Mitochondrial fatty acid oxidation in human liver

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Metabolic diseases are a burden on the European population and health care system. It is increasingly recognised that individual differences with respect to history, lifestyle, and genetic make-up affect disease ...

Considering parameter uncertainty explicitly by generating 50 models.

Following on in silico and in vitro work, the effect of MCAD deficiency on CoA metabolism was investigated. Using a recently published HILIC-MS/MS method, free and acylated CoA species could be measured simultaneously in HepG2 MCAD-KO cells. The levels of CoA biosynthesis intermediates and total CoA was also characterised by HPLC in liver samples from MCAD-KO mice exposed to energetic stress (fasting adn cold). qPCR was applied to investigate changes in the CoA metabolism that might constitute ...

Validation of model's ability to predict oxygen consumption flux as measured usign permeabilised cells in an Oroboros Oxygraph. Generates Fig. 2A in the associated publication.

Download "Model_notebooks.rar", unzip, and run: "2, generate-model-Oroboros-validation-[needs(1)]-20221109.nb" and "4, Fig2A-Oroboros-simulation-data-[needs-(1-2-and-3)]-20221109.nb" after running "1, generate-model-20221109.nb"

Validation of model's ability to predict whole-body ketogeneic flux as extracted form Fletcher et al. (2019). Generates Fig. 2B in the associated publication.

Download "Model_notebooks.rar", unzip, and run: "2, generate-model-Oroboros-validation-[needs(1)]-20221109.nb" and "5, Fig2B-ketogenesis-validation-[needs-(1)]-20221109.nb" after running "1, generate-model-20221109.nb"

Testing the model's ability to predict palmitoyl-CoA and octanoyl-CoA dehydrogenation in human liver lysate, with and without anti-MCAD and anti-VLCAD antibodies. Generates Fig. 2 C and D in the associated publication. Data from Aoyama et al. (1995).

Downoad and unzip "Model_notebooks.rar" and run "6, Fig2C+D-ACAD-partitioning-validation-[needs-(1)]-20221109.nb" after running "1, generate-model-20221109.nb".

Minireviews about each enzyme in the mitochindrial beta-oxidation model on which the final parameter choices (fixed-parameter model) and parameter sampling distributions (ensemble) were based.

The behaviour of a published MCAD-KO HepG2 cell line was compared to that of a wild-type HepG2 cell line in terms of the changes in acyl-CoA and acylcarnitine levels (measured by HILIC-MS/MS) and gene expression pertaining to CoA metabolism (characterised by qPCR). Additionally, the incorporation of label from stable isotope-labelled pantothenate (vitamine B5) was over 24 hours of exposure to an energetic stressor was also investigated.

MCAD-KO mice (C57BL/6J background) were exposed to three different conditions: "fed", "fasting", and "fasting + cold". In this study we determined the concentration of various CoA biosynthetic intermediates (including CoA) and the expression of CoA metabolic genes in liver tissue samples from these groups of mice.

Explicit consideration of kinetic paramater uncertainty as obtained from literature.

Prediction of patient urinary acylcarnitine under metabolic decompensation. Generates Fig. 3, Table 1, and Table S2 in the associated publication.

Download and unzip "Model_notebooks.rar" and run "7, Fig3+4+S1+S3-ACADDs-[needs-(1)]-20221109.nb" after running "1, generate-model-20221109.nb"

Based on odendaal1, a control model is made and compared to model deficient for short-chain acyl-CoA dehydrogenase (SCADD, 0%), medium-chain acyl-CoA dehydrogenase (MCADD, 0%), and very long-chain acyl-CoA dehydrogenase (VLCADD, 10%). With and withou metabolite partitioning, and with a fixed mitohondrial free CoASH. Generates Figures 3, 4, S1, S2, and S3 in the related paper.

Download "Model_notebooks.rar", unzip, and run: "7, Fig3+4+S1+S3-ACADDs-[needs-(1)]-20221109.nb" and "14, ...

Calculation of control and response coefficients. Generates Fig. 5, Fig. S4, and Table S2 in the associated publication.

Download "Model_notebooks.rar", unzip, and run: "8, Fig5-control-coefficients-[needs-(1)]-20221109.nb", "9, TableS2-response-coefficients-[needs-(1)]-20230302.nb", and "15, FigS4-control-coefficients-low-AcetylCoA-[needs-(1)]-20221109.nb" after running "1, generate-model-20221109.nb"

Incrementally increase the activity of some target rescue enzymes from 20% of default expression to 200% of default expression in a control and MCADD model to see if flux and CoASH concentration are rescued. Generates Fig. 6, S5, and S6.

Download "Model_notebooks.rar", unzip, and run "11, Fig6+S5-rescues-[needs-(1-and-10)]-20221109.nb", "10, Fig6B-inset-rescues-(low-acetylCoA)-[needs-(1)]-20221109.nb", and "16, FigS6-rescues-20221109-fixed-[needs-(1)]-CoASH.nb" after running "1, generate-model-20221109.nb" ...

Creation of personalised models of control, symptomatic MCADD, asymptomatic MCADD, and early diagnosis MCADD individuals using fibroblast proteomics to adjust model Vmaxes. Generates Fig. 7 and S7.

Download and unzip "Model_notebooks.rar" and run "13, Fig7-personalised-models-[needs-(1-and-12)]-20221109.nb" after running "1, generate-model-20221109.nb" and "12, Fig7-S7-preprocessing-[needs-(1)-]-20221109.nb".

The expression of various genes related to the CoA metabolism as measured by qPCR.

Incorporation of stable isotope from pantothenate into total and free CoA pools.

Protein measurement used to normalised the oxygen consumption flux measured on an Oroboros oxygraph.

Measured in an Oroboros oxygraph.

Proteomics from MCADD and control individuals' fibroblasts.

Unzip model notebooks and keep in the same folder. Notebook "0, generate_model.nb" needs to be run before the others can be, as the model needs to be exported as Excel files before they cvan be used to make the figures.

Adjusted model to test the model's ability to oxygen consumption rate by permeabilised HepG2 cells in an Oroboros oxygraph. Data from Fletcher et al. (2019).

Adjusted model to test the model's ability to predict palmitoyl-CoA and octanoyl-CoA dehydrogenation in human liver lysate, with and without anti-MCAD and anti-VLCAD antibodies. Data from Aoyama et al. (1995).

Human mitochondrial fatty acid oxidation of saturated, even chain acyl-Coas beginning at C16. See Model description for detail.

Unzip model notebooks and keep in the same folder. Notebook names state which notebooks need to be run before them in order for them to word, e.g. "[needs-(1)]" indicates that the notebook numbered 1 must be run and its exported output generated before the given notebook can work. This has to do with the model being generated in only one notebook to avoid duplication.

Carnitine acetyltransferase - (EC 2.3.1.7)

Medium-/short-chain hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)

Medium-chain ketoacyl-CoA thiolase (EC 2.3.1.16)

Mitochondrial trifunctional protein (UniProt: P40939)

Acyl-CoA thioesterases (EC 3.1.2.1. and EC 3.1.2.2)

L-carnitine as conserved moiety

CoA as conserved moiety

Acetyl-CoA in the mitochondrion as constant metabolite

NADH and NAD+ (Nicotinamide adinine dinucleotide) as constant values.

Oxidised and reduced electron transferring flavoprotein as constant values.

Volume per mg mitochondrial protein of mitochondrion and cytosol.

Carnitine palmitoyltransferase 1 (EC 2.3.1.21)

The theory, calculations, and conditions that went into estimating the Keqs.

Carnitine acylcarnitine translocase (UniProt: O43772)

Carnitine palmitoyltransferase 2 (EC 2.3.1.21)

Carnitine acetyltransferase (EC 2.3.1.7)

Short-chain acyl-CoA dehydrogenase (EC 1.3.99.2)

Medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3)

Very long-chain acyl-CoA dehydrogenase (EC 1.3.8.9)