range Test (DMS) was performed separately for males and females to further identify group differences. Single comparisons between groups were conducted using the Mann–Whitney U test, with asterisks (*) indicating significant differences from the respective vehicle‐treated group and hashtags (#) indicating differences from the respective control group, all at p < 0.05. P values and False Discovery Rate (FDR) for these comparisons are listed from left to right below the graph. C, control; GCR, gestational calorie restriction; Myo, myo‐inositol; V, vehicle.

FIGURE 6

(A) Network illustrating the correlations among metabolite functions significantly changed in males by myo‐inositol supplementation during suckling (considering both C and CR groups). Nodes are mapped with distinct colors corresponding to their betweenness centrality value, providing insights into the relative importance of each function within the network. (B) Normal probability plot of betweenness centrality (Q‐Q plot), highlighting functions deviated significantly from a normal distribution due to myo‐inositol supplementation during suckling in males. CHO, carbohydrates; NO, nitric oxide; TCA, tricarboxylic acid cycle.

FIGURE 7

(A) Network illustrating the correlations among metabolite functions significantly changed in females by myo‐inositol supplementation during suckling (considering both C and CR groups). Nodes are mapped with distinct colors corresponding to their betweenness centrality value, providing insights into the relative importance of each function within the network. (B) Normal probability plot of betweenness centrality (Q‐Q plot), highlighting functions deviated significantly from a normal distribution due to the effect of myo‐inositol supplementation during suckling in females. CHO, carbohydrates; NO, nitric oxide; TCA, tricarboxylic acid cycle.

FIGURE 8

Random forest classification of individuals into vehicle or myo‐inositol groups based on differential metabolite functions in plasma of males (A) and females (B) (Out‐of‐Bag Error: 0.462). Left: Top 15 functions with the highest mean decrease accuracy scores. Right: Normal probability plot of the mean decrease accuracy values (Q‐Q plot) which highlights functions that deviate significantly from a normal distribution due to myoinositol supplementation.