Rodents on a high-fat diet born to mothers with gestational diabetes exhibit sex-specific lipidomic changes in reproductive organs

Abstract

Maternal gestatonal diabetes mellitus (GDM) and offspring high-fat diet (HFD) have been shown to have sex-specific detrimental effects on the health of the offspring. Maternal GDM combined with an offspring HFD alters the lipidomic profiles of offspring reproductive organs with sex hormones and increases insulin signaling, resulting in offspring obesity and diabetes. The pre-pregnancy maternal GDM mice model is established by feeding maternal C57BL/6 mice and their offspring are fed with either a HFD or a low-fat diet (LFD). Testis, ovary and liver are collected from offspring at 20 weeks of age. The lipidomic profiles of the testis and ovary are characterized using gas chromatography-mass spectrometry. Male offspring following a HFD have elevated body weight. In reproductive organs and hormones, male offspring from GDM mothers have decreased testes weights and testosterone levels, while female offspring from GDM mothers show increased ovary weights and estrogen levels. Maternal GDM aggravates the effects of an offspring HFD in male offspring on the AKT pathway, while increasing the risk of developing inflammation when expose to a HFD in female offspring liver. Testes are prone to the effect of maternal GDM, whereas ovarian metabolite profiles are upregulated in maternal GDM and downregulated in offspring following an HFD. Maternal GDM and an offspring HFD have different metabolic effects on offspring reproductive organs, and PUFAs may protect against detrimental outcomes in the offspring, such as obesity and diabetes.

Keywords: hormone; insulin signaling; maternal GDM; metabolomics; mice offspring.

Figure1

Experimental design and maternal characteristics (A) Graphical display of the experimental design of the study. (B) The body weight of normal mothers (n=9) and GDM mothers (n=13) in grams at week 14. (C) Oral glucose tolerance test of normal mothers and GDM mothers (blue line and square; n=13) at 14 weeks. (D) The plasma insulin levels of normal mothers (n=9) and GDM mothers (n=13) at week 14. (E) The relative abundances of fatty acids were plotted using log2 scale. Fold changes of metabolite concentrations compared with their control groups are illustrated in the heatmap. The yellow color indicates decreasing levels. Only the fatty acids with significant P values (Tukey’s HSD: P<0.05) and q values (FDR: q<0.05) are shown. Statistical differences between the normal mother and GDM mother were determined using an unpaired Student’s t-test for B and D or a two-way ANOVA followed by a Tukey’s post hoc test for C. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure2

Characteristics of the offspring (A) Body weight of the offspring. (B) Ovary weight of the female offspring. (C) Testis weight of the male offspring. (D) Blood pressure of the female offspring. (E) Systolic blood pressure of the male offspring. (F) OGTT results from the female offspring. (G) OGTT results from the male offspring. (H) Plasma insulin levels of the female offspring. (I) Plasma insulin levels of the male offspring. (J) Plasma E2 levels of the female offspring. (K) Plasma FSH levels of the female offspring. (L) Plasma testosterone levels of the male offspring. (M) Plasma FSH levels of the male offspring. Statistical differences for the characteristics of offspring were determined using a two-way ANOVA followed by a Tukey’s post hoc test for A to M. *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure3

Effects of maternal GDM and offspring HFD on the liver, gonadal adipose tissue, and reproductive organs of the offspring (A) Protein levels of ESR1 in female offspring ovaries (upper panel). Protein levels of pIRS1, pPI3K, pAKT were normalized against total IRS1, PI3K, AKT and TNFα separately in female offspring livers (lower panel). (B) Protein levels of pIRS1, pPI3K, pAKT were normalized against total IRS1, PI3K, AKT separately and TNFα in female offspring gonadal adipose tissue. (C) Protein levels of AR in male offspring testes (upper panel). Protein levels of pIRS1, pPI3K, pAKT were normalized against total IRS1, PI3K, AKT separately and TNFα in male offspring livers (lower panel). (D) Protein levels of pIRS1, pPI3K, pAKT were normalized against total IRS1, PI3K, AKT separately and TNFα in male offspring gonadal adipose tissue. (E) Representative HE-stained liver section images in female offspring. Scale bar=100 μm. (F) Representative HE-stained liver section images in male offspring. Scale bar=100 μm. Statistical differences for the characteristics of offspring were determined using a two-way ANOVA followed by a Tukey’s post hoc test. *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure4

Principal component analysis (PCA) and lipidomic profiles of ovaries and testes in the offspring (A) The PCA analysis of offspring ovaries. (B) The PCA analysis of offspring testes. The color codes of the balls are listed as follows: purple color represents offspring sex (M=male; F=female)-GDM mother-high-fat diet (M/F-G-H); blue color represents offspring sex-GDM mother-low-fat diet (M/F-G-L); red color represents offspring sex-normal mother-high-fat diet (M/F-N-L); green balls represent offspring sex-normal mother-low-fat diet (M/F-N-H). (C) The heatmap demonstrates the female offspring’s ovary lipidomic profiles. (D) The heatmap demonstrates the male offspring’s testis lipidomic profiles. The maternal obesity indicated that comparisons between the GDM mother normalized against the normal mother (GDM/N) for the offspring fed with the same diet (L=LFD or H=HFD). The offspring diet indicated that comparisons between the offspring HFD normalized against the offspring LFD (H/L) from the same mother (GDM or N). The relative abundances of metabolites were plotted using log2 scale. Fold changes of metabolite concentrations when compared with their control groups are illustrated by purple color (increasing levels) and yellow color (decreasing levels). Only the metabolites with a significant P value (Tukey’s HSD: P<0.05) and q value (FDR: q<0.05) are shown.

Figure5

Receiver operating characteristic curves All fatty acids exhibited an area under the ROC curve greater than 0.95. (A) Comparison between F-G-L and F-N-L for methyl stearate. (B) Comparison between F-N-H and F-N-L for 7,10,13,16-cis-docosatetraenoic acid. (C) Comparison between M-G-H and M-N-H for 7,10,13,16-cis-docosatetraenoic acid and 11-cis-eicosenoic acid. (D) Comparison between M-N-H and M-N-L for cholest-5-ene. (E) Comparison between M-G-L and M-N-L for 6,11-eicosadienoic acid, 11-trans-eicosenoic acid, 5,8,11,14,17-cis-eicosapentaenoic acid and 13-cis-eicosenoic acid. G: GDM mother; N: normal mother; H: high-fat diet; L: low-fat diet.

Figure6

Correlation plots of fatty acids in offspring testes and maternal plasma The blue color represents a positive correlation and the red color represents a negative correlation. The grey numbers are correlation coefficients and only the correlations with P values less than 0.05 are colored.