A Hypomethylating Ketogenic Diet in Apolipoprotein E-Deficient Mice: A Pilot Study on Vascular Effects and Specific Epigenetic Changes

Abstract

Hyperhomocysteneinemia (HHcy) is common in the general population and is a risk factor for atherosclerosis by mechanisms that are still elusive. A hypomethylated status of epigenetically relevant targets may contribute to the vascular toxicity associated with HHcy. Ketogenic diets (KD) are diets with a severely restricted amount of carbohydrates that are being widely used, mainly for weight-loss purposes. However, studies associating nutritional ketosis and HHcy are lacking. This pilot study investigates the effects of mild HHcy induced by nutritional manipulation of the methionine metabolism in the absence of dietary carbohydrates on disease progression and specific epigenetic changes in the apolipoprotein-E deficient (apoE–/–) mouse model. ApoE–/– mice were either fed a KD, a diet with the same macronutrient composition but low in methyl donors (low methyl KD, LMKD), or control diet. After 4, 8 or 12 weeks plasma was collected for the quantification of: (1) nutritional ketosis, (i.e., the ketone body beta-hydroxybutyrate using a colorimetric assay); (2) homocysteine by HPLC; (3) the methylating potential S-adenosylmethionine to S-adenosylhomocysteine ratio (AdoHcy/AdoMet) by LC-MS/MS; and (4) the inflammatory cytokine monocyte chemoattractant protein 1 (MCP1) by ELISA. After 12 weeks, aortas were collected to assess: (1) the vascular AdoHcy/AdoMet ratio; (2) the volume of atherosclerotic lesions by high-field magnetic resonance imaging (14T-MRI); and (3) the content of specific epigenetic tags (H3K27me3 and H3K27ac) by immunofluorescence. The results confirmed the presence of nutritional ketosis in KD and LMKD mice but not in the control mice. As expected, mild HHcy was only detected in the LMKD-fed mice. Significantly decreased MCP1 plasma levels and plaque burden were observed in control mice versus the other two groups, together with an increased content of one of the investigated epigenetic tags (H3K27me3) but not of the other (H3K27ac). Moreover, we are unable to detect any significant differences at the p < 0.05 level for MCP1 plasma levels, vascular AdoMet:AdoHcy ratio levels, plaque burden, and specific epigenetic content between the latter two groups. Nevertheless, the systemic methylating index was significantly decreased in LMKD mice versus the other two groups, reinforcing the possibility that the levels of accumulated homocysteine were insufficient to affect vascular transmethylation reactions. Further studies addressing nutritional ketosis in the presence of mild HHcy should use a higher number of animals and are warranted to confirm these preliminary observations.

A very-low carbohydrate content in a high-fat diet modifies the plasma metabolome and alleviates experimental atherosclerosis

“…The effect of the experimental diets used in this study on the systemic methylation index, the plasma adenosylmethionine to adenosylhomocysteine ratio, has been previously evaluated in ApoE —/— mice 16,17 . A significantly decreased plasma AdoMet:AdoHcy ratio was detected in KD-fed mice but not in HF-fed mice, versus controls, thus suggesting the existence of systemic hypomethylation under nutritional ketosis.…”

“…After 12 weeks, mice were euthanized by carbon dioxide inhalation and aortas were collected, as previously described in detail [16][17][18] . Briefly, aortas were perfused with 10 mL of cold phosphate saline buffer (PBS), followed by 10% neutral buffered formalin (NBF) in PBS, and were then fixed overnight in 10% NBF.…”

“…The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527515 doi: bioRxiv preprint and next transferred into glass tubes (6 mm OD, 4 mm ID and 60 mm length) for magnetic resonance imaging (MRI) analysis. Aortic plaque burden was then quantified using an Agilent 14T micro imaging system, as previously described in detail [16][17][18] . A gradient echo imaging sequence with an imaging time of 9 h 48 min was used to generate 3D datasets of the aortas.…”

Exercise Ameliorates Atherosclerosis via Up-Regulating Serum β-Hydroxybutyrate Levels

“…However, there are many different opinions on the effects of BHB on atherosclerosis. Castro et al, thought that a ketone body supplement may increase the aortic atherosclerosis burden in mice [ 53 ], and, recently, a clinical study conducted by Mu et al demonstrated that an increase in the circulation level of BHB was associated with increased coronary artery disease severity in a Chinese population. However, there are many experimental and clinical studies providing evidence that ketone bodies, especially BHB, have treatment effects for atherosclerosis.…”

Ketosis Prevents Abdominal Aortic Aneurysm Rupture Through CCR2 Downregulation and Enhanced MMP Balance

“…As a physiological process leading to the production of ketone bodies (KBs) such as acetoacetate (AcAc), beta-hydroxybutyrate (βHB) and acetone, ketogenesis not only serves as alternative fuel source, but also activates signaling cascades that can impact cell and organ function. Although high fat diets are linked to increased AAA expansion and aortic plaque formation 19,20 , recent studies suggested that a high fat -low carbohydrate ketogenic diet, as well as exogenous ketone body supplementation, can potentially reduce tissue inflammation and ameliorate the risk of vascular injury and atheroprogression 21,22 . It is unknown whether these potential benefits are limited to atherosclerosis, or whether ketosis can have a broader impact on degenerative aortopathies such as AAA expansion and rupture.…”