Kinetic data for modeling the dynamics of the enzymes involved in animal fatty acid synthesis

The synthesis and modification of fatty acids (FAs) from carbohydrates are paramount for the production of lipids. Simultaneously, lipids are pivotal energy storage in human health. They are associated with various metabolic diseases and their production pathways are for instance candidate therapeutic targets for cancer treatments. The fatty acid de novo synthesis (FADNS) occurs in the cytoplasm, while the microsomal modification of fatty acids (MMFA) happens at the surface of the endoplasmic reticulum (ER). The kinetics and regulation of these complex processes involve several enzymes. In mammals, the main ones are the acetyl-CoA carboxylase (ACC), the fatty acid synthase (FAS), the very-long-chain fatty acid elongases (ELOVL 1-7), and the desaturases (delta family). Their mechanisms and expression in different organs have been studied for more than 50 years. However, modeling them in the context of complex metabolic pathways is still a challenge. Distinct modeling approaches can be implemented. Here, we focus on dynamic modeling using ordinary differential equations (ODEs) based on kinetic rate laws. This requires a combination of knowledge on the enzymatic mechanisms and their kinetics, as well as the interactions between the metabolites, and between enzymes and metabolites. In the present review, after recalling the modeling framework, we support the development of such a mathematical approach by reviewing the available kinetic information of the enzymes involved.</p

Liver steatosis and insulin-resistance : reversal by Sutherlandia frutescens

Type 2 diabetes mellitus (T2DM) is rapidly emerging as one of the greatest global health issues of the 21st century. Insulin-resistance is a condition associated with T2DM and in the cell it is defined as the inadequate strength of insulin signalling from the insulin receptor downstream to the final substrates of insulin action involved in multiple metabolic, gene expression, and mitogenic aspects of cellular function. To investigate the potential mechanisms involved in the development of insulin-resistance, two in vitro liver cell models were established using palmitate or a combination of insulin and fructose as inducers. The development of insulin-resistance was determined via the capacity of the hepatocytes to maintain normal glucose metabolism functionality by measuring hepatic gluconeogenesis and glycogenolysis. It was established that the treatments induced the development of insulinresistance after 24 hours chronic exposure. Previous studies have investigated the potential of Sutherlandia frutescens extracts as therapeutic agents for insulin-resistance. The aim of this study was thus to investigate the ability of a hot aqueous extract of S. frutescens to reverse the insulin-resistant state, via measuring gluconeogenesis and glycogenolysis, the associated changes in cellular physiology (lipid accumulation, oxidative stress, and acetyl- CoA levels), and changes in mRNA expression. The results showed that S. frutescens had a significant effect on reversing the insulin-resistant state in both models of insulin-resistance. Furthermore, S. frutescens was capable of reducing lipid accumulation in the form of triacylglycerol in the high insulin/fructose model, while this was unaffected in the palmitate model. However, S. frutescens did reduce the accumulation of diacylglycerol in the palmitate model. Oxidative stress, seen to be associated with the insulin-resistant state, was successfully treated using the extract, as indicated by a reduction in reactive oxygen species. However no change was seen in the nitric oxide levels, in either model. Interestingly, although S. frutescens had no effect on the level of acetyl-CoA in the insulin/fructose model, it was found to increase this in the palmitate model. It is suggested that this may be due to increased β-oxidation and metabolic activity induced by the extract. The analysis of mRNA expression gave some insight into possible mechanisms by which insulin-resistance develops, although the results were inconclusive due to high variability in samples and the possibility of the RNA being compromised. Future studies will address this issue. The results of this study reflect different proposed clinical causes of insulin-resistance through the responses seen in the two cell models. These indicate that liver steatosis and insulin-resistance are induced by high palmitate as well as high insulin and fructose levels, and reversed by S. frutescens. Therefore the potential of S. frutescens to be used as a therapeutic agent in the treatment of insulin-resistance is indicated by this study

Inborn errors of metabolism with a focus on functional analysis of a special mutation in MCCB causing 3-Methylcrotonyl-CoA carboxylase deficiency, and MMADHC in intracellular vitamin B₁₂ metabolism, a gene in which mutations can lead to three different phenotypes

The present work focuses on two autosomal recessive inborn errors of metabolism, 3- methylcrotonyl-CoA carboxylase (MCC) deficiency, a defect in the catabolism of leucine, and on the cblD defect of intracellular vitamin B12 (cobalamin) metabolism. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing α (MCCA) and smaller β (MCCB) subunits encoded by MCCA and MCCB, respectively. We report studies of the c.1054G→A mutation in exon 11 of MCCB detected in the homozygous state in a patient with MCC deficiency. Sequence analysis of MCCB cDNA revealed two overlapping transcripts, one containing the normal 73 bp of exon 11 including the missense mutation c.1054G→A (p.G352R), the other with exon 11 replaced by a 64 bp sequence from intron 10 (cryptic exon) that maintains the reading frame and is flanked by acceptable splice consensus sites. In expression studies, we show that both transcripts lack detectable MCC activity. Western blot analysis showed slightly reduced levels of MCCB using the transcript containing the missense mutation, whereas no MCCB was detected with the transcript containing the cryptic exon. Analysis of the region harboring the mutation revealed that the c.1054G→A mutation is located in an exon splice enhancer sequence. Using MCCB minigene constructs to transfect MCCB-deficient fibroblasts, we demonstrate that the reduction in utilization of exon 11 associated with the c.1054G→A mutation is due to alteration of this exon splice enhancer. Further, we show that optimization of the weak splice donor site of exon 11 corrects the splicing defect. To our knowledge, this is the first demonstration of a point mutation disrupting an exon splice enhancer that causes exon skipping along with utilization of a cryptic exon. In man, cobalamin is an essential cofactor in only two metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin (AdoCbl) in mitochondria and methylcobalamin (MeCbl) in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. We have identified the MMADHC gene responsible for the cblD defect that can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Various mutations are associated with each of the three biochemical phenotypes of the disorder. Using expression vectors containing the wildtype MMADHC with an enhanced mitochondrial leader sequence, mutations changing possible downstream sites of reinitiation or mutations introducing stop codons, we provide evidence that reinitiation of translation at downstream AUGs explains most of the phenotypes found in cblD patients. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis whereas the sequence between Met62 and Met116 is required for AdoCbl synthesis. Our findings support the hypothesis that a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of the MMADHC protein as a branching point in intracellular cobalamin trafficking. ZUSAMMENFASSUNG Die vorliegende Arbeit beschäftigt sich mit zwei autosomal rezessiv vererbten Stoffwechselkrankheiten, dem 3-Methylcrotonyl-CoA Carboxylase (MCC) Mangel, welches ein Defekt im Leuzin-Abbau ist, und dem cblD Defekt, einer Störung des intrazellulären Vitamin B12 (Cobalamin) Stoffwechsels. MCC ist ein heteromeres mitochondriales Enzym aus Biotin-bindenden α (MCCA) und kleineren β (MCCB) Untereinheiten, welche durch MCCA und MCCB kodiert sind. Wir berichten über die Mutation c.1054G>A (p.G352R) in Exon 11 von MCCB, welche wir homozygot bei einem MCC-Patienten gefunden haben. Die Analyse der cDNA Sequenz von MCCB ergab zwei überlappende Transkripte: eines mit der Mutation c.1054G>A, sowie ein weiteres mit Ersatz der 73 bp von Exon 11 durch 64 bp aus Intron 10. Dieses kryptische Exon verändert das Leseraster nicht und weist akzeptable Spleiss-Stellen auf. Expressionsstudien zeigen, dass beide Transkripte keine MCC Aktivität besitzen bei nur reduzierter Proteinmenge beim mutationstragenden Transkript sowie Proteinverlust des Transkriptes mit kryptischem Exon. Die Mutation c.1054G>A ist Teil eines Exon-Spleiss- Enhancers. Transfektionsexperimente von MCCB Minigen Konstrukten in MCCB defizienten Fibroblasten zeigten bei Veränderung des Exon-Spleiss-Enhancers ein vermindertes Spleissen von Exon 11. Zudem konnte bei Sequenz-Optimierung der Exon 11-Spleiss- Stellen der Spleiss-Defekt korrigiert werden. Unseres Wissens ist dies der erste Nachweis eines durch Punktmutation in einem Exon-Spleiss-Enhancer resultierenden Exonverlustes bei gleichzeitiger Insertion eines kryptischen Exons. Beim Menschen wird Cobalamin für nur zwei Reaktionsschritte des Stoffwechsels benötigt: als mitochondriales Adenosylcobalamin (AdoCbl) für den Abbau von Methylmalonsäure und als zytosolisches Methylcobalamin (MeCbl) für die Methylierung von Homocystein. Bei Vorliegen eines cblD-Defektes besteht eine Methylmalonazidurie, eine Hyperhomocysteinämie oder eine Kombination aus beiden Symptomen. Uns gelang die Klonierung des MMADHC Gens, welches für den cblD Defekt verantwortlich ist. Verschiedene Mutationen sind mit den drei Phänotypen des cblD-Defektes assoziiert. Durch Veränderung der mitochondrialen Importsequenz, durch Korrektur der zusätzlichen AUGs (Transkriptions-Start) und durch Einfügen weiterer Stop-Mutationen konnte gezeigt werden, dass eine Reinitiation der Transkription an weiteren AUGs die in cblD Patienten gefundenen Phänotypen erklären könnte. Weiter haben wir Hinweise darauf, dass ein cblD-Protein mit Beginn bei Met116 weiter MeCbl synthetisieren kann, und dass die Sequenz zwischen Met62 und Met116 notwendig für die Synthese von AdoCbl ist. Unsere Resultate bestätigen die Hypothese eines kritischen Gleichgewichts zwischen zytosolischer MeCbl und mitochondrialer AdoCbl Synthese. Das MMADHC Protein spielt somit eine wichtige Rolle bei der intrazellulären Cobalamin Verteilung

Mito-Nuclear Communication by Mitochondrial Metabolites and Its Regulation by B-Vitamins

Mitochondria are cellular organelles that control metabolic homeostasis and ATP generation, but also play an important role in other processes, like cell death decisions and immune signaling. Mitochondria produce a diverse array of metabolites that act in the mitochondria itself, but also function as signaling molecules to other parts of the cell. Communication of mitochondria with the nucleus by metabolites that are produced by the mitochondria provides the cells with a dynamic regulatory system that is able to respond to changing metabolic conditions. Dysregulation of the interplay between mitochondrial metabolites and the nucleus has been shown to play a role in disease etiology, such as cancer and type II diabetes. Multiple recent studies emphasize the crucial role of nutritional cofactors in regulating these metabolic networks. Since B-vitamins directly regulate mitochondrial metabolism, understanding the role of B-vitamins in mito-nuclear communication is relevant for therapeutic applications and optimal dietary lifestyle. In this review, we will highlight emerging concepts in mito-nuclear communication and will describe the role of B-vitamins in mitochondrial metabolite-mediated nuclear signaling

THE CONCISE GUIDE TO PHARMACOLOGY 2017/18:Enzymes

The Concise Guide to PHARMACOLOGY 2017/18 provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13877/full. Enzymes are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

Investigating the role of nuclear encoded mitochondrial genes in the onset of type 2 diabetes

Mitochondrial dysfunction has long been implicated in Type 2 diabetes (T2D). This rela- tionship appears to be bidirectional, with evidence that mitochondrial dysfunction is both caused by and causal of T2D-related phenotypes. A potential causal role in T2D onset would be supported by evidence of a genetic predisposition to mitochondrial dysfunction, since inherited genetic risk factors precede and contribute to disease onset. Here, a genetic study design is used to investigate the potential role of T2D-associated genetic risk loci (T2D loci) in disrupting mitochondrial function through the altered expression of nuclear- encoded mitochondrial genes (NEMGs). The mitochondria are targeted by multiple T2D drugs and therefore such loci may be informative for effective treatment and prevention measures. The functional cis–genes regulated by T2D loci were identified based on the co-location of T2D loci with adipose tissue expression quantitative trait (eQTL) within a genetic distance of 1 LDU. T2D loci and eQTL were previously mapped using LDU- based gene mapping, which is compared and contrasted in this thesis to other popular tests of association. 50 of the identified T2D cis–genes were NEMGs and implicated a number of pathways in the inherited risk of T2D, including the relevant pathway of branched-chain amino acid catabolism. These same 50 genes were enriched for decreased expression in T2D cases compared to controls in independent gene expression datasets. Compared to the total known NEMGs, the 50 cis-NEMGs showed further enrichment for decreased expression, suggesting that T2D-eQTL co-location may identify specific subsets of causal genes. Finally, a candidate T2D locus associated with the cis–NEMG ACAD11 was fine-mapped using targeted sequence data for 94 T2D cases and 94 controls. Sev- eral candidate causal variants were identified, including two low-frequency haplotypes, one of which contained both an ACAD11 splicing mutation and a mutation predicted to disrupt the observed binding of HNF4A and COUP-TFII within the ACAD11 promoter region.Open Acces

Sodium-dependent multivitamin transporter: a potential novel cause of cardiomyopathy

PhD ThesisCardiomyopathy is a heterogeneous disorder affecting adults and children and is a leading cause of death globally. Paediatric cardiomyopathies affect ~1:100,000 children, with around one third requiring a heart transplant or risk death within two years of diagnosis. A homozygous missense mutation in the human Sodium Multivitamin Transporter (SMVT) gene, SLC5A6, was identified in sisters with paediatric cardiomyopathy. The transporter is a plasma membrane protein that transports biotin, pantothenic acid and lipoic acid throughout several tissues including the brain, intestine and heart. These substrates play an essential role in energy metabolism and homeostasis, suggesting reduced functionality of Slc5a6 within the heart could result in cardiomyopathy. Mouse models were employed to conditionally delete Slc5a6 within cardiomyocytes resulting in the development of cardiomyopathy markers throughout early adulthood leading to sudden death at five months of age. Cardiac functionality was assessed using electrocardiography (ECG) which showed atrioventricular block, and histological analysis demonstrated myocardial fibrosis and cardiomyocyte hypertrophy. Cardiac magnetic resonance imaging (CMR) revealed a reduction in ejection fraction, cardiac output and stroke volume, hallmarks of left ventricular dysfunction. Together, these changes confirm the presence of cardiomyopathy within the cardiomyocyte specific Slc5a6 knockout model. Gross abnormalities in mitochondrial structure and organisation were observed using electron microscopy, and quadruple immunofluorescence staining revealed a reduction in complex I of the mitochondrial electron transport chain. This suggests that loss of Slc5a6 has a negative impact on energy metabolism through deficiency of complex I, causing excess stress upon the heart ultimately resulting in cardiomyopathy. Preliminary data from vitamin supplementation to pregnant females and their Slc5a6 knockout offspring shows a delay in the onset of cardiomyopathy markers including myocardial fibrosis and cardiomyocyte hypertrophy, as shown by ECG and histology. Collectively this data suggests that Slc5a6 is important for normal cardiac structure and function, with potential for therapeutic intervention in patients with variants in SLC5A6

Investigating the role of acetyl-CoA carboxylases in breast cancer progression

Breast cancer is the most commonly diagnosed cancer in women worldwide, thus a need for more effective treatment options is necessary. Deregulation of cellular energetics is a feature of breast cancer, with de novo lipids being recognised as a driver of breast malignancy. The process of de novo fatty acid synthesis and degradation is a tightly regulated process governed by the acetyl-coenzyme A carboxylase (ACC) enzymes; ACC1 and ACC2. Dysregulation of this process has been implicated in various diseases, but its contribution to breast cancer remains relatively unknown. Thus, the aim of this thesis was to investigate the independent and dual roles of ACC1 and ACC2 on breast cancer cell phenotypes. Deletion of ACC1 and/or ACC2 decreased breast cancer cell survival. Subsequent addition of palmitate to ACC1 KO cells rescued this phenotype, highlighting the contribution of de novo lipids. Mass-spectrometric analysis demonstrated shifts in ACC1 KO cell lipids profiles with characterisation of cytoskeletal proteins demonstrating decreased protein integrity. Investigation of human ACC1 expression highlighted the biological relevance of this protein given its upregulation in malignant breast tissue, compared to the almost undetectable levels in corresponding normal breast tissue. The use of ACC inhibitors Soraphen A and PF-05175157 resulted in decreased breast cancer cell survival, with Soraphen A also increasing the efficacy of Tamoxifen, Docetaxel and Doxorubicin. Finally, we demonstrated the selectivity of these compounds to breast cancer cells, and specificity to their intended pharmacological targets of ACC1 and ACC2. Overall, our data highlighted the importance of ACC1 and a novel role for ACC2, in maintaining breast cancer cell viability. We also demonstrated the potential use of ACC inhibitors independently or with Tamoxifen, Docetaxel and Doxorubicin, as a treatment for breast cancer

Role of mitochondria in liver diseases

252 p.La disfunción mitocondrial desempeña un papel clave en el inicio y desarrollo de las enfermedades hepáticas crónicas. La proteína J controlada por metilación (MCJ) es un inhibidor endógeno de laactividad mitocondrial, y previamente hemos podido demostrar niveles significativamente aumentados deMCJ en pacientes con hígado graso, daño hepático inducido por paracetamol y lesión hepática debido a la colestasis, lo que sugiere una posible asociación entre MCJ y la disfunción mitocondrial. La enfermedad hepática alcohólica causa más de 2 millones de fallecimientos en el mundo y es la segunda causa detrasplante hepático. Sin embargo, carece de un tratamiento específico. Por otro lado, las tasas actuales de trasplante hepático cubren menos del 10% de las necesidades globales, y entre las estrategias paraaumentar el grupo de donantes, se ha propuesto el uso de hígado con criterio expandido, aquellos queprovienen de hígados añosos o esteatóticos. Sin embargo, el uso de estos órganos aumenta el fallo hepático post trasplante, ya que su capacidad regenerativa está limitada y sufren una alta susceptibilidad hacia el daño por isquemia. En ambos modelos la disfunción mitocondrial es un indicador temprano deldaño hepático y hemos podido comprobar la sobreexpresión de MCJ en estadios avanzados. De hecho, elsilenciamiento hepático de MCJ (1) recupera la actividad mitocondrial, alivia la esteatosis y evita la inflamación y el estrés oxidativo en modelos preclínicos de enfermedad hepática alcohólica, y (2) acelerala regeneración hepática y reduce la lesión isquémica en ratones jóvenes, pero significativamente,también en ratones añosos y esteatóticos, promoviendo su uso para el trasplante hepático, reduciendo así la escasez existente de donantes. En resumen, este proyecto muestra la contribución de la disfunción mitocondrial, en especial de la proteína MCJ, en el desarrollo de la enfermedad hepática alcohólica y la regeneración limitada junto con mayor susceptibilidad isquémica que se observa en hígados con un metabolismo comprometido, con el objetivo de establecer dicha proteína como futura diana terapéutica para el tratamiento de enfermedades hepáticas crónica