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

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 947-947
Author(s):  
Emanuela Pannia ◽  
Rebecca Simonian ◽  
Rola Hammoud ◽  
Xiucheng Cui ◽  
Ruslan Kubant ◽  
...  
Keyword(s):  
High Throughput ◽  
Lipid Accumulation ◽  
Metabolic Regulation ◽  
Methylenetetrahydrofolate Reductase ◽  
Transcriptional Start Site ◽  
Whole Body ◽  
High Cholesterol Diet ◽  
Zebrafish Model ◽  
Metabolic Dysregulation ◽  
Mthfr Deficiency

Abstract Objectives Methylenetetrahydrofolate 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. Methods Zebrafish 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. Results mthfr 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). Conclusions The 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 Sources CIHR-INMD, EP by NSERC-CGS

scholarly journals Kaempferol-3-O-Glucuronide Ameliorates Non-Alcoholic Steatohepatitis in High-Cholesterol-Diet-Induced Larval Zebrafish and HepG2 Cell Models via Regulating Oxidation Stress

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 445
Author(s):  
Yang Deng ◽  
Ji Ma ◽  
Xin Weng ◽  
Yuqin Wang ◽  
Maoru Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Accumulation ◽  
Hepg2 Cells ◽  
High Cholesterol Diet ◽  
Cholesterol Diet ◽  
High Cholesterol ◽  
Zebrafish Model ◽  
Alcoholic Fatty Liver ◽  
Larval Zebrafish

NAFLD (non-alcoholic fatty liver disease) is one of the most prominent liver diseases in the world. As a metabolic-related disease, the development of NAFLD is closely associated with various degrees of lipid accumulation, oxidation, inflammation, and fibrosis. Ilex chinensis Sims is a form of traditional Chinese medicine which is used to treat bronchitis, burns, pneumonia, ulceration, and chilblains. Kaempferol-3-O-glucuronide (K3O) is a natural chemical present in Ilex chinensis Sims. This study was designed to investigate the antioxidative, fat metabolism-regulating, and anti-inflammatory potential of K3O. A high-cholesterol diet (HCD) was used to establish steatosis in larval zebrafish, whereby 1mM free fatty acid (FFA) was used to induce lipid accumulation in HepG2 cells, while H2O2 was used to induce oxidative stress in HepG2. The results of this experiment showed that K3O reduced lipid accumulation and the level of reactive oxygen species (ROS) both in vivo (K3O, 40μM) and in vitro (K3O, 20μM). Additionally, K3O (40μM) reduced neutrophil aggregation in vivo. K3O (20μM) also decreased the level of malondialdehyde (MDA) and significantly increased the level of glutathione peroxidase (GSH-px) in both the HCD-induced larval zebrafish model and H2O2-exposed HepG2 cells. In the mechanism study, keap1, nrf2, tnf-α, and il-6 mRNA were all significantly reversed by K3O (20μM) in zebrafish. Changes in Keap1 and Nrf2 mRNA expression were also detected in H2O2-exposed HepG2 cells after they were treated with K3O (20μM). In conclusion, K3O exhibited a reduction in oxidative stress and lipid peroxidation, and this may be related to the Nrf2/Keap1 pathway in the NAFLD larval zebrafish model.

scholarly journals Methylenetetrahydrofolate Reductase Deficiency Reduces Brain Microglia in Zebrafish During Embryonic Development and Is Not Corrected by Folic Acid

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 925-925
Author(s):  
Rebecca Simonian ◽  
Emanuela Pannia ◽  
Rola Hammoud ◽  
Xiucheng Cui ◽  
Ruslan Kubant ◽  
...  
Keyword(s):  
Folic Acid ◽  
Embryonic Development ◽  
Methylenetetrahydrofolate Reductase ◽  
Fetal Brain ◽  
Neutral Red ◽  
High Dose ◽  
Head Region ◽  
Zebrafish Model ◽  
Mthfr Deficiency

Abstract Objectives Neuronal development and function is dependent on the interaction between the central nervous system and immune system. Microglia are resident macrophages of the brain critical for regulating neuronal activity during embryonic development. 5-methyltetrahydrofolate (5MTHF), the bioactive folate form, is essential for fetal brain development and immune function. Common variants in methylenetetrahydrofolate reductase (MTHFR), required for conversion of folic acid (FA) to 5-MTHF, limits its production. High dose FA supplementation is recommended but high FA may have the converse effect of reducing MTHFR activity. The objective of this study was to determine the effects of mthfr deficiency and its interaction with FA during embryonic development on microglia in a zebrafish model. Methods The mthfr gene in zebrafish was disrupted using two CRISPR mutagenesis methods. A set of 4 guide RNAs (gRNAs) + cas9 protein or cas9 alone (control) were injected to assay F0 zebrafish, or 2 gRNAs + cas9 mRNA were used to induce a germline mutation. To visualize macrophages at 4 days post fertilization (dpf) in live zebrafish, the transgenic mpeg1: mcherry line was used. In a subset of embryos, FA was added at 0, 50, 75, or 100- μM from 0–4dpf. At 4dpf, live neutral red staining for microglia was performed and the number in the optic tectum was quantified. 5MTHF, s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) were assayed in whole zebrafish at 5dpf. Results In vivo imaging revealed a reduction in macrophage number (∼30%, P < 0.001) in the head region of mthfr disrupted zebrafish, but not in the periphery. mthfr zebrafish also had less microglia compared to controls (15%, P < 0.001). These changes were associated with lower 5MTHF (90%, P < 0.0001) and SAM: SAH (∼50%, P < 0.001) at 5dpf indicative of lower methylation potential. Exposure with FA did not correct the phenotype and at 100µM FA, control zebrafish also showed a decrease in microglia similar to mthfr zebrafish, confirming inhibitory effects of the high FA dose. Conclusions mthfr deficiency reduces microglia in zebrafish but supplementation with FA does not prevent and may exacerbate the negative effects. The 5MTHF form of folate may be a better alternative to FA for brain health in patients with underlying genetic conditions. Funding Sources Supported by CIHR-INMD.

scholarly journals The Zebrafish (Danio Rerio) as a Novel Model to Study Folate-mthfr Interactions During Embryonic Development and Effect(s) on Long-Term Health

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1269-1269
Author(s):  
Emanuela Pannia ◽  
Xiucheng Cui ◽  
Rebecca Simonian ◽  
Rola Hammoud ◽  
Paula Ashcraft ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Danio Rerio ◽  
Metabolic Regulation ◽  
Whole Body ◽  
High Cholesterol Diet ◽  
Global Methylation ◽  
Mthfr Genotype ◽  
Cas9 Protein

Abstract Objectives Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of the bioactive folate form, 5-methyltetrahydrofolate (5MTHF). Common polymorphisms in MTHFR limits the availability of 5MTHF and high intakes of folic acid (FA, synthetic form) may exacerbate these effects. 5MTHF is an alternative supplement to FA, but the interaction of folate form and MTHFR genotype and role in programming metabolic health has not been determined. The zebrafish (Danio rerio) is a proven model system in nutrient and drug discovery studies. The objectives of this study were to: 1) develop a mthfr zebrafish mutant model using CRISPR-Cas9 technology; and 2) identify in zebrafish the relationship between folate form (FA vs 5MTHF) and mthfr genotype on early-life development and metabolic regulation. Methods To assay the function of mthfr in vivo, we used a rapid transient knock-out approach reported to recapitulate germline zebrafish loss-of-function phenotypes. Embryos were co-injected with Cas9 protein and a set of 4 guide RNAs (4gRNA) or Cas9 protein alone (control). Zero to 5 days post-fertilization (dpf), embryos were raised in standard conditions then fed a high cholesterol diet (HCD) up to 15 dpf with a fluorescent cholesteryl BODIPY-C12. The HCD was used to induce obesity as previously reported. Global DNA methylation (5-mC%) was measured at 5 dpf and whole-body and hepatic lipid accumulation and live imaging analyses performed at 15 dpf. Results Compared to control, 4gRNA mthfr zebrafish on an HCD had enlarged liver, greater accumulation of hepatic and whole body lipids and altered lipid transport. They also had 80% lower mthfr mRNA than control zebrafish. Global methylation was ∼15% higher (P = 0.06) in 4gRNA zebrafish suggesting a compensatory dependency on an alternative methyl donor pathway during embryonic development. We are now screening germline mutant carriers and assessing folate and methylation metabolites and the interaction of folate form and genotype. Conclusions The zebrafish mthfr mutant will be a valuable model to examine the mechanisms underlining mthfr-related pathologies and provide a high-throughput in vivo system to ascertain the role of different folate forms on embryonic development and long-term health. Funding Sources CIHR-INMD; EP supported by NSERC-CGS D.

scholarly journals Zebrafish Model for Screening Antiatherosclerosis Drugs

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Jichun Han ◽  
Rui Zhang ◽  
Xiaofeng Zhang ◽  
Jing Dong ◽  
Minghan Chen ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
High Throughput ◽  
High Throughput Screening ◽  
Early Stage ◽  
Vascular Inflammation ◽  
Fibrous Tissue ◽  
High Cholesterol Diet ◽  
Zebrafish Model ◽  
Lipid Metabolism Disorder ◽  
Lipid Metabolism Disorders

This study is aimed at establishing a zebrafish model of AS, which can be applied for high-throughput screening anti-AS drugs. A zebrafish AS model was induced by high cholesterol diet (HCD) and lipopolysaccharide (LPS). In the early stage of modeling, HCD induced zebrafish to show some early symptoms similar to human AS, mainly cholesterol accumulation, vascular inflammation, lipid metabolism disorder, and oxidative stress. In addition to lipid metabolism disorders, LPS also induced the same symptoms. And when HCD and LPS exist at the same time, these AS symptoms in zebrafish become more severe. When the modeling time reached 45 days, HCD and LPS induce the formation of plaques in zebrafish blood vessels, and these plaques contain fibrous tissue and lipids, which are similar to human AS plaques. We also evaluated the efficacy of some anti-AS drugs (atorvastatin, aspirin, and vitamin C) through these zebrafish AS models. The results found that atorvastatin can significantly reduce the symptoms of AS induced by HCD and LPS, and aspirin and vitamins can significantly reduce the symptoms of AS induced by LPS. It is feasible to use zebrafish to establish an AS model, and the zebrafish AS model can be used for high-throughput screening of anti-AS drugs.

scholarly journals High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica

2021 ◽  
Author(s):  
Christian Südfeld ◽  
Michal Hubáček ◽  
Daniel Rodrigues Figueiredo ◽  
Mihris I.S. Naduthodi ◽  
John van der Oost ◽  
...  
Keyword(s):  
High Throughput ◽  
Lipid Accumulation ◽  
Insertional Mutagenesis ◽  
Nannochloropsis Oceanica ◽  
Novel Targets

scholarly journals Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dasol Kim ◽  
Hui-Yun Hwang ◽  
Eun Sun Ji ◽  
Jin Young Kim ◽  
Jong Shin Yoo ◽  
...  
Keyword(s):  
Metabolic Regulation ◽  
Elongation Factor ◽  
Dependent Manner ◽  
Diet Induced Obesity ◽  
Metabolic Dysregulation ◽  
Autophagy Induction ◽  
Transcription Factor Eb ◽  
Mitochondrial Elongation

AbstractDisorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

scholarly journals Distinct Changes in Gut Microbiota Are Associated with Estradiol-Mediated Protection from Diet-Induced Obesity in Female Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 499
Author(s):  
Kalpana D. Acharya ◽  
Hye L. Noh ◽  
Madeline E. Graham ◽  
Sujin Suk ◽  
Randall H. Friedline ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Energy Homeostasis ◽  
Whole Body ◽  
Glucose Turnover ◽  
Female Mice ◽  
Adult Mice ◽  
Diet Induced Obesity ◽  
Metabolic Dysregulation ◽  
Regulation Of Metabolism ◽  
Junction Protein

A decrease in ovarian estrogens in postmenopausal women increases the risk of weight gain, cardiovascular disease, type 2 diabetes, and chronic inflammation. While it is known that gut microbiota regulates energy homeostasis, it is unclear if gut microbiota is associated with estradiol regulation of metabolism. In this study, we tested if estradiol-mediated protection from high-fat diet (HFD)-induced obesity and metabolic changes are associated with longitudinal alterations in gut microbiota in female mice. Ovariectomized adult mice with vehicle or estradiol (E2) implants were fed chow for two weeks and HFD for four weeks. As reported previously, E2 increased energy expenditure, physical activity, insulin sensitivity, and whole-body glucose turnover. Interestingly, E2 decreased the tight junction protein occludin, suggesting E2 affects gut epithelial integrity. Moreover, E2 increased Akkermansia and decreased Erysipleotrichaceae and Streptococcaceae. Furthermore, Coprobacillus and Lactococcus were positively correlated, while Akkermansia was negatively correlated, with body weight and fat mass. These results suggest that changes in gut epithelial barrier and specific gut microbiota contribute to E2-mediated protection against diet-induced obesity and metabolic dysregulation. These findings provide support for the gut microbiota as a therapeutic target for treating estrogen-dependent metabolic disorders in women.

scholarly journals Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency

2015 ◽  
Vol 39 (1) ◽  
pp. 115-124 ◽  
Author(s):  
Martina Huemer ◽  
Regina Mulder-Bleile ◽  
Patricie Burda ◽  
D. Sean Froese ◽  
Terttu Suormala ◽  
...  
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Residual Activity ◽  
Clinical Pattern ◽  
Mthfr Deficiency

Abstract P428: A New Mouse Model Of Myocardial Lipids, Fibrosis, Arrhythmia And Diastolic Dysfunction Induced By Hyperlipidemia And Cardiac LDLR

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Monique Williams ◽  
Camila Iansen Irion ◽  
Jose Manuel Condor Capcha ◽  
Guerline Lambert ◽  
Grace Seo ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Cardiac Function ◽  
Diastolic Dysfunction ◽  
Diastolic Function ◽  
Lipid Accumulation ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Poloxamer 407 ◽  
Whole Body ◽  
Subsequent Increase

Background: Hyperlipidemia is a major risk factor for CVD. Patients with HF with preserved ejection fraction (HFpEF) have more myocardial lipid accumulation than patients with reduced EF (HFrEF). RNASeq data from cardiac biopsies showed downregulation of the gene for lipoprotein lipase (LPL) that degrades triglycerides, in HFpEF patients compared to healthy and HFrEF controls. Poloxamer-407 (p407) induces hyperlipidemia by blocking LPL and subsequent increase in plasma triglycerides and low-density lipoprotein (LDL) cholesterol. We hypothesized that mice treated with p407 and cardiac LDL-Receptor (LDLR) over-expression (OE) develop hyperlipidemia, myocardial lipid accumulation, and diastolic dysfunction resulting in HFpEF and arrhythmias. Methods: Baseline cardiac function was assessed by echo for male and female C57Bl6 mice (n=9) for 2 groups: 4wk biweekly i.p. p407-injections with (n=4) or without (n=3) single i.v. injection with AAV9-cTnT-LDLR. Cardiac function was assessed by echocardiography at 3 and 4 wks. Blood Pressure (BP) and Whole Body Plethysmography (WBP) were assessed during wk4. Ttest was used for statistics. PR and ORO staining and telemetry were performed at wk4. Results: At wk3, P407 and LDLR OE led to alterations in diastolic function (increased IVCT, IVRT, MV E/E’, MPI, and NFT) and increased LV wall thickness, p<0.05. At wk4, there was pulmonary hypertension (increased mean pulmonary arterial pressure, decreased pulmonary acceleration time p <.05).Histology showed excessive myocardial lipids and fibrosis, and telemetry showed incidents of second-degree and higher-degree AV block. The group injected solely with p407 show e d alterations in diastolic function (increased IVCT, IVRT, NFT, LVMPI, LVMPI NFT p<.05 ) and decreased EDV, ESV, EDLVM, ESLVM, p<.05 at wk4. All groups had preserved %EF and no abnormalities in BP or WBP. Conclusions: P407 and cardiac LDLR OE induce a drastic decline in cardiac diastolic function over a shorter period of time compared to p407 alone. Diastolic dysfunction was observed in wk3 followed by pulmonary hypertension, arrhythmia, myocardial lipid accumulation and fibrosis in wk4. This new model may allow for more rapid investigations of cardiac abnormalities seen in HFpEF patients.

scholarly journals Testing Tuberculosis Drug Efficacy in a Zebrafish High-Throughput Translational Medicine Screen

2014 ◽  
Vol 59 (2) ◽  
pp. 753-762 ◽  
Author(s):  
Anita Ordas ◽  
Robert-Jan Raterink ◽  
Fraser Cunningham ◽  
Hans J. Jansen ◽  
Malgorzata I. Wiweger ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Drug Efficacy ◽  
Drug Uptake ◽  
Spectrometric Analysis ◽  
Major Advance ◽  
Zebrafish Model ◽  
Content Type ◽  
Screening Platform

ABSTRACTThe translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data fromMycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models ofin vivoMycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

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