Perinatal Exposure to a High-Fat Diet Is Associated with Reduced Hepatic Sympathetic Innervation in One-Year Old Male Japanese Macaques

<div><p>Our group recently demonstrated that maternal high-fat diet (HFD) consumption is associated with non-alcoholic fatty liver disease, increased apoptosis, and changes in gluconeogenic gene expression and chromatin structure in fetal nonhuman primate (NHP) liver. However, little is known about the long-term effects that a HFD has on hepatic nervous system development in offspring, a system that plays an important role in regulating hepatic metabolism. Utilizing immunohistochemistry and Real-Time PCR, we quantified sympathetic nerve fiber density, apoptosis, inflammation, and other autonomic components in the livers of fetal and one-year old Japanese macaques chronically exposed to a HFD. We found that HFD exposure <em>in-utero</em> and throughout the postnatal period (HFD/HFD), when compared to animals receiving a CTR diet for the same developmental period (CTR/CTR), is associated with a 1.7 fold decrease in periportal sympathetic innervation, a 5 fold decrease in parenchymal sympathetic innervation, and a 2.5 fold increase in hepatic apoptosis in the livers of one-year old male animals. Additionally, we observed an increase in hepatic inflammation and a decrease in a key component of the cholinergic anti-inflammatory pathway in one-year old HFD/HFD offspring. Taken together, these findings reinforce the impact that continuous exposure to a HFD has in the development of long-term hepatic pathologies in offspring and highlights a potential neuroanatomical basis for hepatic metabolic dysfunction.</p> </div

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

Summary of Significant Results in Fetal and Juvenile Liver.

1<p>PERIPORTAL.</p>2<p>PARENCHYMA.</p>3<p>Grant <i>et al.</i> PLoS One. 2011 Feb 25;6(2): e17261.</p>4<p>McCurdy <i>et al.</i> J Clin Invest. 2009 Feb;119(2): 323–35.</p>5<p>NPYY1R Significantly Decreased vs. HFD.</p

Visualization and quantification of the median density of portal NPY nerve fibers between maternal diet groups in the fetal macaque liver.

<p>(A) Tissue autofluorescence of a representative fetal portal region. (B) NPY immunofluorescence in the same portal region. (C) Overlay of NPY immunoreactivity with portal region. The volume of NPY immunoreactive fibers in each portal region (B) was quantified and normalized to the volume of hepatic tissue in each image (A). (D) No differences were observed in the density of NPY peptidergic innervation in fetal liver between maternal diet groups. Scale bar = 20 µm. CTR; n = 6, HFD; n = 8, REV; n = 7.</p

Hepatic sympathetic innervation in the one-year old juvenile macaque.

<p>(A-B) Representative image of TH immunoreactive nerve fibers in the portal region (A) and parenchyma (B) in juvenile liver. Scale bar = 20 µm. (C-E) Colocalization of NPY and TH immunoreactivity in the hepatic parenchyma of a one-year old juvenile macaque. (C) NPY immunoreactive fibers in the hepatic parenchyma. (D) TH immunoreactive fibers in the hepatic parenchyma. (E) Overlay of C and D demonstrates that TH and NPY are sympathetic in origin and are tightly colocalized in the juvenile macaque liver. Scale bar = 8 µm.</p

Peripheral Nervous System mRNA expression in Juvenile Macaque Liver.¹

1<p>All values are means ± SEMs and are expressed as relative fold compared to CTR/CTR. (n = 7−10 for CTR/CTR, n = 8−10 for HFD/HFD).</p>2<p>Overall significance as determined by Kruskal-Wallis rank sum test.</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

Peripheral Nervous System mRNA expression in Fetal Macaque Liver.¹

1<p>All values are means ± SEMs and are expressed as relative fold compared to CTR. n = 7 for CTR, n = 8 for HFD, n = 7 for REV.</p>2<p>Overall significance as determined by Kruskal-Wallis rank sum test.</p>a<p>Significantly different from CTR, p<.0167, Bonferroni adjusted α.</p>b<p>Significantly different from HFD, p<.0167, Bonferroni adjusted α.</p>c<p>Significantly different from REV, p<.0167, Bonferroni adjusted α.</p

Quantification of hepatic apoptosis between CTR/CTR and HFD/HFD juvenile macaques as determined by TUNEL staining.

<p>(A) Quantification of hepatic apoptosis between entire cohort of juvenile macaques. (B) Comparison of apoptosis in female juvenile macaque liver between CTR/CTR and HFD/HFD diet groups. (C) Quantification of apoptosis in male juvenile macaque liver between CTR/CTR and HFD/HFD diet groups. CTR/CTR n = 11, 6 males 5 females; HFD/HFD n = 10, 6 males, 4 females. (<b>* = </b><i>p</i><0.05, <b>**</b> = <i>p</i><0.01).</p

Quantification of the density of TH nerve fibers between CTR/CTR and HFD/HFD diet groups in the juvenile macaque liver.

<p>(A,C and E) Quantification of TH nerve fibers in the periportal region. (B,D and F) Quantification of TH nerve fibers in the hepatic parenchyma. TH immunofluorescence was acquired in each region by laser scanning confocal microscopy. The volume of TH immunoreactive fibers was normalized to the volume of hepatic tissue in each image. Data are expressed as the median normalized density for each juvenile diet group. (A-B) No differences were observed in the density of sympathetic innervation in juvenile liver between diet groups. CTR/CTR; n = 12, HFD/HFD; n = 9. (C-D) Quantification of the density of TH nerve fibers between CTR/CTR and HFD/HFD diet groups in the female juvenile macaque liver. A nonsignificant trend for higher sympathetic innervation was observed in the female juvenile liver between diet groups. CTR/CTR; n = 5, HFD/HFD; n = 4. (E-F) Quantification of the density of TH nerve fibers between CTR/CTR and HFD/HFD diet groups in the male juvenile macaque liver. Significantly reduced sympathetic innervation was observed between diet groups in both portal (E) and parenchymal regions (F) in the male juvenile liver. CTR/CTR; n = 7, HFD/HFD; n = 5. (* = <i>p</i><0.05, ** = <i>p</i><0.01). (G-J) Representative images of TH immunoreactivity in the portal region for CTR/CTR (G) and HFD/HFD (H) males. Representative images of TH immunoreactivity in the hepatic parenchyma for CTR/CTR (I) and HFD/HFD (J) males. Scale bar = 20 µm.</p