Transgenic Increase in N-3/N-6 Fatty Acid Ratio Reduces Maternal Obesity-Associated Inflammation and Limits Adverse Developmental Programming in Mice

<div><p>Maternal and pediatric obesity has risen dramatically over recent years, and is a known predictor of adverse long-term metabolic outcomes in offspring. However, which particular aspects of obese pregnancy promote such outcomes is less clear. While maternal obesity increases both maternal and placental inflammation, it is still unknown whether this is a dominant mechanism in fetal metabolic programming. In this study, we utilized the Fat-1 transgenic mouse to test whether increasing the maternal n-3/n-6 tissue fatty acid ratio could reduce the consequences of maternal obesity-associated inflammation and thereby mitigate downstream developmental programming. Eight-week-old WT or hemizygous Fat-1 C57BL/6J female mice were placed on a high-fat diet (HFD) or control diet (CD) for 8 weeks prior to mating with WT chow-fed males. Only WT offspring from Fat-1 mothers were analyzed. WT-HFD mothers demonstrated increased markers of infiltrating adipose tissue macrophages (<i>P<</i>0.02), and a striking increase in 12 serum pro-inflammatory cytokines (<i>P</i><0.05), while Fat1-HFD mothers remained similar to WT-CD mothers, despite equal weight gain. E18.5 Fetuses from WT-HFD mothers had larger placentas (<i>P</i><0.02), as well as increased placenta and fetal liver TG deposition (<i>P</i><0.01 and <i>P</i><0.02, respectively) and increased placental LPL TG-hydrolase activity (<i>P</i><0.02), which correlated with degree of maternal insulin resistance (<i>r</i> = 0.59, <i>P</i><0.02). The placentas and fetal livers from Fat1-HFD mothers were protected from this excess placental growth and fetal-placental lipid deposition. Importantly, maternal protection from excess inflammation corresponded with improved metabolic outcomes in adult WT offspring. While the offspring from WT-HFD mothers weaned onto CD demonstrated increased weight gain (<i>P</i><0.05), body and liver fat (<i>P</i><0.05 and <i>P</i><0.001, respectively), and whole body insulin resistance (<i>P</i><0.05), these were prevented in WT offspring from Fat1-HFD mothers. Our results suggest that reducing excess maternal inflammation may be a promising target for preventing adverse fetal metabolic outcomes in pregnancies complicated by maternal obesity.</p></div

Effects of maternal HFD and Fat-1 transgene on measures of placental inflammation.

<p>(A) Flow cytometry for F4/80+ and CD11c+ macrophages in collagenase-digested whole placentas (<i>n = </i>6 mothers per group, average of 2 placentas per mother). (B) Placental gene expression of select pro-inflammatory cytokines and macrophage markers by quantitative PCR (<i>n</i> = 9 mothers per group, 3 RNA pools of 3 mothers each). Data represented as mean ± SEM with only WT placentas included in maternal averages for the Fat1-HFD group; *<i>P</i><0.05.</p

Effect of maternal HFD and Fat-1 transgene on placental fatty acid transporter expression and lipoprotein lipase activity.

<p>(A) Placental gene expression of select genes involved in fatty acid transport by quantitative PCR (<i>n = </i>9 mothers per group, 3 RNA pools of 3 mothers each). (B) Placental LPL hydrolase activity. Activity was measured in 3–4 placentas from each mother and averaged, with <i>n</i> = 6 mothers per experimental group. (C) Correlation of average maternal placental LPL activity and maternal insulin resistance, as measured by HOMA-IR. All data are expressed as mean ± SEM with only WT placentas included in maternal averages for the Fat1-HFD group; *<i>P</i><0.05.</p

Effect of maternal HFD and Fat-1 transgene on WT adult male offspring weight gain and body composition.

<p>(A) WT male offspring weight gain. Offspring from each maternal group (<i>n</i> = 10 mothers per group) were weaned onto CD, and weighed every other week for 16 weeks. (B) Adult male offspring body composition (fat and lean mass) at 16 wks post-wean, as determined by Echo MRI. (C and D) Adult male offspring liver TG deposition. TG were quantitated in (C) by colorimetric assay on tissue lipid extracts, and visualized in (D) by Oil Red O staining of representative liver cryosections for neutral lipids (lipid droplet = red, cell nuclei = blue, 20X magnification). All offspring measures are averaged per mother, with siblings treated as replicates. For Fat-1 mothers, only WT offspring were included in analyses. All data are expressed as mean ± SEM; *<i>P</i><0.05.</p

Impact of maternal HFD and Fat-1 transgene on maternal weight gain and inflammatory markers.

<p>(A) Maternal weight gain prior to mating, and at E18.5 (late gestation). Gestational weight was estimated by subtracting the complete fetal-placental litter weight from the weight of the pregnant mother. (B) Adipose tissue (AT) macrophage quantitation by flow cytometry. Antibodies against mouse macrophage marker F4/80 and M1 macrophage marker CD11c were used to determine the percent of macrophages present and their relative M1 polarization in maternal AT stroma at E18.5. (C) Relative maternal serum pro-inflammatory cytokine levels at E18.5 by membrane-bound antibody array. Results are the average of three membranes per maternal group, with each membrane incubated with the pooled serum from four separate mothers. (A–C) Results are the average of <i>n</i> = 12−14 mothers per experimental group. Data represented as mean ± SEM; *<i>P</i><0.05.</p

Fetal E18.5 fasting serum measurements.

<p>Measurements were conducted on pooled litter serum for each mother. Data are expressed as mean ± SEM for the fetal serum of <i>n</i> = 12−14 mothers per maternal group.</p

Effect of maternal HFD and Fat-1 transgene on WT adult male offspring metabolic inflammation and insulin resistance.

<p>(A) Adipose tissue (AT) macrophage quantitation by flow cytometry. Antibodies against mouse macrophage marker F4/80 and M1 macrophage marker CD11c were used to determine the percent of macrophages present and their relative M1 polarization in adult offspring AT at 16 wks post-weaning. (B) Blood glucose curve in response to insulin tolerance test on fasted adult male offspring 16 wks post-weaning. All offspring measures are averaged per mother, with siblings treated as replicates. For Fat-1 mothers, only WT offspring were included in analyses. All data are expressed as mean ± SEM; *<i>P</i><0.05.</p

Clinical characteristics of the subjects for Study 2.

<p>Data are mean ± SEM. OGTT, oral glucose tolerance test. Independent <i>t</i>-test *<i>P</i><0.05.</p><p>Clinical characteristics of the subjects for Study 2.</p

Work flow of statistical analysis of single proteins.

<p>Work flow of statistical analysis of single proteins.</p

Mitochondrial respiratory chain complex proteins are not different between ONGT and OGDM women.

<p>Complex I (<i>A</i>), complex II (<i>B</i>), complex III (<i>C</i>), complex IV (<i>D</i>). Representative Western blots where calnexin is used as loading control (<i>E</i>).</p