Development of a Zebrafish Model for Studies of the Interaction of Methylenetetrahydrofolate Reductase Deficiency and Dietary Folates on Metabolic Regulation

Abstract

ObjectivesMethylenetetrahydrofolate reductase (MTHFR) is required for 5-methyltetrahydrofolate (5MTHF) synthesis, and common variants reduces its efficiency and associate with metabolic disorders. High folic acid (FA) intakes, commonly consumed by pregnant women in North America, may further inhibit MTHFR enzyme; programming long-term metabolic dysregulation in offspring. The zebrafish (Danio rerio) is a valuable model for study of embryonic development and high-throughput nutrient × gene interactions. The objective of this study was to characterize a zebrafish model of mthfr deficiency and assess the interaction between mthfr and FA intakes on early-life metabolic dysregulation. MethodsZebrafish were co-injected with a set of 4 guide RNAs (gRNAs) or cas9 protein alone and F0 embryos were assayed for a high-throughput phenotypic screen. Germline F1 knock-out homozygous mutants (mthfr –/–) were made by co-injecting cas9 mRNA with 2 gRNAs targeting the transcriptional start site of the mthfr gene. Embryos were raised up to 5 days post-fertilization (dpf) and folate and 1-carbon metabolites measured by LC-MS/MS. Lipid accumulation was assessed at 5dpf and after feeding a high cholesterol diet (HDC) with cholesteryl-ester (CE)-BoDipy-C12® from 5–15dpf. A subset of embryos were exposed to no (0μM) or high (100μM) FA from 0–5dpf and whole-body lipids measured. Resultsmthfr disruption in zebrafish reduced (80%) mthfr mRNA and 5MTHF levels (90%) compared to controls (P < 0.0001). They had lower 1-carbon metabolites including betaine, methionine, s-adenosylmethionine, and higher choline, s-adenosylhomocysteine, cystathionine and homocysteine (P < 0.01). As well, neutral lipid accumulation was higher in liver, heart and vasculature at 5 and 15 dpf along with higher CE altered cholesterol transport/metabolism. High FA exposure ameliorated lipid accumulation in mthfr mutants at 5 dpf (P = 0.06), but increased lipids accumulation in controls compared to no exposure (P = 0.03). ConclusionsThe zebrafish mthfr deficient model exhibits a similar alteration to 1-carbon metabolites as in humans with severe MTHFR deficiency. This zebrafish model has potential for understanding the interaction of mthfr deficiency and dietary folates on metabolism. Funding SourcesCIHR-INMD, EP by NSERC-CGS