Characterization of Blood DNA Methylation Changes in a Murine Model of Prediabetes Induced by Fructose and Its Reversal

Abstract

Background: DNA methylation is implicated in type 2 diabetes (T2D); however, its role in prediabetes remains poorly understood. Objectives: This study identified blood DNA methylation changes in a rodent model of prediabetes induced by fructose and tracked its evolution during prediabetes reversal after removing the fructose supplement. Methods: Adult male Sprague–Dawley rats were divided into 3 groups (n = 8/group): control, prediabetic (PD), and reversal groups. Animals were fed a standard nonpurified chow. The control group consumed water, while PD group received 10% fructose solution for 70 d. The reversal group received fructose for 21 d to induce prediabetes, followed by water. Glucose tolerance test was performed in a subgroup of rats. Whole-genome bisulfite sequencing was conducted on whole blood samples collected at day 70. DNA methylation data were analyzed using Rfastp and Bismark. Differential methylation analysis was performed using methylKit. Genomation package and STRING website were used for annotation and functional analysis. Results: PD and reversal (21 d) animals showed higher triglyceride and insulin resistance index and impaired glucose tolerance (P < 0.05 compared with the control group), confirming the establishment of prediabetes. On day 70, 4339 differentially methylated (DM) cytosine–guanine dinucleotide (CpG) sites were identified across PD compared with control, reversal compared with PD, and reversal compared with control, associated with 842 genes. Hypermethylation predominated in PD compared with control (54%), reversal compared with control (69%), and reversal compared with PD (65%). Most changes occurred in intergenic regions and outside CpG islands. Oxidative phosphorylation was the main affected pathway in prediabetes and persisted in reversal, accompanied by altered expression of mitochondrial genes (mt-Nd2 and mt-Cytb). Under more stringent criteria, 41 DM CpG sites associated with 17 genes were identified, with most alterations (95%) observed in reversal condition, particularly in Uxs1 and Emb. Conclusions: These findings identify novel DNA methylation changes associated with fructose-induced prediabetes and its reversal, providing insights into early epigenetic alterations involved in T2D pathogenesis.