FFA2 Contribution to Gestational Glucose Tolerance Is Not Disrupted by Antibiotics

<div><p>During the insulin resistant phase of pregnancy, the mRNA expression of free fatty acid 2 receptor (<i>Ffar2</i>) is upregulated and as we recently reported, this receptor contributes to insulin secretion and pancreatic beta cell mass expansion in order to maintain normal glucose homeostasis during pregnancy. As impaired gestational glucose levels can affect metabolic health of offspring, we aimed to explore the role of maternal <i>Ffar2</i> expression during pregnancy on the metabolic health of offspring and also the effects of antibiotics, which have been shown to disrupt gut microbiota fermentative activity (the source of the FFA2 ligands) on gestational glucose homeostasis. We found that maternal <i>Ffar2</i> expression and impaired glucose tolerance during pregnancy had no effect on the growth rates, <i>ad lib</i> glucose and glucose tolerance in the offspring between 3 and 6 weeks of age. To disrupt short chain fatty acid production, we chronically treated WT mice and <i>Ffar2</i>^<i>-/-</i> mice with broad range antibiotics and further compared their glucose tolerance prior to pregnancy and at gestational day 15, and also quantified cecum and plasma SCFAs. We found that during pregnancy antibiotic treatment reduced the levels of SCFAs in the cecum of the mice, but resulted in elevated levels of plasma SCFAs and altered concentrations of individual SCFAs. Along with these changes, gestational glucose tolerance in WT mice, but not <i>Ffar2</i>^<i>-/-</i> mice improved while on antibiotics. Additional data showed that gestational glucose tolerance worsened in <i>Ffar2</i>^<i>-/-</i> mice during a second pregnancy. Together, these results indicate that antibiotic treatment alone is inadequate to deplete plasma SCFA concentrations, and that modulation of gut microbiota by antibiotics does not disrupt the contribution of FFA2 to gestational glucose tolerance.</p></div

<i>Ffar2</i>^+/- mice exhibit normal glucose tolerance at gestational day 15 (G15).

<p>(<b>a</b>) Plasma glucose concentrations from WT, <i>Ffar2</i>^+/- and <i>Ffar2</i>^-/- female mice during an IPGTT at G15. (<b>b</b>) The corresponding area under the curve (AUC) for the IPGTT. WT circles and white bars; <i>Ffar2</i>^+/-, squares and gray bars; <i>Ffar2</i>^-/-, triangles and black bars. Data in (a) were compared by 2-way ANOVA with Bonferroni post-hoc analyses. Data in (b) were compared by Student’s t-test (*, p<0.05; **, p<0.01; ***p<0.001), n = 4–5 mice/ group.</p

Effects of antibiotic treatment on gestational glucose tolerance before and during pregnancy.

<p>(<b>a</b>) Timeline of antibiotic treatment, pregnancy and reconstitution of the gut microbiota of female mice, where the control group was WT (<i>Ffar2</i>^+/+) mice and the experimental group was <i>Ffar2</i>^-/- mice. (<b>b-c</b>) Plasma glucose concentrations during an IPGTT in antibiotic-treated mice at G0 (<b>b</b>) and at G15 (<b>c</b>). (<b>d-e</b>) Plasma glucose concentrations during an IPGTT in mice after gut microbiome reconstitution at G0 (<b>d</b>) and G15 (<b>e</b>). (<b>f-g</b>) Comparison of AUC values for plasma glucose concentrations during an IPGTT administered to control, antibiotic-treated and gut microbiota reconstituted WT and <i>Ffar2</i>^-/- mice prior to pregnancy (<b>f</b>) and at G15 (<b>g</b>). (<b>h</b>) Area under the curve (AUC) values for plasma glucose concentrations during an IPGTT in WT and <i>Ffar2</i>^-/- mice on G15 of their first and second pregnancy. WT, circles and white bars; <i>Ffar2</i>^-/-, triangles and black bars. Data in (<b>b-e</b>) were compared by 2-way ANOVA with Bonferroni post-hoc analyses. Data in (<b>f-h</b>) were compared by Student’s t-test. (*, p<0.05; **, p<0.01; ***p<0.001), n = 7–16, mice/group.</p

Antibiotics alter the relative abundance of individual SCFAs in circulation during pregnancy.

<p>Total plasma SCFA levels which includes acetate, propionate, and butyrate measured in WT and <i>Ffar2</i>^-/- mice at G0 <b>(a)</b> and G15 <b>(b)</b> under control vs antibiotic-treated conditions. (<b>b-h</b>) Relative abundance of individual SCFAs (acetate, <b>c-d</b>; propionate, <b>e-f</b>; and butyrate, <b>g-h</b>) in WT and <i>Ffar2</i>^-/- mice at G0 (<b>c, e</b> and <b>g</b>) and G15 (<b>d, f</b> and <b>h</b>) under control vs antibiotic-treated conditions. WT, white bars; <i>Ffar2</i>^-/-, black bars. Data are represented as mean ± SEM n = 6–15, and were analyzed by Student’s t-test (*p ≤ 0.05).</p

Antibiotic treatment substantially alters GLP-1 secretion independent of the mouse genotype.

<p>Insulin sensitivity, as measured by the insulin tolerance test in control female WT (<b>a</b>) and female <i>Ffar2</i>^-/- mice (<b>b</b>), treated with antibiotics (open circles) or untreated (filled circles), where the y axis shows the relative glucose level at each time point as compared to the glucose level at 0 min. (<b>c-d</b>) The serum insulin response to a glucose challenge in antibiotic treated mice (open symbols) compared with control mice (filled symbols) in both WT mice (<b>c</b>) and <i>Ffar2</i>^-/- mice (<b>d</b>). Plasma GLP-1 levels in antibiotic treated mice (white bars) compared to control mice (black bars) at 0 min and 30 min for the WT (<b>e</b>) and <i>Ffar2</i>^-/- mice (<b>f</b>). Data are represented as mean ± SEM. Data in (<b>a-d</b>) were compared by 2-way ANOVA with Bonferroni post-hoc analyses. Data in (<b>e, f</b>) were compared by Student’s t-test. (*, p<0.05; **, p<0.01; ***p<0.001), n = 5–8 mice/group.</p

<i>WT</i>, <i>Ffar2</i>^-/- and *<i>Ffar2</i>^-/- mice exhibit similar weight gain, random glucose levels and glucose tolerance at 6 weeks of age.

<p>(<b>a</b>) Breeding scheme to generate these offspring is shown. For <b>b-g</b>, changes in body weight (<b>b-c</b>), <i>ad lib</i> plasma glucose concentrations (<b>d-e</b>) and plasma glucose concentrations during an IPGTT (<b>f-g</b>) with male (<b>b, d</b> and <b>f</b>) and female (<b>c, e</b> and <b>g</b>) <i>WT</i>, <i>Ffar2</i>^-/- and *<i>Ffar2</i>^-/- offspring are shown. Inserts for <b>f</b> and <b>g</b> represent the area under the curve for the IPGTT. WT, circles and white bars; <i>Ffar2</i>^-/-, squares and gray bars; *<i>Ffar2</i>^-/-, triangles and black bars. Data are represented as mean ± SEM (*p ≤ 0.05), n = 3–12. Data in (b-g) were compared by 2-way ANOVA with Bonferroni post-hoc analyses.</p

Litter size and morbidity rate

<p>Litter size and morbidity rate</p