In Silico Evidence for Gluconeogenesis from Fatty Acids in Humans

The question whether fatty acids can be converted into glucose in humans has a long standing tradition in biochemistry, and the expected answer is “No”. Using recent advances in Systems Biology in the form of large-scale metabolic reconstructions, we reassessed this question by performing a global investigation of a genome-scale human metabolic network, which had been reconstructed on the basis of experimental results. By elementary flux pattern analysis, we found numerous pathways on which gluconeogenesis from fatty acids is feasible in humans. On these pathways, four moles of acetyl-CoA are converted into one mole of glucose and two moles of CO2. Analyzing the detected pathways in detail we found that their energetic requirements potentially limit their capacity. This study has many other biochemical implications: effect of starvation, sports physiology, practically carbohydrate-free diets of inuit, as well as survival of hibernating animals and embryos of egg-laying animals. Moreover, the energetic loss associated to the usage of gluconeogenesis from fatty acids can help explain the efficiency of carbohydrate reduced and ketogenic diets such as the Atkins diet

Genome-scale metabolic model of the rat liver predicts effects of diet restriction.

Mapping network analysis in cells and tissues can provide insights into metabolic adaptations to changes in external environment, pathological conditions, and nutrient deprivation. Here, we reconstructed a genome-scale metabolic network of the rat liver that will allow for exploration of systems-level physiology. The resulting in silico model (iRatLiver) contains 1,882 reactions, 1,448 metabolites, and 994 metabolic genes. We then used this model to characterize the response of the liver\u27s energy metabolism to a controlled perturbation in diet. Transcriptomics data were collected from the livers of Sprague Dawley rats at 4 or 14 days of being subjected to 15%, 30%, or 60% diet restriction. These data were integrated with the iRatLiver model to generate condition-specific metabolic models, allowing us to explore network differences under each condition. We observed different pathway usage between early and late time points. Network analysis identified several highly connected hub genes (Pklr, Hadha, Tkt, Pgm1, Tpi1, and Eno3) that showed differing trends between early and late time points. Taken together, our results suggest that the liver\u27s response varied with short- and long-term diet restriction. More broadly, we anticipate that the iRatLiver model can be exploited further to study metabolic changes in the liver under other conditions such as drug treatment, infection, and disease

The evolutionary rewiring of ubiquitination targets has reprogrammed the regulation of carbon assimilation in the pathogenic yeast Candida albicans

Date of Acceptance: 13/11/2012 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Correction for Sandai et al., The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans published 20-01-2015 DOI: 10.1128/mBio.02489-14Peer reviewedPublisher PD

An in Silico Liver: Model of Gluconeogenesis

An in silico liver was developed in attempt to represent the in vivo state of the fasted liver. It featured two conceptual models. The first one represented carbohydrate metabolism of the human liver, which included the heterogeneous nature of the liver by incorporating spatial variation of key enzyme activities. This model was able to predict the overall fluxes in tissue and the effect of high intensity exercise on the various hepatic fluxes. A second model of hepatic metabolism was developed to represent the complex interplay between gluconeogenesis, lipid metabolism, and alcohol metabolism in the fasted rat liver. Biochemical pathways are represented by key kinetic reactions that include allosteric and substrates effectors, and phosphorylation/dephosphorylation enzymes regulation. The model also incorporates the compartmentation and intercompartmental transports between the cytosol and the mitochondria, and transport of metabolites between blood compartment and the tissue. The model is based on the experimental set-up of fasted perfused rat livers. The model was used to simulate the effects of the two main gluconeogenic substrates available during the fasting state-- lactate and pyruvate--along with the addition of fatty acids and/or ethanol. The model predicts successfully the rates of glucose and ketone production, substrate uptake, and citric acid cycle. Parameter estimations were performed in order to obtain a set of physiological parameters capable of representing the liver under various combinations of nutrients. Parameter sensitivity analysis was generated to quantify the contribution of each parameter to the model output. The model was validated with data available in the published literature from ex vivo studies. The in silico liver constitutes a tool that can be used to predict the effect of physiological stimuli on flux and concentration distributions. This will provide an increase in the understanding of such effects and to determine what parameters, enzymes, and fluxes are responsible

Metabolic adaptation of two in silico mutants of Mycobacterium tuberculosis during infection

ABSTRACT: Background: Up to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb’s vulnerability and metabolic adaptation have been carried out. Results: A genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports. Conclusions: This study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas

Regulation of genes involved in carnitine homeostasis by PPARa across different species (rat, mouse, pig, cattle, chicken, and human)

Recent studies in rodents convincingly demonstrated that PPAR-alpha is a key regulator of genes involved in carnitine homeostasis, which serves as a reasonable explanation for the phenomenon that energy deprivation and fibrate treatment, both of which cause activation of hepatic PPAR-alpha, causes a strong increase of hepatic carnitine concentration in rats. The present paper aimed to comprehensively analyse available data from genetic and animal studies with mice, rats, pigs, cows, and laying hens and from human studies in order to compare the regulation of genes involved in carnitine homeostasis by PPAR-alpha across different species. Overall, our comparative analysis indicates that the role of PPAR-alpha as a regulator of carnitine homeostasis is well conserved across different species. However, despite demonstrating a well-conserved role of PPAR-alpha as a key regulator of carnitine homeostasis in general, our comprehensive analysis shows that this assumption particularly applies to the regulation by PPAR-alpha of carnitine uptake which is obviously highly conserved across species, whereas regulation by PPAR-alpha of carnitine biosynthesis appears less well conserved across species

Short chain fatty acids in human gut and metabolic health

Peer reviewedPublisher PD

Personal model-assisted identification of NAD(+) and glutathione metabolism as intervention target in NAFLD

To elucidate the molecular mechanisms underlying non-alcoholic fatty liver disease (NAFLD), we recruited 86 subjects with varying degrees of hepatic steatosis (HS). We obtained experimental data on lipoprotein fluxes and used these individual measurements as personalized constraints of a hepatocyte genome-scale metabolic model to investigate metabolic differences in liver, taking into account its interactions with other tissues. Our systems level analysis predicted an altered demand for NAD(+) and glutathione (GSH) in subjects with high HS. Our analysis and metabolomic measurements showed that plasma levels of glycine, serine, and associated metabolites are negatively correlated with HS, suggesting that these GSH metabolism precursors might be limiting. Quantification of the hepatic expression levels of the associated enzymes further pointed to altered de novo GSH synthesis. To assess the effect of GSH and NAD(+) repletion on the development of NAFLD, we added precursors for GSH and NAD(+) biosynthesis to the Western diet and demonstrated that supplementation prevents HS in mice. In a proof-of-concept human study, we found improved liver function and decreased HS after supplementation with serine (a precursor to glycine) and hereby propose a strategy for NAFLD treatment.Peer reviewe

Strategies for wheat stripe rust pathogenicity identified by transcriptome sequencing

Stripe rust caused by the fungus Puccinia striiformis f.sp. tritici (Pst) is a major constraint to wheat production worldwide. The molecular events that underlie Pst pathogenicity are largely unknown. Like all rusts, Pst creates a specialized cellular structure within host cells called the haustorium to obtain nutrients from wheat, and to secrete pathogenicity factors called effector proteins. We purified Pst haustoria and used next-generation sequencing platforms to assemble the haustorial transcriptome as well as the transcriptome of germinated spores. 12,282 transcripts were assembled from 454-pyrosequencing data and used as reference for digital gene expression analysis to compare the germinated uredinospores and haustoria transcriptomes based on Illumina RNAseq data. More than 400 genes encoding secreted proteins which constitute candidate effectors were identified from the haustorial transcriptome, with two thirds of these up-regulated in this tissue compared to germinated spores. RT-PCR analysis confirmed the expression patterns of 94 effector candidates. The analysis also revealed that spores rely mainly on stored energy reserves for growth and development, while haustoria take up host nutrients for massive energy production for biosynthetic pathways and the ultimate production of spores. Together, these studies substantially increase our knowledge of potential Pst effectors and provide new insights into the pathogenic strategies of this important organism.J.P.R. is an ARC Future Fellow (FT0992129). This project has been supported by Bioplatforms Australia through funding from the Commonwealth Government NCRIS and Education Investment Fund Super Science programs

Hepatic insulin-degrading enzyme regulation and its role on glucagon signaling

Insulin-degrading enzyme (IDE) is a highly conserved and ubiquitously expressed Zn2+-metalloendopeptidase that degrades insulin and glucagon among other substrates. By decades, its main function has been attributed to hepatic insulin clearance, a process that regulates availability of circulating insulin levels, but recent studies performed by our group indicate a more important role of this protein regulating hepatic insulin sensitivity and glucose homeostasis. However, its regulation in response to nutritional state and the fasting-to-postprandial transition is poorly understood and much less attention has been dedicated to its role on regulating glucagon signal transduction and mitochondrial function in hepatocytes. In this thesis, we studied the regulation of IDE mRNA and protein levels as well as its proteolytic activity in the liver under fasting (18 h) and refeeding (30 min and 3 h) conditions, in mice fed a standard (SD) or high-fat (HFD) diets. Likewise, we aim to elucidate the role of IDE on glucagon signaling and its impact on energy metabolism in hepatocytes using a loss-of-function approach. Livers from L-IDE-KO and WT mice were used to obtain tissue extracts and primary hepatocytes for culture. The mouse hepatocyte cell line (AML12) was transduced with an shRNA targeting Ide mRNA by means of a lentiviral vector and obtaining a stable line (AML12-shRNA-IDE). L-IDE-KO primary hepatocytes and AML12-shRNA-IDE with their respective controls were stimulated with glucagon and the signaling pathway was analyzed by western blot and ELISA. Mitochondrial function and energy metabolism of AML12-shRNA-IDE and control cells were assessed by Seahorse XFe24 Analyzer with a Mito Stress Assay. In the liver of mice fed a HFD, fasting reduced IDE protein levels (~30%); whereas refeeding increased its activity (~45%) in both mice fed an SD and HFD. Circulating lactate concentrations directly correlated with hepatic IDE activity and protein levels. Of note, L-lactate in liver lysates augmented IDE activity in a dose-dependent manner. Additionally, IDE protein levels in liver, but not its activity, inversely correlated (R2 = 0.3734, p < 0.01) with a surrogate marker of insulin resistance (HOMA index). Liver extracts and primary hepatocytes from L-IDE-KO mice, compared to WT, showed decreased expression of glucagon receptor (~60%), CREB protein (~40%), and diminished phosphorylation of CREB (~50%) upon glucagon stimulation. Ide expression and IDE protein levels were reduced by ~50% in AML12-shRNA-IDE cells. At basal state, glucagon receptor, FoxO1 and CREB protein were significantly lower in AML12-shRNA-IDE cells than in control cells, (~40%, ~75% and ~75%, respectively). Glucagon stimulation resulted in less (~30%) cAMP levels and changes in the kinetic of glucagon-mediated phosphorylation of CREB and other PKA substrates in AML12-shRNA-IDE. Seahorse analyses showed that both oxygen consumption and extracellular acidification rates increased 2-fold in AML12-shRNA-IDE with a 2-fold increment of mitochondrial and glycolytic adenosine triphosphate (ATP) production. Finally, we generated, using homology modeling by satisfaction of spatial restrains technique, complete 3D structures of human and murine IDE isoforms with 15a and 15b exons. Our results highlight that the nutritional regulation of IDE in liver is more complex than previously expected in mice. Reduced IDE expression in mouse hepatocytes has a deleterious effect on glucagon signaling, affecting this intracellular pathway in parallel with a shift to a more energetic phenotype. These findings suggest that IDE is necessary for proper glucagon signal transduction and regulation of the energy production in hepatocytes.La enzima degradadora de insulina (IDE) es una Zn2+-metaloendopeptidasa altamente conservada y expresada de forma ubicua que degrada la insulina y el glucagón, entre otros sustratos. Durante décadas, se ha atribuido su función principal al aclaramiento de insulina hepática, proceso que regula la disponibilidad de los niveles de insulina circulante, pero estudios recientes realizados por nuestro grupo indican un papel más importante de esta proteína en la regulación de la sensibilidad a la insulina hepática y la homeostasis de la glucosa. Sin embargo, su regulación en respuesta al estado nutricional y la transición de ayuno a posprandial es poco conocida y se ha dedicado mucha menos atención a su papel en la regulación de la transducción de señales de glucagón y la función mitocondrial en los hepatocitos. En esta tesis estudiamos la regulación de los niveles de mRNA y proteína de IDE así como su actividad proteolítica en el hígado en condiciones de ayuno (18 h) y realimentación (30 min y 3 h), en ratones alimentados con un estándar (SD) o alto dietas ricas en grasas (HFD). Asimismo, nuestro objetivo es dilucidar el papel de IDE en la señalización de glucagón y su impacto en el metabolismo energético en los hepatocitos utilizando un enfoque de pérdida de función. Se usaron hígados de ratones L-IDE-KO y WT para obtener extractos de tejido y hepatocitos primarios para cultivo. La línea celular de hepatocitos de ratón (AML12) se transdujo con un shRNA dirigido a Ide mRNA mediante un vector lentiviral y se obtuvo una línea estable (AML12-shRNA-IDE). Los hepatocitos primarios L-IDE-KO y AML12-shRNA-IDE con sus respectivos controles se estimularon con glucagón y la vía de señalización se analizó mediante western blot y ELISA. La función mitocondrial y el metabolismo energético de AML12-shRNA-IDE y las células de control se evaluaron mediante Seahorse XFe24 Analyzer con un Mito Stress Assay. En el hígado de ratones alimentados con HFD, el ayuno redujo los niveles de proteína IDE (~30%); mientras que la realimentación aumentó su actividad (~45%) en ambos ratones alimentados con SD y HFD. Las concentraciones de lactato circulante se correlacionaron directamente con la actividad hepática de IDE y los niveles de proteína. Es de destacar que el L-lactato en los lisados ​​de hígado aumentó la actividad de IDE de una manera dependiente de la dosis. Además, los niveles de proteína IDE en el hígado, pero no su actividad, se correlacionaron inversamente (R2 = 0,3734, p < 0,01) con un marcador sustituto de resistencia a la insulina (índice HOMA). Los extractos de hígado y hepatocitos primarios de ratones L-IDE-KO, en comparación con WT, mostraron una expresión reducida del receptor de glucagón (~60 %), la proteína CREB (~40 %) y una fosforilación disminuida de CREB (~50 %) tras la estimulación con glucagón . La expresión de ide y los niveles de proteína IDE se redujeron en ~ 50% en células AML12-shRNA-IDE. En el estado basal, el receptor de glucagón, FoxO1 y la proteína CREB fueron significativamente más bajos en las células AML12-shRNA-IDE que en las células de control (~40 %, ~75 % y ~75 %, respectivamente). La estimulación con glucagón resultó en menos (~ 30 %) niveles de cAMP y cambios en la cinética de la fosforilación mediada por glucagón de CREB y otros sustratos de PKA en AML12-shRNA-IDE. Los análisis de caballitos de mar mostraron que tanto el consumo de oxígeno como las tasas de acidificación extracelular aumentaron dos veces en AML12-shRNA-IDE con un incremento de dos veces en la producción de trifosfato de adenosina (ATP) mitocondrial y glucolítico. Finalmente, generamos, utilizando modelos de homología mediante la técnica de satisfacción de restricciones espaciales, estructuras 3D completas de isoformas IDE humanas y murinas con los exones 15a y 15b. Nuestros resultados destacan que la regulación nutricional de IDE en el hígado es más compleja de lo esperado previamente en ratones. La expresión reducida de IDE en hepatocitos de ratón tiene un efecto nocivo sobre la señalización de glucagón, lo que afecta esta vía intracelular en paralelo con un cambio hacia un fenotipo más energético. Estos hallazgos sugieren que la IDE es necesaria para la transducción adecuada de señales de glucagón y la regulación de la producción de energía en los hepatocitos.Escuela de DoctoradoDoctorado en Investigación Biomédic