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<h2>Introduction</h2>
<p>The global rise in obesity and metabolic syndrome has prompted investigation into developmental origins of health and disease [1,2]. Maternal nutrition during critical windows of development can program offspring metabolism, increasing susceptibility to obesity, insulin resistance, and non-alcoholic fatty liver disease (NAFLD) [3,4]. Rodent models have demonstrated that maternal high-fat diet (HFD) leads to hepatic steatosis, dyslipidemia, and altered expression of genes involved in lipid metabolism in offspring [5,6]. However, the underlying epigenetic mechanisms remain incompletely understood.</p><p>Epigenetic modifications, including DNA methylation and histone modifications, are key mediators of gene-environment interactions [7,8]. Histone modifications such as H3K4me3 (active mark) and H3K9me3 (repressive mark) regulate chromatin structure and gene expression [9]. Maternal HFD has been shown to alter histone marks in fetal liver, but the persistence and functional consequences in postnatal offspring are less clear [10,11].</p><p>In this study, we hypothesized that maternal HFD induces persistent histone modifications at lipogenic gene promoters in offspring liver, leading to increased expression of lipogenic enzymes and hepatic lipid accumulation. We used a rat model to examine hepatic lipid content, gene expression, and histone modifications in male and female offspring at weaning.</p>
<h2>Literature Review</h2>
<p>Maternal overnutrition has been extensively studied in animal models. White et al. [1] reported that maternal saturated fat diet impaired hepatic lipid metabolism and detoxifying enzymes in offspring. Ingvorsen et al. [2] found that maternal high-fat/high-sucrose diet altered hepatic lipid metabolism in rat offspring, with sex-specific effects. Huang et al. [3] demonstrated that maternal HFD during pregnancy and lactation affected hepatic lipid metabolism in early life of offspring rats. These studies collectively indicate that maternal diet programs offspring liver function.</p><p>Epigenetic mechanisms are increasingly recognized as mediators. Suter et al. [22] showed that in utero exposure to maternal HFD alters the epigenetic histone code in a murine model. McCurdy et al. [23] demonstrated lipotoxicity in fetal livers of nonhuman primates exposed to maternal HFD. Aagaard-Tillery et al. [25] reported that maternal diet modifies the primate fetal epigenome. Additionally, Kusuyama [6] found that maternal exercise-induced placental SOD3 reverses deleterious effects of maternal HFD through stabilization of H3K4me3, highlighting the reversibility of epigenetic marks.</p><p>Sex-specific differences are prominent. Nguyen et al. [5] observed SIRT1 reduction associated with sex-specific dysregulation of renal lipid metabolism in offspring by maternal HFD. Harmancıoğlu and Kabaran [20] reviewed maternal high fat diets and impacts on offspring obesity and epigenetic hypothalamic programming. However, few studies have simultaneously examined histone modifications and gene expression in liver at weaning.</p>
<h2>Methodology</h2>
<h4>Animal model</h4><p>All procedures were approved by the Institutional Animal Care and Use Committee. Female Sprague-Dawley rats (8 weeks old) were randomly assigned to control diet (CD, 10% kcal from fat, n=8) or high-fat diet (HFD, 45% kcal from fat, n=8) ad libitum for 2 weeks before mating and throughout gestation and lactation. At postnatal day 21, male and female offspring were weaned and sacrificed. Liver tissues were collected, snap-frozen, and stored at -80°C.</p><h4>Hepatic lipid measurement</h4><p>Triglycerides (TG) and total cholesterol (TC) were measured using commercial kits (Sigma-Aldrich) following manufacturer's instructions. Results were normalized to protein content.</p><h4>Gene expression analysis</h4><p>Total RNA was extracted using TRIzol reagent. cDNA was synthesized and real-time PCR performed using SYBR Green. Relative expression was calculated by the 2^-ΔΔCt method using β-actin as housekeeping gene. Primers for lipogenic genes: Fasn, Scd1, Acc1, Srebp1c, and Pparγ.</p><h4>Chromatin immunoprecipitation (ChIP)</h4><p>ChIP was performed using antibodies against H3K4me3 (Abcam) and H3K9me3 (Abcam). Enrichment at promoters of Fasn, Scd1, and Acc1 was quantified by qPCR and normalized to input.</p><h4>Statistical analysis</h4><p>Data are presented as mean ± SEM. Comparisons between groups were performed using unpaired two-tailed Student's t-test or Mann-Whitney U test for non-normal data. Multiple testing correction used Benjamini-Hochberg false discovery rate. Significance set at p<0.05. Analyses were conducted in R version 4.2.</p>
<h2>Results</h2>
<h4>Hepatic lipid accumulation</h4><p>Offspring from HFD dams exhibited significantly higher hepatic triglycerides compared to CD offspring (Table 1). Total cholesterol did not differ significantly. Male offspring showed more pronounced increases than females.</p><figure class="table-figure"><table><thead><tr><th>Parameter</th><th>CD Male (n=8)</th><th>HFD Male (n=8)</th><th>CD Female (n=8)</th><th>HFD Female (n=8)</th></tr></thead><tbody><tr><td>Triglycerides (mg/g)</td><td>28.7 ± 3.2</td><td>45.2 ± 4.1*</td><td>25.3 ± 2.9</td><td>36.8 ± 3.8*</td></tr><tr><td>Total cholesterol (mg/g)</td><td>5.1 ± 0.6</td><td>5.8 ± 0.7</td><td>4.9 ± 0.5</td><td>5.3 ± 0.6</td></tr></tbody></table><figcaption>Table 1. Hepatic lipid content in offspring at postnatal day 21. Values are mean ± SEM. *p<0.05 vs. CD of same sex.</figcaption></figure><h4>Gene expression</h4><p>Expression of lipogenic genes Fasn, Scd1, and Acc1 was significantly upregulated in HFD offspring, with fold changes ranging from 1.5 to 2.8 (Table 2). Srebp1c and Pparγ showed modest increases. Male offspring had higher expression levels overall.</p><figure class="table-figure"><table><thead><tr><th>Gene</th><th>CD Male</th><th>HFD Male</th><th>CD Female</th><th>HFD Female</th></tr></thead><tbody><tr><td>Fasn</td><td>1.00 ± 0.12</td><td>2.34 ± 0.28*</td><td>1.00 ± 0.15</td><td>1.82 ± 0.22*</td></tr><tr><td>Scd1</td><td>1.00 ± 0.10</td><td>2.81 ± 0.35*</td><td>1.00 ± 0.13</td><td>2.10 ± 0.26*</td></tr><tr><td>Acc1</td><td>1.00 ± 0.09</td><td>1.92 ± 0.21*</td><td>1.00 ± 0.11</td><td>1.65 ± 0.18*</td></tr><tr><td>Srebp1c</td><td>1.00 ± 0.08</td><td>1.45 ± 0.16*</td><td>1.00 ± 0.10</td><td>1.28 ± 0.14</td></tr><tr><td>Pparγ</td><td>1.00 ± 0.07</td><td>1.38 ± 0.15*</td><td>1.00 ± 0.09</td><td>1.22 ± 0.13</td></tr></tbody></table><figcaption>Table 2. Relative mRNA expression of lipogenic genes in offspring liver. Values are mean ± SEM relative to CD group of same sex. *p<0.05 vs. CD.</figcaption></figure><h4>Histone modifications</h4><p>ChIP-qPCR revealed increased H3K4me3 enrichment at promoters of Fasn, Scd1, and Acc1 in HFD offspring, while H3K9me3 was reduced (Table 3). These changes were more pronounced in males.</p><figure class="table-figure"><table><thead><tr><th>Gene</th><th>Mark</th><th>CD Male</th><th>HFD Male</th><th>CD Female</th><th>HFD Female</th></tr></thead><tbody><tr><td>Fasn</td><td>H3K4me3</td><td>1.00 ± 0.11</td><td>1.89 ± 0.22*</td><td>1.00 ± 0.13</td><td>1.54 ± 0.19*</td></tr><tr><td>Fasn</td><td>H3K9me3</td><td>1.00 ± 0.09</td><td>0.62 ± 0.08*</td><td>1.00 ± 0.10</td><td>0.75 ± 0.09*</td></tr><tr><td>Scd1</td><td>H3K4me3</td><td>1.00 ± 0.12</td><td>2.12 ± 0.25*</td><td>1.00 ± 0.14</td><td>1.71 ± 0.20*</td></tr><tr><td>Scd1</td><td>H3K9me3</td><td>1.00 ± 0.08</td><td>0.55 ± 0.07*</td><td>1.00 ± 0.11</td><td>0.68 ± 0.08*</td></tr><tr><td>Acc1</td><td>H3K4me3</td><td>1.00 ± 0.10</td><td>1.73 ± 0.19*</td><td>1.00 ± 0.12</td><td>1.45 ± 0.17*</td></tr><tr><td>Acc1</td><td>H3K9me3</td><td>1.00 ± 0.09</td><td>0.70 ± 0.08*</td><td>1.00 ± 0.10</td><td>0.82 ± 0.09</td></tr></tbody></table><figcaption>Table 3. Histone modification enrichment at lipogenic gene promoters. Values are fold enrichment over input, normalized to CD group of same sex. *p<0.05 vs. CD.</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 1. bar chart comparing H3K4me3 and H3K9me3 enrichment at Fasn, Scd1, Acc1 promoters in male offspring from CD vs HFD groups</figcaption></figure></p><h4>Epigenetic regulators</h4><p>Expression of Sirt1, a histone deacetylase, was significantly reduced in HFD offspring (male: 0.62-fold, female: 0.74-fold, p<0.05). Dnmt3a expression was also decreased, while Hdac1 and Hdac3 showed no significant changes.</p>
<h2>Discussion</h2>
<p>Our results demonstrate that maternal HFD programs hepatic lipid metabolism in offspring through epigenetic modifications that activate lipogenic gene expression. The increased H3K4me3 and reduced H3K9me3 at promoters of Fasn, Scd1, and Acc1 provide a mechanism for sustained upregulation of these genes, leading to hepatic steatosis.</p><p>These findings align with previous studies showing maternal HFD alters histone modifications in fetal liver [22,25]. However, we extend these observations to weaning, indicating persistence of epigenetic marks after birth. The reduction in Sirt1 expression is notable, as Sirt1 deacetylates histones and promotes repressive chromatin [5]. Downregulation of Sirt1 may contribute to the observed increase in H3K4me3.</p><p>Sex-specific differences were evident, with male offspring more susceptible to hepatic lipid accumulation and epigenetic changes. This is consistent with reports of sexual dimorphism in developmental programming [5,26]. Estrogen may protect females by modulating lipid metabolism and epigenetic regulation.</p><p>Our study has limitations. We only examined histone modifications at selected loci; genome-wide analysis would provide broader insights. Additionally, we did not investigate DNA methylation, which may also contribute. Future studies should explore intergenerational effects and potential interventions such as maternal exercise or dietary supplementation [6].</p>
<h2>Conclusion</h2>
<p>Maternal high-fat diet induces epigenetic reprogramming of hepatic lipid metabolism in offspring, characterized by altered histone modifications at lipogenic gene promoters and increased expression of lipogenic enzymes. These changes are more pronounced in males and are associated with downregulation of Sirt1. Our findings highlight potential epigenetic targets for early intervention to prevent NAFLD in offspring exposed to maternal overnutrition.</p>
<h2>References</h2>
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