Diet-Induced Mild Hyperhomocysteinemia in Mice Fed a Ketogenic Diet Does Not Alter the Development of Atherosclerosis nor Specific Epigenetic Content

Abstract

ObjectivesElevated plasma homocysteine (Hcy), or hyperhomocysteinemia (HHcy), is a risk factor for atherosclerosis by mechanisms still elusive. A possibility includes the alteration of specific epigenetic tags at lysine 27 of histone H3 (H3K27) due to hypomethylating stress. Similarly, ketogenic diets (KD), or very-low carbohydrate diets, which stimulate ketosis, and may also affect the epigenetic content on the H3K27 residue. Studies connecting the effects of dietary ketosis, mild HHcy, and specific epigenetic dysregulation are lacking. We hypothesize that diet-induced HHcy and ketosis will induce H3K27 hypomethylation combined with increased acetylation to produce a pro-atherogenic phenotype. MethodsSeven-week-old male apoe–/– (apolipoprotein E-deficient) mice, a model for human atherosclerosis, were fed ad libitum a KD (in %kcal: fat, 81; carbohydrate, 1; protein, 18; n = 4–6) or HHcy-KD (same macronutrients, with added methionine and reduced methyl donors; n = 4). After 4, 8 and 12 wk of diet treatment, plasma was collected to quantify ketosis via beta-hydroxybutyrate levels (OH-But) by a colorimetric assay, and measure Hcy by HPLC. At the endpoint, mice were euthanized and aortas were collected for quantification of the vascular methylation index, S-adenosylmethionine to S-adenosylHcy ratio by LC-MS-MS; 3-D analysis of the atherosclerotic plaque burden by magnetic resonance imaging; and quantification of the epigenetic tags H3K27me3 and H3K27ac using immunohistochemistry. ResultsA sustained ketosis was detected through elevated OH-But levels in both KD and HHcy-KD mice. HHcy was mildly but significantly (P < 0.05) elevated in HHcy-KD-mice compared to KD-mice after 4 wk (19.5 ± 2.3 vs 4.5 ± 0.6 μM) and 12 wk (17.2 ± 2.1 vs 4.4 ± 0.9 μM). Nevertheless, no significant differences were observed in aortic methylation index, plaque accumulation, or content of the H3K27me3 or H3K27ac epigenetic tags between the two groups of mice. ConclusionsWhile mild HHcy was achieved in HHcy-KD mice, this phenotype failed to induce vascular hypomethylation, atherosclerosis progression or specific epigenetic dysregulation, suggesting that a more severe Hcy accumulation may be necessary to cause vascular toxicity and specific epigenetic dysregulation. Funding SourcesHuck Institutes of the Life Sciences