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

Quantification of hepatic glycogen content in the one-year old juvenile macaque.

<p>Quantification of hepatic glycogen between CTR/CTR and HFD/HFD diet groups (A). Quantification of hepatic glycogen in female juvenile livers between CTR/CTR and HFD/HFD juvenile diet groups (B). Quantification of hepatic glycogen in male juvenile livers between CTR/CTR and HFD/HFD juvenile diet groups (C). CTR/CTR n = 17, 9 males 8 females; HFD/HFD n = 14, 7 males, 7 females. (<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

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

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

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

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

PA female infant islets had an increase in their beta to alpha cell ratio (<i>P</i><0.05).

<p>(a) Immunofluorescent images representing the beta (blue) versus alpha (green) cell predominant islets of the PA infant (c) verses controls (b). Regression analysis demonstrates a trend for correlation between increased relative beta cell mass and basal insulin levels (d).</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