Early-life stress perturbs the epigenetics of Cd36 concurrent with adult onset of NAFLD in mice

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the U.S. and worldwide. The roles of early postnatal life stress (EPLS) and the fatty acid translocase (CD36) on the pathogenesis of adult-onset NAFLD remain unknown. We hypothesized that EPLS, in the form of neonatal maternal separation (NMS), would predispose mice towards developing adult NAFLD, increase hepatic CD36 expression, and differentially methylate Cd36 promoter concurrently.

Methods: NMS was performed on mice from postnatal day 1 to 21 and a high-fat/high-sucrose (HFS) diet was started at 4 weeks of age to generate four experimental groups: Naive-control diet (CD), Naive-HFS, NMS-CD, and NMS-HFS.

Results: NMS alone caused NAFLD in adult male mice at 25 weeks of age. The effects of NMS and HFS were generally additive in terms of NAFLD, hepatic Cd36 mRNA levels, and hepatic Cd36 promoter DNA hypomethylation. Cd36 promoter methylation negatively correlated with Cd36 mRNA levels. Two differentially methylated regions (DMRs) within Cd36 promoter regions appeared to be vulnerable to NMS in the mouse.

Conclusions: Our findings suggest that NMS increases the risk of an individual, particularly male, towards NAFLD when faced with a HFS diet later in life.

Impact: The key message of this article is that neonatal maternal separation and a postweaning high-fat/high-sucrose diet increased the risk of an individual, particularly male, towards NAFLD in adult life. What this study adds to the existing literature includes the identification of two vulnerable differentially methylated regions in hepatic Cd36 promoters whose methylation levels very strongly negatively correlated with Cd36 mRNA. The impact of this article is that it provides an early-life environment-responsive gene/promoter methylation model and an animal model for furthering the mechanistic study on how the insults in early-life environment are "transmitted" into adulthood and caused NAFLD.

Fig. 1. NMS and HFS increased the prevalence and severity of hepatic steatosis in adult mice in a sex-specific manner.

NAFLD activity scores in male (a) and female (b) mice. Labeled groups without a common letter differ (steatosis severity A < B < C < D), p < 0.05. Hepatic TG levels in male (c) and female (d) mice. Values are means ± SDs. n = 8. § and * denote significant NMS and diet effects, respectively. &, &&, &&& p < 0.05, 0.01, 0.001 HFS vs CD. CD control diet, HFS high-fat/high-sucrose diet, Naive no stress control group, NMS neonatal maternal separation, NAS NAFLD activity score, TG triglycerides.

Fig. 2. HFS diet significantly upregulated hepatic CD36 membrane protein in both sexes.

CD36 membrane protein expression and immunoblot image (a, b) and correlation analysis (c, d) in livers of 25-week-old male and female mice from the Naive and NMS groups and on the postweaning diet of CD and HFS. a, b Values are means ± SDs. n = 8. *Denotes significant diet effect. ^&p < 0.05 HFS vs CD. CD control diet, HFS high-fat/high-sucrose diet, Naive no stress control group, NMS neonatal maternal separation, R Pearson’s r, TG triglycerides.

Fig. 3. NMS and HFS diet synergistically upregulated Cd36 total mRNA and P2-initiated transcripts in male livers.

Hepatic mRNA expression of Cd36 total mRNA, P1, P2, and P3 transcripts in male (a) and female (b) livers of 25-week-old mice from the Naive and NMS groups and on the postweaning diet of CD and HFS. Values are mRNA expression levels relative to internal control Ppia and expressed as means ± SDs. n = 8. §, *, and ‡ denote significant effects of NMS, diet, and a NMS/diet interaction, respectively. &, &&, &&& p < 0.05, 0.01, 0.001 HFS vs CD. ### p < 0.001 NMS-HFS vs Naive-HFS. CD control diet, HFS high-fat/high-sucrose diet, Naive no stress control group, NMS neonatal maternal separation, P promoter.

Fig. 4. Cd36 total mRNA, P2- and P3-initiated transcripts were significantly positively correlated with the hepatic TG contents.

Correlations between hepatic TG contents and Cd36 total mRNA, P1, P2, and P3 transcripts in 25-week-old male (a) and female (b) mice from the Naive and NMS groups and on the postweaning diet of CD or HFS. CD control diet, HFS high-fat/high-sucrose diet, Naive no stress control group, NMS neonatal maternal separation, R Pearson’s r, TG triglycerides.

Fig. 5. NMS and HFS diet significantly hypomethylated promoters 2 and 3 of Cd36 in male liver.

Percent of DNA CpG methylation of Cd36 promoters 1, 2, and 3 in the livers of 25-week-old male (a) and female (b) mice from the Naive and NMS groups and on the postweaning diet of CD or HFS. The negative number below each CpG site indicates the number of base pair upstream relative to the transcription start site of the corresponding promoter, respectively. Values are means ± SDs. n = 8. §, *, and ‡ denote significant effects of NMS, diet, and a NMS/diet interaction, respectively. &, &&, &&&, &&&& p < 0.05, 0.01, 0.001, 0.0001 HFS vs CD. #, ## p < 0.05, 0.01 NMS-HFS vs Naive-HFS. CD control diet, HFS high-fat/high-sucrose diet, Naive no stress control group, NMS neonatal maternal separation.

Fig. 6. Cd36 total mRNA, P2- and P3-initiated transcripts were strongly and negatively correlated with promoter methylation in a CpG- and RNA variant-specific manner.

Pearson correlation score obtained from comparisons of promoter CpG methylation with Cd36 total mRNA and promoter transcripts in male (a) and female (b) livers at 25 weeks of age. The negative number below the graphs indicates the number of base pair upstream relative to the transcription start site of the corresponding exon, respectively. Values are Pearson’s r. P promoter.