Adverse Maternal Environment and Postweaning Western Diet Alter Hepatic CD36 Expression and Methylation Concurrently with Nonalcoholic Fatty Liver Disease in Mouse Offspring

Abstract

Background: The role of an adverse maternal environment (AME) in conjunction with a postweaning Western diet (WD) in the development of nonalcoholic fatty liver disease (NAFLD) in adult offspring has not been explored. Likewise, the molecular mechanisms associated with AME-induced NAFLD have not been studied. The fatty acid translocase or cluster of differentiation 36 (CD36) has been implicated to play a causal role in the pathogenesis of WD-induced steatosis. However, it is unknown if CD36 plays a role in AME-induced NAFLD.

Objective: This study was designed to evaluate the isolated and additive impact of AME and postweaning WD on the expression and DNA methylation of hepatic Cd36 in association with the development of NAFLD in a novel mouse model.

Methods: AME constituted maternal WD and maternal stress, whereas the control (Con) group had neither. Female C57BL/6J mice were fed a WD [40% fat energy, 29.1% sucrose energy, and 0.15% cholesterol (wt/wt)] 5 wk prior to pregnancy and throughout lactation. Non invasive variable stressors (random frequent cage changing, limited bedding, novel object, etc.) were applied to WD dams during the last third of pregnancy to produce an AME. Con dams consumed the control diet (CD) (10% fat energy, no sucrose or cholesterol) and were not exposed to stress. Male offspring were weaned onto either CD or WD, creating 4 experimental groups: Con-CD, Con-WD, AME-CD, and AME-WD, and evaluated for metabolic and molecular parameters at 120 d of age.

Results: AME and postweaning WD independently and additively increased the development of hepatic steatosis in adult male offspring. AME and WD independently and additively upregulated hepatic CD36 protein and mRNA expression and hypomethylated promoters 2 and 3 of the Cd36 gene.

Conclusions: Using a mouse AME model together with postweaning WD, this study demonstrates a role for CD36 in AME-induced NAFLD in offspring and reveals 2 regions of environmentally induced epigenetic heterogeneity within Cd36.

Keywords: CD36; DNA; NAFLD; maternal environment; methylation; perinatal.

FIGURE 1

Liver histology (A), diagnosis of hepatic steatosis (B), and serum ALT (C) in d120 mice from the Con and AME maternal groups and on the postweaning diet of CD or WD. Representative images were H&E-stained liver sections from each group and are shown as 100× (large images) and 200× (small images) magnifications (A). Values are number of mice diagnosed with varying degrees of hepatic steatosis based on the evaluation results of NAFLD activity scoring. Labeled groups without a common letter differ, P < 0.05 (B). Values are means ± SDs. n = 6. *Different from corresponding postweaning CD group, P < 0.05 (C). ALT, alanine transaminase; AME, adverse maternal environment; CD, control diet; Con, control; H&E, hematoxylin and eosin; WD, Western diet.

FIGURE 2

Hepatic mRNA expression of genes for FA transport (A), FA synthesis (B), and FA oxidation (C) in d120 mice from the Con and AME maternal groups and on the postweaning diet of CD or WD. Values are means ± SDs. n = 6. *Different from corresponding postweaning CD group, P < 0.05; ^#Different from corresponding maternal Con group, P < 0.05; When the diet × maternal environment interaction was significant, labeled means without a common letter differ, P < 0.05. AME, adverse maternal environment; CD, control diet; Con, control group; FA, fatty acid; WD, western diet.

FIGURE 3

CD36 membrane protein expression and immunoblot image (A), CD36 protein immunohistochemistry staining (B), and expression of Cd36 mRNA variants (C) in the livers of d120 mice from the Con and AME groups and on the postweaning diet of CD or WD. Values are means ± SDs. n = 6. *Different from corresponding postweaning CD group, P < 0.05; ^#Different from corresponding maternal Con group, P < 0.05; The diet x maternal environment interaction was not significant, P ≥ 0.05 (A) and (C). Representative images were immunohistochemically stained for CD36 protein and negative control on liver sections from each group and are shown as 50× (large images) and 200× (small images) magnifications (B). Arrows, macrophages strongly expressed CD36. AME, adverse maternal environment; C, the central vein; CD, control diet; Con, control; P, the portal; WD, western diet.

FIGURE 4

DNA CpG methylation of Cd36 promoters 1, 2, and 3 regions in livers of d120 mouse from the Con and AME maternal groups and on the postweaning diet of CD or WD (n = 6) (A) and in laser-capture micro-dissected CD36⁻ and CD36⁺ hepatocytes (n = 3–4) (B). The negative number below each CpG site indicates the number of base pair upstream relative to the transcription start site of the corresponding exon, respectively. Values are means ± SDs. *Different from corresponding postweaning CD group, P < 0.05; ^#Different from corresponding maternal Con group, P < 0.05; The diet x maternal environment interaction was not significant, P ≥ 0.05; ^$Different between CD36⁻ and CD36⁺ hepatocytes, P < 0.05. AME, adverse maternal environment; CD, control diet; Con, control group; WD, western diet.