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A comparison between different human hepatocyte models reveals profound differences in net glucose production, lipid composition and metabolism in vitro.

Abstract (Expand)

Hepatocytes are responsible for maintaining a stable blood glucose concentration during periods of nutrient scarcity. The breakdown of glycogen and de novo synthesis of glucose are crucial metabolic … pathways deeply interlinked with lipid metabolism. Alterations in these pathways are often associated with metabolic diseases with serious clinical implications. Studying energy metabolism in human cells is challenging. Primary hepatocytes are still considered the golden standard for in vitro studies and have been instrumental in elucidating key aspects of energy metabolism found in vivo. As a result of several limitations posed by using primary cells, a multitude of alternative hepatocyte cellular models emerged as potential substitutes. Yet, there remains a lack of clarity regarding the precise applications for which these models accurately reflect the metabolic competence of primary hepatocytes. In this study, we compared primary hepatocytes, stem cell-derived hepatocytes, adult donor-derived liver organoids, immortalized Upcyte-hepatocytes and the hepatoma cell line HepG2s in their response to a glucose production challenge. We observed the highest net glucose production in primary hepatocytes, followed by organoids, stem-cell derived hepatocytes, Upcyte-hepatocytes and HepG2s. Glucogenic gene induction was observed in all tested models, as indicated by an increase in G6PC and PCK1 expression. Lipidomic analysis revealed considerable differences across the models, with organoids showing the closest similarity to primary hepatocytes in the common lipidome, comprising 347 lipid species across 19 classes. Changes in lipid profiles as a result of the glucose production challenge showed a variety of, and in some cases opposite, trends when compared to primary hepatocytes.

Authors: F. Bonanini, M. Singh, H. Yang, D. Kurek, A. C. Harms, A. Mardinoglu, T. Hankemeier

Date Published: 1st Apr 2024

Publication Type: Journal

Hepatic ChREBP orchestrates intrahepatic carbohydrate metabolism to limit hepatic glucose 6-phosphate and glycogen accumulation in a mouse model for acute Glycogen Storage Disease type Ib

Abstract (Expand)

Carbohydrate Response Element Binding Protein (ChREBP) is a glucose 6-phosphate (G6P)-sensitive transcription factor that acts as a metabolic switch to maintain intracellular glucose and phosphate … homeostasis. Hepatic ChREBP is well-known for its regulatory role in glycolysis, the pentose phosphate pathway, and de novo lipogenesis. The physiological role of ChREBP in hepatic glycogen metabolism and blood glucose regulation has not been assessed in detail, and ChREBP's contribution to carbohydrate flux adaptations in hepatic Glycogen Storage Disease type 1 (GSD I) requires further investigation.

Authors: K.A. Krishnamurthy, M.G.S. Rutten, J.A. Hoogerland, T.H. van Dijk, T. Bos, M. Koehorst, M.P. de Vries, N.J. Kloosterhuis, H. Havinga, B.V. Schomakers, M. van Weeghel, J.C. Wolters, B.M. Bakker, M.H. Oosterveer

Date Published: 2024

Publication Type: Journal

Systematic transcriptional analysis of human cell lines for gene expression landscape and tumor representation

Abstract (Expand)

Abstract Cell lines are valuable resources as model for human biology and translational medicine. It is thus important to explore the concordance between the expression in various cell lines vis-à-vis …ous cell lines vis-à-vis human native and disease tissues. In this study, we investigate the expression of all human protein-coding genes in more than 1,000 human cell lines representing 27 cancer types by a genome-wide transcriptomics analysis. The cell line gene expression is compared with the corresponding profiles in various tissues, organs, single-cell types and cancers. Here, we present the expression for each cell line and give guidance for the most appropriate cell line for a given experimental study. In addition, we explore the cancer-related pathway and cytokine activity of the cell lines to aid human biology studies and drug development projects. All data are presented in an open access cell line section of the Human Protein Atlas to facilitate the exploration of all human protein-coding genes across these cell lines.

Authors: Han Jin, Cheng Zhang, Martin Zwahlen, Kalle von Feilitzen, Max Karlsson, Mengnan Shi, Meng Yuan, Xiya Song, Xiangyu Li, Hong Yang, Hasan Turkez, Linn Fagerberg, Mathias Uhlén, Adil Mardinoglu

Date Published: 1st Dec 2023

Publication Type: Journal

Development of HILIC-MS/MS method for acyl-CoAs covering short- to long-chain species in a single analytical run

Abstract (Expand)

Acyl-CoAs play a significant role in numerous physiological and metabolic processes making it important to assess their concentration levels for evaluating metabolic health. Considering the important …he important role of acyl-CoAs, it is crucial to develop an analytical method that can analyze these compounds. Due to the structural variations of acyl-CoAs, multiple analytical methods are often required for comprehensive analysis of these compounds, which increases complexity and the analysis time. In this study, we have developed a method using a zwitterionic HILIC column that enables the coverage of free CoA and short- to long-chain acyl-CoA species in one analytical run. Initially, we developed the method on a QTOF instrument for the identification of acyl-CoA species, optimizing their chromatography and retention times. Later, a targeted HILIC-MS/MS method was created in scheduled multiple reaction monitoring mode on a QTRAP instrument. The performance of the method was evaluated based on various parameters such as linearity, precision, recovery and matrix effect. This method was applied to identify the difference in acyl-CoA profiles in HepG2 cells cultured in different conditions. Our findings revealed an increase in levels of acetyl-CoA, medium- and long-chain acyl-CoA while a decrease in the profiles of free CoA in the starved state, indicating a clear alteration in the fatty acid oxidation process.

Authors: Madhulika Singh, Ligia Akemi Kiyuna, Christoff Odendaal, Barbara M. Bakker, Amy C Harms, Thomas Hankemeier

Date Published: 11th Sep 2023

Publication Type: Journal

Personalised modelling of clinical heterogeneity between medium-chain acyl-CoA dehydrogenase patients.

Abstract (Expand)

BACKGROUND: Monogenetic inborn errors of metabolism cause a wide phenotypic heterogeneity that may even differ between family members carrying the same genetic variant. Computational modelling of … metabolic networks may identify putative sources of this inter-patient heterogeneity. Here, we mainly focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common inborn error of the mitochondrial fatty acid oxidation (mFAO). It is an enigma why some MCADD patients-if untreated-are at risk to develop severe metabolic decompensations, whereas others remain asymptomatic throughout life. We hypothesised that an ability to maintain an increased free mitochondrial CoA (CoASH) and pathway flux might distinguish asymptomatic from symptomatic patients. RESULTS: We built and experimentally validated, for the first time, a kinetic model of the human liver mFAO. Metabolites were partitioned according to their water solubility between the bulk aqueous matrix and the inner membrane. Enzymes are also either membrane-bound or in the matrix. This metabolite partitioning is a novel model attribute and improved predictions. MCADD substantially reduced pathway flux and CoASH, the latter due to the sequestration of CoA as medium-chain acyl-CoA esters. Analysis of urine from MCADD patients obtained during a metabolic decompensation showed an accumulation of medium- and short-chain acylcarnitines, just like the acyl-CoA pool in the MCADD model. The model suggested some rescues that increased flux and CoASH, notably increasing short-chain acyl-CoA dehydrogenase (SCAD) levels. Proteome analysis of MCADD patient-derived fibroblasts indeed revealed elevated levels of SCAD in a patient with a clinically asymptomatic state. This is a rescue for MCADD that has not been explored before. Personalised models based on these proteomics data confirmed an increased pathway flux and CoASH in the model of an asymptomatic patient compared to those of symptomatic MCADD patients. CONCLUSIONS: We present a detailed, validated kinetic model of mFAO in human liver, with solubility-dependent metabolite partitioning. Personalised modelling of individual patients provides a novel explanation for phenotypic heterogeneity among MCADD patients. Further development of personalised metabolic models is a promising direction to improve individualised risk assessment, management and monitoring for inborn errors of metabolism.

Authors: C. Odendaal, E. A. Jager, A. M. F. Martines, M. A. Vieira-Lara, N. C. A. Huijkman, L. A. Kiyuna, A. Gerding, J. C. Wolters, R. Heiner-Fokkema, K. van Eunen, T. G. J. Derks, B. M. Bakker

Date Published: 4th Sep 2023

Publication Type: Journal

Development of a targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry based lipidomics platform applied to a coronavirus disease severity study.

Abstract (Expand)

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative … and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R(2)) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.

Authors: Z. Zhang, M. Singh, A. Kindt, A. B. Wegrzyn, M. J. Pearson, A. Ali, A. C. Harms, P. Baker, T. Hankemeier

Date Published: 31st Aug 2023

Publication Type: Journal

Chemical identification and indexing in full-text articles: an overview of the NLM-Chem track at BioCreative VII

Abstract (Expand)

Abstract The BioCreative National Library of Medicine (NLM)-Chem track calls for a community effort to fine-tune automated recognition of chemical names in the biomedical literature. Chemicals are one …erature. Chemicals are one of the most searched biomedical entities in PubMed, and—as highlighted during the coronavirus disease 2019 pandemic—their identification may significantly advance research in multiple biomedical subfields. While previous community challenges focused on identifying chemical names mentioned in titles and abstracts, the full text contains valuable additional detail. We, therefore, organized the BioCreative NLM-Chem track as a community effort to address automated chemical entity recognition in full-text articles. The track consisted of two tasks: (i) chemical identification and (ii) chemical indexing. The chemical identification task required predicting all chemicals mentioned in recently published full-text articles, both span [i.e. named entity recognition (NER)] and normalization (i.e. entity linking), using Medical Subject Headings (MeSH). The chemical indexing task required identifying which chemicals reflect topics for each article and should therefore appear in the listing of MeSH terms for the document in the MEDLINE article indexing. This manuscript summarizes the BioCreative NLM-Chem track and post-challenge experiments. We received a total of 85 submissions from 17 teams worldwide. The highest performance achieved for the chemical identification task was 0.8672 F-score (0.8759 precision and 0.8587 recall) for strict NER performance and 0.8136 F-score (0.8621 precision and 0.7702 recall) for strict normalization performance. The highest performance achieved for the chemical indexing task was 0.6073 F-score (0.7417 precision and 0.5141 recall). This community challenge demonstrated that (i) the current substantial achievements in deep learning technologies can be utilized to improve automated prediction accuracy further and (ii) the chemical indexing task is substantially more challenging. We look forward to further developing biomedical text–mining methods to respond to the rapid growth of biomedical literature. The NLM-Chem track dataset and other challenge materials are publicly available at https://ftp.ncbi.nlm.nih.gov/pub/lu/BC7-NLM-Chem-track/. Database URL https://ftp.ncbi.nlm.nih.gov/pub/lu/BC7-NLM-Chem-track/

Authors: Robert Leaman, Rezarta Islamaj, Virginia Adams, Mohammed A Alliheedi, João Rafael Almeida, Rui Antunes, Robert Bevan, Yung-Chun Chang, Arslan Erdengasileng, Matthew Hodgskiss, Ryuki Ida, Hyunjae Kim, Keqiao Li, Robert E Mercer, Lukrécia Mertová, Ghadeer Mobasher, Hoo-Chang Shin, Mujeen Sung, Tomoki Tsujimura, Wen-Chao Yeh, Zhiyong Lu

Date Published: 2023

Publication Type: Journal

WeLT: Improving Biomedical Fine-tuned Pre-trained Language Models with Cost-sensitive Learning

Abstract

Not specified

Authors: Ghadeer Mobasher, Wolfgang Müller, Olga Krebs, Michael Gertz

Date Published: 2023

Publication Type: InProceedings

Cardiac PI3K p110alpha attenuation delays aging and extends lifespan.

Abstract (Expand)

Phosphoinositide 3-kinase (PI3K) is a key component of the insulin signaling pathway that controls cellular me-tabolism and growth. Loss-of-function mutations in PI3K signaling and other downstream … effectors of the insulin signaling pathway extend the lifespan of various model organisms. However, the pro-longevity effect appears to be sex-specific and young mice with reduced PI3K signaling have increased risk of cardiac disease. Hence, it remains elusive as to whether PI3K inhibition is a valid strategy to delay aging and extend healthspan in humans. We recently demonstrated that reduced PI3K activity in cardiomyocytes delays cardiac growth, causing subnormal contractility and cardiopulmonary functional capacity, as well as increased risk of mortality at young age. In stark contrast, in aged mice, experi-mental attenuation of PI3K signaling reduced the age-dependent decline in cardiac function and extended maximal lifespan, suggesting a biphasic effect of PI3K on cardiac health and survival. The cardiac anti-aging effects of reduced PI3K activity coincided with enhanced oxida-tive phosphorylation and required increased autophagic flux. In humans, explanted failing hearts showed in-creased PI3K signaling, as indicated by increased phos-phorylation of the serine/threonine-protein kinase AKT. Hence, late-life cardiac-specific targeting of PI3K might have a therapeutic potential in cardiac aging and related diseases.

Authors: M. Abdellatif, T. Eisenberg, A. M. Heberle, K. Thedieck, G. Kroemer, S. Sedej

Date Published: 30th Nov 2022

Publication Type: Journal

In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed.

Abstract (Expand)

With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we … introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.

Authors: F. Bonanini, D. Kurek, S. Previdi, A. Nicolas, D. Hendriks, S. de Ruiter, M. Meyer, M. Clapes Cabrer, R. Dinkelberg, S. B. Garcia, B. Kramer, T. Olivier, H. Hu, C. Lopez-Iglesias, F. Schavemaker, E. Walinga, D. Dutta, K. Queiroz, K. Domansky, B. Ronden, J. Joore, H. L. Lanz, P. J. Peters, S. J. Trietsch, H. Clevers, P. Vulto

Date Published: 16th Jun 2022

Publication Type: Journal

A network-based approach reveals the dysregulated transcriptional regulation in non-alcoholic fatty liver disease.

Abstract (Expand)

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease worldwide. We performed network analysis to investigate the dysregulated biological processes in the disease … progression and revealed the molecular mechanism underlying NAFLD. Based on network analysis, we identified a highly conserved disease-associated gene module across three different NAFLD cohorts and highlighted the predominant role of key transcriptional regulators associated with lipid and cholesterol metabolism. In addition, we revealed the detailed metabolic differences between heterogeneous NAFLD patients through integrative systems analysis of transcriptomic data and liver-specific genome-scale metabolic model. Furthermore, we identified transcription factors (TFs), including SREBF2, HNF4A, SREBF1, YY1, and KLF13, showing regulation of hepatic expression of genes in the NAFLD-associated modules and validated the TFs using data generated from a mouse NAFLD model. In conclusion, our integrative analysis facilitates the understanding of the regulatory mechanism of these perturbed TFs and their associated biological processes.

Authors: H. Yang, M. Arif, M. Yuan, X. Li, K. Shong, H. Turkez, J. Nielsen, M. Uhlen, J. Boren, C. Zhang, A. Mardinoglu

Date Published: 19th Nov 2021

Publication Type: Journal

Bistability in fatty-acid oxidation resulting from substrate inhibition.

Abstract (Expand)

In this study we demonstrated through analytic considerations and numerical studies that the mitochondrial fatty-acid beta-oxidation can exhibit bistable-hysteresis behavior. In an experimentally … validated computational model we identified a specific region in the parameter space in which two distinct stable and one unstable steady state could be attained with different fluxes. The two stable states were referred to as low-flux (disease) and high-flux (healthy) state. By a modular kinetic approach we traced the origin and causes of the bistability back to the distributive kinetics and the conservation of CoA, in particular in the last rounds of the beta-oxidation. We then extended the model to investigate various interventions that may confer health benefits by activating the pathway, including (i) activation of the last enzyme MCKAT via its endogenous regulator p46-SHC protein, (ii) addition of a thioesterase (an acyl-CoA hydrolysing enzyme) as a safety valve, and (iii) concomitant activation of a number of upstream and downstream enzymes by short-chain fatty-acids (SCFA), metabolites that are produced from nutritional fibers in the gut. A high concentration of SCFAs, thioesterase activity, and inhibition of the p46Shc protein led to a disappearance of the bistability, leaving only the high-flux state. A better understanding of the switch behavior of the mitochondrial fatty-acid oxidation process between a low- and a high-flux state may lead to dietary and pharmacological intervention in the treatment or prevention of obesity and or non-alcoholic fatty-liver disease.

Authors: F. Abegaz, A. M. F. Martines, M. A. Vieira-Lara, M. Rios-Morales, D. J. Reijngoud, E. C. Wit, B. M. Bakker

Date Published: 13th Aug 2021

Publication Type: Journal

Welcome to the Family: Identification of the NAD(+) Transporter of Animal Mitochondria as Member of the Solute Carrier Family SLC25.

Abstract (Expand)

Subcellular compartmentation is a fundamental property of eukaryotic cells. Communication and metabolic and regulatory interconnectivity between organelles require that solutes can be transported across … their surrounding membranes. Indeed, in mammals, there are hundreds of genes encoding solute carriers (SLCs) which mediate the selective transport of molecules such as nucleotides, amino acids, and sugars across biological membranes. Research over many years has identified the localization and preferred substrates of a large variety of SLCs. Of particular interest has been the SLC25 family, which includes carriers embedded in the inner membrane of mitochondria to secure the supply of these organelles with major metabolic intermediates and coenzymes. The substrate specificity of many of these carriers has been established in the past. However, the route by which animal mitochondria are supplied with NAD(+) had long remained obscure. Only just recently, the existence of a human mitochondrial NAD(+) carrier was firmly established. With the realization that SLC25A51 (or MCART1) represents the major mitochondrial NAD(+) carrier in mammals, a long-standing mystery in NAD(+) biology has been resolved. Here, we summarize the functional importance and structural features of this carrier as well as the key observations leading to its discovery.

Authors: M. Ziegler, M. Monne, A. Nikiforov, G. Agrimi, I. Heiland, F. Palmieri

Date Published: 14th Jun 2021

Publication Type: Journal

Finding new edges: systems approaches to MTOR signaling.

Abstract (Expand)

Cells have evolved highly intertwined kinase networks to finely tune cellular homeostasis to the environment. The network converging on the mechanistic target of rapamycin (MTOR) kinase constitutes a … central hub that integrates metabolic signals and adapts cellular metabolism and functions to nutritional changes and stress. Feedforward and feedback loops, crosstalks and a plethora of modulators finely balance MTOR-driven anabolic and catabolic processes. This complexity renders it difficult - if not impossible - to intuitively decipher signaling dynamics and network topology. Over the last two decades, systems approaches have emerged as powerful tools to simulate signaling network dynamics and responses. In this review, we discuss the contribution of systems studies to the discovery of novel edges and modulators in the MTOR network in healthy cells and in disease.

Authors: A. M. Heberle, U. Rehbein, M. Rodriguez Peiris, K. Thedieck

Date Published: 26th Feb 2021

Publication Type: Journal

Transcriptome analysis suggests a compensatory role of the cofactors coenzyme A and NAD+ in medium-chain acyl-CoA dehydrogenase knockout mice

Abstract (Expand)

Abstract During fasting, mitochondrial fatty-acid β-oxidation (mFAO) is essential for the generation of glucose by the liver. Children with a loss-of-function deficiency in the mFAO enzyme medium-chain … acyl-Coenzyme A dehydrogenase (MCAD) are at serious risk of life-threatening low blood glucose levels during fasting in combination with intercurrent disease. However, a subset of these children remains asymptomatic throughout life. In MCAD-deficient (MCAD-KO) mice, glucose levels are similar to those of wild-type (WT) mice, even during fasting. We investigated if metabolic adaptations in the liver may underlie the robustness of this KO mouse. WT and KO mice were given a high- or low-fat diet and subsequently fasted. We analyzed histology, mitochondrial function, targeted mitochondrial proteomics, and transcriptome in liver tissue. Loss of MCAD led to a decreased capacity to oxidize octanoyl-CoA. This was not compensated for by altered protein levels of the short- and long-chain isoenzymes SCAD and LCAD. In the transcriptome, we identified subtle adaptations in the expression of genes encoding enzymes catalyzing CoA- and NAD(P)(H)-involving reactions and of genes involved in detoxification mechanisms. We discuss how these processes may contribute to robustness in MCAD-KO mice and potentially also in asymptomatic human subjects with a complete loss of MCAD activity.

Authors: Anne-Claire M. F. Martines, Albert Gerding, Sarah Stolle, Marcel A. Vieira-Lara, Justina C. Wolters, Angelika Jurdzinski, Laura Bongiovanni, Alain de Bruin, Pieter van der Vlies, Gerben van der Vries, Vincent W. Bloks, Terry G. J. Derks, Dirk-Jan Reijngoud, Barbara M. Bakker

Date Published: 1st Dec 2019

Publication Type: Journal

Hepatocytes contribute to residual glucose production in a mouse model for glycogen storage disease type Ia.

Abstract (Expand)

It is a long-standing enigma how glycogen storage disease (GSD) type I patients retain a limited capacity for endogenous glucose production despite the loss of glucose-6-phosphatase activity. Insight … into the source of residual endogenous glucose production is of clinical importance given the risk of sudden death in these patients, but so far contradictory mechanisms have been proposed. We investigated glucose-6-phosphatase-independent endogenous glucose production in hepatocytes isolated from a liver-specific GSD Ia mouse model (L-G6pc(-/-) mice) and performed real-time analysis of hepatic glucose fluxes and glycogen metabolism in L-G6pc(-/-) mice using state-of-the-art stable isotope methodologies. Here we show that G6pc-deficient hepatocytes are capable of producing glucose. In vivo analysis of hepatic glucose metabolism revealed that the hepatic glucokinase flux was decreased by 95% in L-G6pc(-/-) mice. It also showed increased glycogen phosphorylase flux in L-G6pc(-/-) mice, which is coupled to the release of free glucose through glycogen debranching. Although the ex vivo activities of debranching enzyme and lysosomal acid maltase, two major hepatic alpha-glucosidases, were unaltered in L-G6pc(-/-) mice, pharmacological inhibition of alpha-glucosidase activity almost completely abolished residual glucose production by G6pc-deficient hepatocytes. CONCLUSION: Our data indicate that hepatocytes contribute to residual glucose production in GSD Ia. We show that alpha-glucosidase activity, i.e. glycogen debranching and/or lysosomal glycogen breakdown, contributes to residual glucose production by GSD Ia hepatocytes. A strong reduction in hepatic GCK flux in L-G6pc-/- mice furthermore limits the phosphorylation of free glucose synthesized by G6pc-deficient hepatocytes, allowing the release of glucose into the circulation. The almost complete abrogation of GCK flux in G6pc-deficient liver also explains the contradictory reports on residual glucose production in GSD Ia patients. (Hepatology 2017;66:2042-2054).

Authors: B. S. Hijmans, A. Boss, T. H. van Dijk, M. Soty, H. Wolters, E. Mutel, A. K. Groen, T. G. J. Derks, G. Mithieux, A. Heerschap, D. J. Reijngoud, F. Rajas, M. H. Oosterveer

Date Published: 21st Jul 2017

Publication Type: Journal

Living on the edge: substrate competition explains loss of robustness in mitochondrial fatty-acid oxidation disorders.

Abstract (Expand)

BACKGROUND: Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different … chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders. RESULTS: First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients' metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach. CONCLUSION: We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

Authors: K. van Eunen, C. M. Volker-Touw, A. Gerding, A. Bleeker, J. C. Wolters, W. J. van Rijt, A. M. Martines, K. E. Niezen-Koning, R. M. Heiner, H. Permentier, A. K. Groen, D. J. Reijngoud, T. G. Derks, B. M. Bakker

Date Published: 7th Dec 2016

Publication Type: Journal

A systems study reveals concurrent activation of AMPK and mTOR by amino acids.

Abstract (Expand)

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known … about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

Authors: P. Dalle Pezze, S. Ruf, A. G. Sonntag, M. Langelaar-Makkinje, P. Hall, A. M. Heberle, P. Razquin Navas, K. van Eunen, R. C. Tolle, J. J. Schwarz, H. Wiese, B. Warscheid, J. Deitersen, B. Stork, E. Fassler, S. Schauble, U. Hahn, P. Horvatovich, D. P. Shanley, K. Thedieck

Date Published: 21st Nov 2016

Publication Type: Journal

Translational Targeted Proteomics Profiling of Mitochondrial Energy Metabolic Pathways in Mouse and Human Samples.

Abstract (Expand)

Absolute measurements of protein abundance are important in the understanding of biological processes and the precise computational modeling of biological pathways. We developed targeted LC-MS/MS assays … in the selected reaction monitoring (SRM) mode to quantify over 50 mitochondrial proteins in a single run. The targeted proteins cover the tricarboxylic acid cycle, fatty acid beta-oxidation, oxidative phosphorylation, and the detoxification of reactive oxygen species. Assays used isotopically labeled concatemers as internal standards designed to target murine mitochondrial proteins and their human orthologues. Most assays were also suitable to quantify the corresponding protein orthologues in rats. After exclusion of peptides that did not pass the selection criteria, we arrived at SRM assays for 55 mouse, 52 human, and 51 rat proteins. These assays were optimized in isolated mitochondrial fractions from mouse and rat liver and cultured human fibroblasts and in total liver extracts from mouse, rat, and human. The developed proteomics approach is suitable for the quantification of proteins in the mitochondrial energy metabolic pathways in mice, rats, and humans as a basis for translational research. Initial data show that the assays have great potential for elucidating the adaptive response of human patients to mutations in mitochondrial proteins in a clinical setting.

Authors: J. C. Wolters, J. Ciapaite, K. van Eunen, K. E. Niezen-Koning, A. Matton, R. J. Porte, P. Horvatovich, B. M. Bakker, R. Bischoff, H. P. Permentier

Date Published: 2nd Sep 2016

Publication Type: Journal

Parallel DNA Polymerase Chain Reaction (PD-PCR)

Abstract (Expand)

This protocol describe an approach in which the first primer binds in a parallel complementary orientation to the single-stranded DNA, leading to synthesis in a parallel direction. Further reactions … happened in a conventional way leading to the synthesis of PCR product having polarity opposite to the template used. Here in FAIRDOM we use this SOP as an example/template

Authors: vikash bhardwaj, Vikash bhardwaj, Kulbhushan Sharma

Date Published: 5th May 2016

Publication Type: Journal

Genome-scale metabolic modelling of hepatocytes reveals serine deficiency in patients with non-alcoholic fatty liver disease.

Abstract (Expand)

Several liver disorders result from perturbations in the metabolism of hepatocytes, and their underlying mechanisms can be outlined through the use of genome-scale metabolic models (GEMs). Here we … reconstruct a consensus GEM for hepatocytes, which we call iHepatocytes2322, that extends previous models by including an extensive description of lipid metabolism. We build iHepatocytes2322 using Human Metabolic Reaction 2.0 database and proteomics data in Human Protein Atlas, which experimentally validates the incorporated reactions. The reconstruction process enables improved annotation of the proteomics data using the network centric view of iHepatocytes2322. We then use iHepatocytes2322 to analyse transcriptomics data obtained from patients with non-alcoholic fatty liver disease. We show that blood concentrations of chondroitin and heparan sulphates are suitable for diagnosing non-alcoholic steatohepatitis and for the staging of non-alcoholic fatty liver disease. Furthermore, we observe serine deficiency in patients with NASH and identify PSPH, SHMT1 and BCAT1 as potential therapeutic targets for the treatment of non-alcoholic steatohepatitis.

Authors: A. Mardinoglu, R. Agren, C. Kampf, A. Asplund, M. Uhlen, J. Nielsen

Date Published: 15th Jan 2014

Publication Type: Journal

Know when your numbers are significant

Abstract (Expand)

Experimental biologists, their reviewers and their publishers must grasp basic statistics, urges David L. Vaux, or sloppy science will continue to grow. "And, once in the lab, people generally …y just do what everyone else does, without always understanding why." (D. Vaux)

Author: David L. Vaux

Date Published: 1st Dec 2012

Publication Type: Journal

Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening.

Abstract (Expand)

Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel … missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the beta-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.

Authors: E. M. Maier, S. W. Gersting, K. F. Kemter, J. M. Jank, M. Reindl, D. D. Messing, M. S. Truger, C. P. Sommerhoff, A. C. Muntau

Date Published: 1st May 2009

Publication Type: Journal

Disturbed hepatic carbohydrate management during high metabolic demand in medium-chain acyl-CoA dehydrogenase (MCAD)-deficient mice.

Abstract (Expand)

UNLABELLED: Medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) catalyzes crucial steps in mitochondrial fatty acid oxidation, a process that is of key relevance for maintenance of energy homeostasis, … especially during high metabolic demand. To gain insight into the metabolic consequences of MCAD deficiency under these conditions, we compared hepatic carbohydrate metabolism in vivo in wild-type and MCAD(-/-) mice during fasting and during a lipopolysaccharide (LPS)-induced acute phase response (APR). MCAD(-/-) mice did not become more hypoglycemic on fasting or during the APR than wild-type mice did. Nevertheless, microarray analyses revealed increased hepatic peroxisome proliferator-activated receptor gamma coactivator-1alpha (Pgc-1alpha) and decreased peroxisome proliferator-activated receptor alpha (Ppar alpha) and pyruvate dehydrogenase kinase 4 (Pdk4) expression in MCAD(-/-) mice in both conditions, suggesting altered control of hepatic glucose metabolism. Quantitative flux measurements revealed that the de novo synthesis of glucose-6-phosphate (G6P) was not affected on fasting in MCAD(-/-) mice. During the APR, however, this flux was significantly decreased (-20%) in MCAD(-/-) mice compared with wild-type mice. Remarkably, newly formed G6P was preferentially directed toward glycogen in MCAD(-/-) mice under both conditions. Together with diminished de novo synthesis of G6P, this led to a decreased hepatic glucose output during the APR in MCAD(-/-) mice; de novo synthesis of G6P and hepatic glucose output were maintained in wild-type mice under both conditions. APR-associated hypoglycemia, which was observed in wild-type mice as well as MCAD(-/-) mice, was mainly due to enhanced peripheral glucose uptake. CONCLUSION: Our data demonstrate that MCAD deficiency in mice leads to specific changes in hepatic carbohydrate management on exposure to metabolic stress. This deficiency, however, does not lead to reduced de novo synthesis of G6P during fasting alone, which may be due to the existence of compensatory mechanisms or limited rate control of MCAD in murine mitochondrial fatty acid oxidation.

Authors: H. Herrema, T. G. Derks, T. H. van Dijk, V. W. Bloks, A. Gerding, R. Havinga, U. J. Tietge, M. Muller, G. P. Smit, F. Kuipers, D. J. Reijngoud

Date Published: 7th May 2008

Publication Type: Not specified

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