Publications by authors named "Michel van Weeghel"

53 Publications

Reduced nicotinamide mononucleotide is a new and potent NAD precursor in mammalian cells and mice.

FASEB J 2021 Apr;35(4):e21456

Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology, Endocrinology, and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Nicotinamide adenine dinucleotide (NAD ) homeostasis is constantly compromised due to degradation by NAD -dependent enzymes. NAD replenishment by supplementation with the NAD precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can alleviate this imbalance. However, NMN and NR are limited by their mild effect on the cellular NAD pool and the need of high doses. Here, we report a synthesis method of a reduced form of NMN (NMNH), and identify this molecule as a new NAD precursor for the first time. We show that NMNH increases NAD levels to a much higher extent and faster than NMN or NR, and that it is metabolized through a different, NRK and NAMPT-independent, pathway. We also demonstrate that NMNH reduces damage and accelerates repair in renal tubular epithelial cells upon hypoxia/reoxygenation injury. Finally, we find that NMNH administration in mice causes a rapid and sustained NAD surge in whole blood, which is accompanied by increased NAD levels in liver, kidney, muscle, brain, brown adipose tissue, and heart, but not in white adipose tissue. Together, our data highlight NMNH as a new NAD precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD precursors and establish NMNH as a member of the new family of reduced NAD precursors.
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http://dx.doi.org/10.1096/fj.202001826RDOI Listing
April 2021

Aging selectively dampens oscillation of lipid abundance in white and brown adipose tissue.

Sci Rep 2021 Mar 15;11(1):5932. Epub 2021 Mar 15.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC-AMC, University of Amsterdam, Amsterdam Gastroenterology and Metabolism, Amsterdam Cardiovascular Sciences, Meibergdreef 9, 1105 AZ, Amsterdam, the Netherlands.

Lipid metabolism is under the control of the circadian system and circadian dysregulation has been linked to obesity and dyslipidemia. These factors and outcomes have also been associated to, or affected by, the process of aging. Here, we investigated whether murine white (WAT) and brown (BAT) adipose tissue lipids exhibit rhythmicity and if this is affected by aging. To this end, we have measured the 24 h lipid profiles of WAT and BAT using a global lipidomics analysis of > 1100 lipids. We observed rhythmicity in nearly all lipid classes including glycerolipids, glycerophospholipids, sterol lipids and sphingolipids. Overall, ~ 22% of the analyzed lipids were considered rhythmic in WAT and BAT. Despite a general accumulation of lipids upon aging the fraction of oscillating lipids decreased in both tissues to 14% and 18%, respectively. Diurnal profiles of lipids in BAT appeared to depend on the lipid acyl chain length and this specific regulation was lost in aged mice. Our study revealed how aging affects the rhythmicity of lipid metabolism and could contribute to the quest for targets that improve diurnal lipid homeostasis to maintain cardiometabolic health during aging.
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http://dx.doi.org/10.1038/s41598-021-85455-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961067PMC
March 2021

Metabolic rerouting via SCD1 induction impacts X-linked adrenoleukodystrophy.

J Clin Invest 2021 Mar 9. Epub 2021 Mar 9.

Department of Pediatric Neurology, University of Amsterdam, Amsterdam, Netherlands.

X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift towards mono-unsaturated fatty acids relieved toxicity. In human ALD fibroblasts chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts whereas mono-unsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated towards mono-unsaturated VLCFA, and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to mono-unsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.
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http://dx.doi.org/10.1172/JCI142500DOI Listing
March 2021

Metabolomics and lipidomics in using a single sample preparation.

Dis Model Mech 2021 Mar 2. Epub 2021 Mar 2.

Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology, Endocrinology and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands

Comprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand, for instance in research related to nutrition and aging. The nematode is a key model organism in these fields, due to the large repository of available mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in , using a single sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for instance, glycolysis and the TCA cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.
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http://dx.doi.org/10.1242/dmm.047746DOI Listing
March 2021

Metabolic differences between bronchial epithelium from healthy individuals and patients with asthma and the effect of bronchial thermoplasty.

J Allergy Clin Immunol 2021 Feb 6. Epub 2021 Feb 6.

Department of Respiratory Medicine, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Electronic address:

Background: Asthma is a heterogeneous disease with differences in onset, severity, and inflammation. Bronchial epithelial cells (BECs) contribute to asthma pathophysiology.

Objective: We determined whether transcriptomes of BECs reflect heterogeneity in inflammation and severity in asthma, and whether this was affected in BECs from patients with severe asthma after their regeneration by bronchial thermoplasty.

Methods: RNA sequencing was performed on BECs obtained by bronchoscopy from healthy controls (n = 16), patients with mild asthma (n = 17), patients with moderate asthma (n = 5), and patients with severe asthma (n = 17), as well as on BECs from treated and untreated airways of the latter (also 6 months after bronchial thermoplasty) (n = 23). Lipidome and metabolome analyses were performed on cultured BECs from healthy controls (n = 7); patients with severe asthma (n = 9); and, for comparison, patients with chronic obstructive pulmonary disease (n = 7).

Results: Transcriptome analysis of BECs from patients showed a reduced expression of oxidative phosphorylation (OXPHOS) genes, most profoundly in patients with severe asthma but less profoundly and more heterogeneously in patients with mild asthma. Genes related to fatty acid metabolism were significantly upregulated in asthma. Lipidomics revealed enhanced levels of lipid species (phosphatidylcholines, lysophosphatidylcholines. and bis(monoacylglycerol)phosphate), whereas levels of OXPHOS metabolites were reduced in BECs from patients with severe asthma. BECs from patients with mild asthma characterized by hyperresponsive production of mediators implicated in neutrophilic inflammation had decreased expression of OXPHOS genes compared with that in BECs from patients with mild asthma with normoresponsive production. BECs obtained after thermoplasty had significantly increased expression of OXPHOS genes and decreased expression of fatty acid metabolism genes compared with BECs obtained from untreated airways.

Conclusion: BECs in patients with asthma are metabolically different from those in healthy individuals. These differences are linked with inflammation and asthma severity, and they can be reversed by bronchial thermoplasty.
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http://dx.doi.org/10.1016/j.jaci.2020.12.653DOI Listing
February 2021

Hypoxia-inducible lipid droplet-associated induces DGAT1 and promotes lipid storage in hepatocytes.

Mol Metab 2021 Jan 16;47:101168. Epub 2021 Jan 16.

Nutrition, Metabolism, and Genomics Group, Division of Human Nutrition and Health, Wageningen University, Stippeneng 4, Wageningen, 6708 WE, the Netherlands. Electronic address:

Objective: Storage of triglycerides in lipid droplets is governed by a set of lipid droplet-associated proteins. One of these lipid droplet-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair lipid droplet breakdown in macrophages and cancer cells by inhibiting adipose triglyceride lipase. Here, we aimed to better characterize the role and mechanism of action of HILPDA in hepatocytes.

Methods: We performed studies in HILPDA-deficient and HILPDA-overexpressing liver cells, liver slices, and mice. The functional role and physical interactions of HILPDA were investigated using a variety of biochemical and microscopic techniques, including real-time fluorescence live-cell imaging and Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM).

Results: Levels of HILPDA were markedly induced by fatty acids in several hepatoma cell lines. Hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglycerides in mice with non-alcoholic steatohepatitis. Similarly, deficiency of HILPDA in mouse liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labeled fatty acids in lipid droplets, respectively, which was independent of adipose triglyceride lipase. Fluorescence microscopy showed that HILPDA partly colocalizes with lipid droplets and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging further revealed that HILPDA preferentially localizes to lipid droplets that are being remodeled. Overexpression of HILPDA in liver cells increased the activity of diacylglycerol acyltransferases (DGAT) and DGAT1 protein levels, concurrent with increased lipid storage. Confocal microscopy coupled to FRET-FLIM analysis demonstrated that HILPDA physically interacts with DGAT1 in living liver cells. The stimulatory effect of HILPDA on lipid storage via DGAT1 was corroborated in adipocytes.

Conclusions: Our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity. Our findings suggest a novel regulatory mechanism by which fatty acids promote triglyceride synthesis and storage.
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http://dx.doi.org/10.1016/j.molmet.2021.101168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881268PMC
January 2021

Adherence Affects Monocyte Innate Immune Function and Metabolic Reprogramming after Lipopolysaccharide Stimulation In Vitro.

J Immunol 2021 Feb 6;206(4):827-838. Epub 2021 Jan 6.

Center for Experimental and Molecular Medicine, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.

Circulating nonadherent monocytes can migrate to extravascular sites by a process that involves adherence. Alterations in intracellular metabolism shape the immunological phenotype of phagocytes upon activation. To determine the effect of adherence on their metabolic and functional response human monocytes were stimulated with LPS under nonadherent and adherent conditions. Adherent monocytes (relative to nonadherent monocytes) produced less TNF and IL-1β (proinflammatory) and more IL-10 (anti-inflammatory) upon LPS stimulation and had an increased capacity to phagocytose and produce reactive oxygen species. RNA sequencing analysis confirmed that adherence modified the LPS-induced response of monocytes, reducing expression of proinflammatory genes involved in TLR signaling and increasing induction of genes involved in pathogen elimination. Adherence resulted in an increased glycolytic response as indicated by lactate release, gene set enrichment, and [C]-glucose flux analysis. To determine the role of glycolysis in LPS-induced immune responses, this pathway was inhibited by glucose deprivation or the glucose analogue 2-deoxy-d-glucose (2DG). Although both interventions equally inhibited glycolysis, only 2DG influenced monocyte functions, inhibiting expression of genes involved in TLR signaling and pathogen elimination, as well as cytokine release. 2DG, but not glucose deprivation, reduced expression of genes involved in oxidative phosphorylation. Inhibition of oxidative phosphorylation affected TNF and IL-10 release in a similar way as 2DG. Collectively, these data suggest that adherence may modify the metabolic and immunological profile of monocytes and that inhibition of glycolysis and oxidative phosphorylation, but not inhibition of glycolysis alone, has a profound effect on immune functions of monocytes exposed to LPS.
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http://dx.doi.org/10.4049/jimmunol.2000702DOI Listing
February 2021

NLRX1 Deletion Increases Ischemia-Reperfusion Damage and Activates Glucose Metabolism in Mouse Heart.

Front Immunol 2020 11;11:591815. Epub 2020 Dec 11.

Laboratory of Experimental Intensive Care and Anesthesiology, Department of Anesthesiology, Amsterdam UMC, University of Amsterdam, Amsterdam Cardiovascular Sciences, Amsterdam, Netherlands.

Background: NOD-like receptors (NLR) are intracellular sensors of the innate immune system, with the NLRP3 being a pro-inflammatory member that modulates cardiac ischemia-reperfusion injury (IRI) and metabolism. No information is available on a possible role of anti-inflammatory NLRs on IRI and metabolism in the intact heart. Here we hypothesize that the constitutively expressed, anti-inflammatory mitochondrial NLRX1, affects IRI and metabolism of the isolated mouse heart.

Methods: Isolated C57Bl/6J and NLRX1 knock-out (KO) mouse hearts were perfused with a physiological mixture of the essential substrates (lactate, glucose, pyruvate, fatty acid, glutamine) and insulin. For the IRI studies, hearts were subjected to either mild (20 min) or severe (35 min) ischemia and IRI was determined at 60 min reperfusion. Inflammatory mediators (IL-6, TNFα) and survival pathways (mito-HKII, p-Akt, p-AMPK, p-STAT3) were analyzed at 5 min of reperfusion. For the metabolism studies, hearts were perfused for 35 min with either 5.5 mM C-glucose or 0.4 mM C-palmitate under normoxic conditions, followed by LC-MS analysis and integrated, stepwise, mass-isotopomeric flux analysis (MIMOSA).

Results: NLRX1 KO significantly increased IRI (infarct size from 63% to 73%, end-diastolic pressure from 59 mmHg to 75 mmHg, and rate-pressure-product recovery from 15% to 6%), following severe, but not mild, ischemia. The increased IRI in NLRX1 KO hearts was associated with depressed Akt signaling at early reperfusion; other survival pathways or inflammatory parameters were not affected. Metabolically, NLRX1 KO hearts displayed increased lactate production and glucose oxidation relative to fatty acid oxidation, associated with increased pyruvate dehydrogenase flux and 10% higher cardiac oxygen consumption.

Conclusion: Deletion of the mitochondrially-located NOD-like sensor NLRX1 exacerbates severe cardiac IR injury, possibly through impaired Akt signaling, and increases cardiac glucose metabolism.
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http://dx.doi.org/10.3389/fimmu.2020.591815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759503PMC
December 2020

Empagliflozin Decreases Lactate Generation in an NHE-1 Dependent Fashion and Increases α-Ketoglutarate Synthesis From Palmitate in Type II Diabetic Mouse Hearts.

Front Cardiovasc Med 2020 4;7:592233. Epub 2020 Dec 4.

Laboratory of Experimental Intensive Care and Anesthesiology, Department of Anesthesiology, Amsterdam Cardiovascular Sciences, Amsterdam Universitair Medische Centra, University of Amsterdam, Amsterdam, Netherlands.

Changes in cardiac metabolism and ion homeostasis precede and drive cardiac remodeling and heart failure development. We previously demonstrated that sodium/glucose cotransporter 2 inhibitors (SGLT2i's) have direct cardiac effects on ion homeostasis, possibly through inhibition of the cardiac sodium/hydrogen exchanger (NHE-1). Here, we hypothesize that Empagliflozin (EMPA) also possesses direct and acute cardiac effects on glucose and fatty acid metabolism of isolated type II diabetes mellitus () mouse hearts. In addition, we explore whether direct effects on glucose metabolism are nullified in the presence of an NHE-1 inhibitor. Langendorff-perfused type II diabetic db/db mouse hearts were examined in three different series: : C glucose perfusions ( = 32); : C palmitate perfusions ( = 13); and : C glucose + 10 μM Cariporide (specific NHE-1 inhibitor) perfusions ( = 17). Within each series, EMPA treated hearts (1 μM EMPA) were compared with vehicle-perfused hearts (0.02% DMSO). Afterwards, hearts were snap frozen and lysed for stable isotope analysis and metabolomics using LC-MS techniques. Hearts from series 1 were also analyzed for phosphorylation status of AKT, STAT3, AMPK, ERK, and eNOS ( = 8 per group). Cardiac mechanical performance, oxygen consumption and protein phosphorylation were not altered by 35 min EMPA treatment. EMPA was without an overall acute and direct effect on glucose or fatty acid metabolism. However, EMPA did specifically decrease cardiac lactate labeling in the C glucose perfusions (C labeling of lactate: 58 ± 2% vs. 50 ± 3%, for vehicle and EMPA, respectively; = 0.02), without changes in other glucose metabolic pathways. In contrast, EMPA increased cardiac labeling in α-ketoglutarate derived from C palmitate perfusions (C labeling of α-KG: 79 ± 1% vs. 86 ± 1% for vehicle and EMPA, respectively; = 0.01). Inhibition of the NHE by Cariporide abolished EMPA effects on lactate labeling from C glucose. The present study shows for the first time that the SGLT2 inhibitor Empagliflozin has acute specific metabolic effects in isolated diabetic hearts, i.e., decreased lactate generation from labeled glucose and increased α-ketoglutarate synthesis from labeled palmitate. The decreased lactate generation by EMPA seems to be mediated through NHE-1 inhibition.
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http://dx.doi.org/10.3389/fcvm.2020.592233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746656PMC
December 2020

Identification of Metabolic Biomarkers in Relation to Methotrexate Response in Early Rheumatoid Arthritis.

J Pers Med 2020 Dec 10;10(4). Epub 2020 Dec 10.

Amsterdam Gastroenterology and Metabolism, Department of Clinical Chemistry, Amsterdam UMC, VUmc, 1081 HV Amsterdam, The Netherlands.

This study aimed to identify baseline metabolic biomarkers for response to methotrexate (MTX) therapy in rheumatoid arthritis (RA) using an untargeted method. In total, 82 baseline plasma samples (41 insufficient responders and 41 sufficient responders to MTX) were selected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, trial number: ISRCTN26791028) based on patients' EULAR response at 3 months. Metabolites were assessed using high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Differences in metabolite concentrations between insufficient and sufficient responders were assessed using partial least square regression discriminant analysis (PLS-DA) and Welch's -test. The predictive performance of the most significant findings was assessed in a receiver operating characteristic plot with area under the curve (AUC), sensitivity and specificity. Finally, overrepresentation analysis was performed to assess if the best discriminating metabolites were enriched in specific metabolic events. Baseline concentrations of homocystine, taurine, adenosine triphosphate, guanosine diphosphate and uric acid were significantly lower in plasma of insufficient responders versus sufficient responders, while glycolytic intermediates 1,3-/2,3-diphosphoglyceric acid, glycerol-3-phosphate and phosphoenolpyruvate were significantly higher in insufficient responders. Homocystine, glycerol-3-phosphate and 1,3-/2,3-diphosphoglyceric acid were independent predictors and together showed a high AUC of 0.81 (95% CI: 0.72-0.91) for the prediction of insufficient response, with corresponding sensitivity of 0.78 and specificity of 0.76. The Warburg effect, glycolysis and amino acid metabolism were identified as underlying metabolic events playing a role in clinical response to MTX in early RA. New metabolites and potential underlying metabolic events correlating with MTX response in early RA were identified, which warrant validation in external cohorts.
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http://dx.doi.org/10.3390/jpm10040271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768454PMC
December 2020

Macrophage ATP citrate lyase deficiency stabilizes atherosclerotic plaques.

Nat Commun 2020 12 8;11(1):6296. Epub 2020 Dec 8.

Department of Medical Biochemistry, Experimental Vascular Biology, Amsterdam Infection and Immunity, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

Macrophages represent a major immune cell population in atherosclerotic plaques and play central role in the progression of this lipid-driven chronic inflammatory disease. Targeting immunometabolism is proposed as a strategy to revert aberrant macrophage activation to improve disease outcome. Here, we show ATP citrate lyase (Acly) to be activated in inflammatory macrophages and human atherosclerotic plaques. We demonstrate that myeloid Acly deficiency induces a stable plaque phenotype characterized by increased collagen deposition and fibrous cap thickness, along with a smaller necrotic core. In-depth functional, lipidomic, and transcriptional characterization indicate deregulated fatty acid and cholesterol biosynthesis and reduced liver X receptor activation within the macrophages in vitro. This results in macrophages that are more prone to undergo apoptosis, whilst maintaining their capacity to phagocytose apoptotic cells. Together, our results indicate that targeting macrophage metabolism improves atherosclerosis outcome and we reveal Acly as a promising therapeutic target to stabilize atherosclerotic plaques.
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http://dx.doi.org/10.1038/s41467-020-20141-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722882PMC
December 2020

IgG Subclasses Shape Cytokine Responses by Human Myeloid Immune Cells through Differential Metabolic Reprogramming.

J Immunol 2020 12 13;205(12):3400-3407. Epub 2020 Nov 13.

Amsterdam Rheumatology and Immunology Center, Department of Rheumatology and Clinical Immunology, Amsterdam University Medical Centers, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands;

IgG Abs are crucial for various immune functions, including neutralization, phagocytosis, and Ab-dependent cellular cytotoxicity. In this study, we identified another function of IgG by showing that IgG immune complexes elicit distinct cytokine profiles by human myeloid immune cells, which are dependent on FcγR activation by the different IgG subclasses. Using monoclonal IgG subclasses with identical Ag specificity, our data demonstrate that the production of Th17-inducing cytokines, such as TNF, IL-1β, and IL-23, is particularly dependent on IgG2, whereas type I IFN responses are controlled by IgG3, and IgG1 is able to regulate both. In addition, we identified that subclass-specific cytokine production is orchestrated at the posttranscriptional level through distinct glycolytic reprogramming of human myeloid immune cells. Combined, these data identify that IgG subclasses provide pathogen- and cell type-specific immunity through differential metabolic reprogramming by FcγRs. These findings may be relevant for future design of Ab-related therapies in the context of infectious diseases, chronic inflammation, and cancer.
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http://dx.doi.org/10.4049/jimmunol.2000263DOI Listing
December 2020

Probing metabolic memory in the hepatic response to fasting.

Physiol Genomics 2020 12 19;52(12):602-617. Epub 2020 Oct 19.

Nutrition, Metabolism and Genomics Group, Division of Human Nutrition and Health, Wageningen University, Wageningen, The Netherlands.

Tissues may respond differently to a particular stimulus if they have been previously exposed to that same stimulus. Here, we tested the hypothesis that a strong metabolic stimulus such as fasting may influence the hepatic response to a subsequent fast and thus elicit a memory effect. Overnight fasting in mice significantly increased plasma free fatty acids, glycerol, β-hydroxybutyrate, and liver triglycerides, and decreased plasma glucose, plasma triglycerides, and liver glycogen levels. In addition, fasting dramatically changed the liver transcriptome, upregulating genes involved in gluconeogenesis and in uptake, oxidation, storage, and mobilization of fatty acids, and downregulating genes involved in fatty acid synthesis, fatty acid elongation/desaturation, and cholesterol synthesis. Fasting also markedly impacted the liver metabolome, causing a decrease in the levels of numerous amino acids, glycolytic-intermediates, TCA cycle intermediates, and nucleotides. However, these fasting-induced changes were unaffected by two previous overnight fasts. Also, no significant effect was observed of prior fasting on glucose tolerance. Finally, analysis of the effect of fasting on the transcriptome in hepatocyte humanized mouse livers indicated modest similarity in gene regulation in mouse and human liver cells. In general, genes involved in metabolic pathways were upregulated or downregulated to a lesser extent in human liver cells than in mouse liver cells. In conclusion, we found that previous exposure to fasting in mice did not influence the hepatic response to a subsequent fast, arguing against the concept of metabolic memory in the liver. Our data provide a useful resource for the study of liver metabolism during fasting.
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http://dx.doi.org/10.1152/physiolgenomics.00117.2020DOI Listing
December 2020

Lipidomics in Nonalcoholic Fatty Liver Disease: Exploring Serum Lipids as Biomarkers for Pediatric Nonalcoholic Fatty Liver Disease.

J Pediatr Gastroenterol Nutr 2020 10;71(4):433-439

Department of Pediatric Gastroenterology and Nutrition, Amsterdam University Medical Centers, Location Academic Medical Center/Emma Children's Hospital, University of Amsterdam.

Objectives: Disturbances in lipid metabolism play an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Using lipidomics, an analytical technique that is used to broadly survey lipid metabolism, we searched for biomarkers in plasma that are correlated with the presence of hepatic steatosis in children with obesity.

Methods: Lipidomics was performed in plasma samples of 21 children with obesity in whom steatosis was detected using proton magnetic resonance spectroscopy (H-MRS) and were compared with the lipidome of 21 samples of nonsteatotic subjects with obesity.

Results: Forty-two samples were analyzed (57% boys; median age 15 years). A total of 18 lipid classes constituting 839 different lipid species were identified. A statistically significant increase in alkyldiacylglycerol (TG[O]) and phosphatidylethanolamine (PE) species and a significant decrease in alkyl/alkenyl-phosphatidylethanolamine (PE[O]), alkyl/alkenyl-lysophosphatidylethanolamine (LPE[O]) and alkyl/alkenyl-phosphatidylcholine (PC[O]) was observed in children with hepatic steatosis compared with controls. Twelve individual lipid species of 3 lipid classes were significantly increased in steatotic subjects compared with controls.

Conclusions: In this pilot study, we found statistically significant alterations in 5 major lipid classes and 12 individual lipid species in children with steatosis. These might be potential biomarkers for pediatric NAFLD. Lipidomic studies in larger cohorts of children are needed to determine the diagnostic value of these lipids and determine whether results can be generalized for different age groups and ethnic backgrounds.
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http://dx.doi.org/10.1097/MPG.0000000000002875DOI Listing
October 2020

Succinate Is an Inflammation-Induced Immunoregulatory Metabolite in Macrophages.

Metabolites 2020 Sep 15;10(9). Epub 2020 Sep 15.

Department of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.

Immunometabolism revealed the crucial role of cellular metabolism in controlling immune cell phenotype and functions. Macrophages, key immune cells that support progression of numerous inflammatory diseases, have been well described as undergoing vast metabolic rewiring upon activation. The immunometabolite succinate particularly gained a lot of attention and emerged as a crucial regulator of macrophage responses and inflammation. Succinate was originally described as a metabolite that supports inflammation via distinct routes. Recently, studies have indicated that succinate and its receptor SUCNR1 can suppress immune responses as well. These apparent contradictory effects might be due to specific experimental settings and particularly the use of distinct succinate forms. We therefore compared the phenotypic and functional effects of distinct succinate forms and receptor mouse models that were previously used for studying succinate immunomodulation. Here, we show that succinate can suppress secretion of inflammatory mediators IL-6, tumor necrosis factor (TNF) and nitric oxide (NO), as well as inhibit mRNA expression of inflammatory macrophages in a SUCNR1-independent manner. We also observed that macrophage SUCNR1 deficiency led to an enhanced inflammatory response without addition of exogenous succinate. While our study does not reveal new mechanistic insights into how succinate elicits different inflammatory responses, it does indicate that the inflammatory effects of succinate and its receptor SUCNR1 in macrophages are clearly context dependent.
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http://dx.doi.org/10.3390/metabo10090372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569821PMC
September 2020

The effect of mirabegron on energy expenditure and brown adipose tissue in healthy lean South Asian and Europid men.

Diabetes Obes Metab 2020 11 29;22(11):2032-2044. Epub 2020 Jul 29.

Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, the Netherlands.

Aim: To compare the effects of cold exposure and the β3-adrenergic receptor agonist mirabegron on plasma lipids, energy expenditure and brown adipose tissue (BAT) activity in South Asians versus Europids.

Materials And Methods: Ten lean Dutch South Asian (aged 18-30 years; body mass index [BMI] 18-25 kg/m ) and 10 age- and BMI-matched Europid men participated in a randomized, double-blinded, cross-over study consisting of three interventions: short-term (~ 2 hours) cold exposure, mirabegron (200 mg one dose p.o.) and placebo. Before and after each intervention, we performed lipidomic analysis in serum, assessed resting energy expenditure (REE) and skin temperature, and measured BAT fat fraction by magnetic resonance imaging.

Results: In both ethnicities, cold exposure increased the levels of several serum lipid species, whereas mirabegron only increased free fatty acids. Cold exposure increased lipid oxidation in both ethnicities, while mirabegron increased lipid oxidation in Europids only. Cold exposure and mirabegron enhanced supraclavicular skin temperature in both ethnicities. Cold exposure decreased BAT fat fraction in both ethnicities. After the combination of data from both ethnicities, mirabegron decreased BAT fat fraction compared with placebo.

Conclusions: In South Asians and Europids, cold exposure and mirabegron induced beneficial metabolic effects. When combining both ethnicities, cold exposure and mirabegron increased REE and lipid oxidation, coinciding with a higher supraclavicular skin temperature and lower BAT fat fraction.
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http://dx.doi.org/10.1111/dom.14120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771034PMC
November 2020

Inhibition of Hepatic Bile Acid Uptake by Myrcludex B Promotes Glucagon-Like Peptide-1 Release and Reduces Obesity.

Cell Mol Gastroenterol Hepatol 2020 21;10(3):451-466. Epub 2020 Apr 21.

Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Department of Gastroenterology and Hepatology, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Background & Aims: Bile acids are important metabolic signaling molecules. Bile acid receptor activation promotes body weight loss and improves glycemic control. The incretin hormone GLP-1 and thyroid hormone activation of T4 to T3 have been suggested as important contributors. Here, we identify the hepatic bile acid uptake transporter Na taurocholate co-transporting polypeptide (NTCP) as target to prolong postprandial bile acid signaling.

Methods: Organic anion transporting polypeptide (OATP)1a/1b KO mice with or without reconstitution with human OATP1B1 in the liver were treated with the NTCP inhibitor Myrcludex B for 3.5 weeks after the onset of obesity induced by high fat diet-feeding. Furthermore, radiolabeled T4 was injected to determine the role of NTCP and OATPs in thyroid hormone clearance from plasma.

Results: Inhibition of NTCP by Myrcludex B in obese Oatp1a/1b KO mice inhibited hepatic clearance of bile acids from portal and systemic blood, stimulated GLP-1 secretion, reduced body weight, and decreased (hepatic) adiposity. NTCP inhibition did not affect hepatic T4 uptake nor lead to increased thyroid hormone activation. Myrcludex B treatment increased fecal energy output, explaining body weight reductions amongst unaltered food intake and energy expenditure.

Conclusions: Pharmacologically targeting hepatic bile acid uptake to increase bile acid signaling is a novel approach to treat obesity and induce GLP1- secretion.
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http://dx.doi.org/10.1016/j.jcmgh.2020.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363705PMC
April 2020

Skeletal muscle in healthy humans exhibits a day-night rhythm in lipid metabolism.

Mol Metab 2020 07 6;37:100989. Epub 2020 Apr 6.

Department of Nutrition and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, P.O. Box 616, 6200 MD Maastricht, the Netherlands. Electronic address:

Objective: Human energy metabolism is under the regulation of the molecular circadian clock; we recently reported that mitochondrial respiration displays a day-night rhythm under study conditions that are similar to real life. Mitochondria are interconnected with lipid droplets, which are of importance in fuel utilization and play a role in muscle insulin sensitivity. Here, we investigated if skeletal muscle lipid content and composition also display day-night rhythmicity in healthy, lean volunteers.

Methods: Skeletal muscle biopsies were obtained from 12 healthy lean male volunteers every 5 h over a 24 h period. Volunteers were provided with standardized meals, and biopsies were taken 4.5 h after each last meal. Lipid droplet size and number were investigated by confocal microscopy. Additionally, the muscle lipidome was assessed using UPLC/HRMS-based semi-targeted lipidomics.

Results: Confocal microscopy revealed diurnal differences in intramyocellular lipid content (P < 0.05) and lipid droplet size in oxidative type 1 muscle fibers (P < 0.01). Lipidomics analysis revealed that 13% of all detected lipids displayed significant day-night rhythmicity. The most rhythmic lipid species were glycerophospholipids and diacylglycerols (DAG), with the latter being the largest fraction (>50% of all rhythmic species). DAG levels showed a day-night pattern with a trough at 1 PM and a peak at 4 AM.

Conclusions: Using two distinct methods, our findings show that myocellular lipid content and whole muscle lipid composition vary across the day-night cycle under normal living conditions. In particular, day-night rhythmicity was present in over half of the DAG lipid species. Future studies are needed to investigate whether rhythmicity in DAG is functionally related to insulin sensitivity and how this might be altered in prediabetes.
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http://dx.doi.org/10.1016/j.molmet.2020.100989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217992PMC
July 2020

Mitochondrial translation and dynamics synergistically extend lifespan in C. elegans through HLH-30.

J Cell Biol 2020 06;219(6)

Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands.

Mitochondrial form and function are closely interlinked in homeostasis and aging. Inhibiting mitochondrial translation is known to increase lifespan in C. elegans, and is accompanied by a fragmented mitochondrial network. However, whether this link between mitochondrial translation and morphology is causal in longevity remains uncharacterized. Here, we show in C. elegans that disrupting mitochondrial network homeostasis by blocking fission or fusion synergizes with reduced mitochondrial translation to prolong lifespan and stimulate stress response such as the mitochondrial unfolded protein response, UPRMT. Conversely, immobilizing the mitochondrial network through a simultaneous disruption of fission and fusion abrogates the lifespan increase induced by mitochondrial translation inhibition. Furthermore, we find that the synergistic effect of inhibiting both mitochondrial translation and dynamics on lifespan, despite stimulating UPRMT, does not require it. Instead, this lifespan-extending synergy is exclusively dependent on the lysosome biogenesis and autophagy transcription factor HLH-30/TFEB. Altogether, our study reveals the mechanistic crosstalk between mitochondrial translation, mitochondrial dynamics, and lysosomal signaling in regulating longevity.
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http://dx.doi.org/10.1083/jcb.201907067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265311PMC
June 2020

Atherogenic Lipoprotein(a) Increases Vascular Glycolysis, Thereby Facilitating Inflammation and Leukocyte Extravasation.

Circ Res 2020 05 12;126(10):1346-1359. Epub 2020 Mar 12.

From the Experimental Vascular Medicine (J.G.S., L.A., J.C.B., M.V., A.K.G., J.K.), Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, the Netherlands.

Rationale: Patients with elevated levels of lipoprotein(a) [Lp(a)] are hallmarked by increased metabolic activity in the arterial wall on positron emission tomography/computed tomography, indicative of a proinflammatory state.

Objective: We hypothesized that Lp(a) induces endothelial cell inflammation by rewiring endothelial metabolism.

Methods And Results: We evaluated the impact of Lp(a) on the endothelium and describe that Lp(a), through its oxidized phospholipid content, activates arterial endothelial cells, facilitating increased transendothelial migration of monocytes. Transcriptome analysis of Lp(a)-stimulated human arterial endothelial cells revealed upregulation of inflammatory pathways comprising monocyte adhesion and migration, coinciding with increased 6-phophofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)-3-mediated glycolysis. ICAM (intercellular adhesion molecule)-1 and PFKFB3 were also found to be upregulated in carotid plaques of patients with elevated levels of Lp(a). Inhibition of PFKFB3 abolished the inflammatory signature with concomitant attenuation of transendothelial migration.

Conclusions: Collectively, our findings show that Lp(a) activates the endothelium by enhancing PFKFB3-mediated glycolysis, leading to a proadhesive state, which can be reversed by inhibition of glycolysis. These findings pave the way for therapeutic agents targeting metabolism aimed at reducing inflammation in patients with cardiovascular disease.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7208285PMC
May 2020

Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism.

Nat Commun 2020 02 28;11(1):1128. Epub 2020 Feb 28.

Department of Medical Biochemistry, Amsterdam UMC, Amsterdam Cardiovascular Sciences and Gastroenterology and Metabolism, University of Amsterdam, Meibergdreef 9, 1105AZ, Amsterdam, The Netherlands.

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRING cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRING cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.
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http://dx.doi.org/10.1038/s41467-020-14811-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048761PMC
February 2020

A Conserved Mito-Cytosolic Translational Balance Links Two Longevity Pathways.

Cell Metab 2020 03 20;31(3):549-563.e7. Epub 2020 Feb 20.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, Amsterdam Gastroenterology and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands. Electronic address:

Slowing down translation in either the cytosol or the mitochondria is a conserved longevity mechanism. Here, we found a non-interventional natural correlation of mitochondrial and cytosolic ribosomal proteins (RPs) in mouse population genetics, suggesting a translational balance. Inhibiting mitochondrial translation in C. elegans through mrps-5 RNAi repressed cytosolic translation. Transcriptomics integrated with proteomics revealed that this inhibition specifically reduced translational efficiency of mRNAs required in growth pathways while increasing stress response mRNAs. The repression of cytosolic translation and extension of lifespan from mrps-5 RNAi were dependent on atf-5/ATF4 and independent from metabolic phenotypes. We found the translational balance to be conserved in mammalian cells upon inhibiting mitochondrial translation pharmacologically with doxycycline. Lastly, extending this in vivo, doxycycline repressed cytosolic translation in the livers of germ-free mice. These data demonstrate that inhibiting mitochondrial translation initiates an atf-5/ATF4-dependent cascade leading to coordinated repression of cytosolic translation, which could be targeted to promote longevity.
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http://dx.doi.org/10.1016/j.cmet.2020.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214782PMC
March 2020

Hepatic Carbohydrate Response Element Binding Protein Activation Limits Nonalcoholic Fatty Liver Disease Development in a Mouse Model for Glycogen Storage Disease Type 1a.

Hepatology 2020 11 30;72(5):1638-1653. Epub 2020 Oct 30.

Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

Background And Aims: Glycogen storage disease (GSD) type 1a is an inborn error of metabolism caused by defective glucose-6-phosphatase catalytic subunit (G6PC) activity. Patients with GSD 1a exhibit severe hepatomegaly due to glycogen and triglyceride (TG) accumulation in the liver. We have shown that the activity of carbohydrate response element binding protein (ChREBP), a key regulator of glycolysis and de novo lipogenesis, is increased in GSD 1a. In the current study, we assessed the contribution of ChREBP to nonalcoholic fatty liver disease (NAFLD) development in a mouse model for hepatic GSD 1a.

Approach And Results: Liver-specific G6pc-knockout (L-G6pc ) mice were treated with adeno-associated viruses (AAVs) 2 or 8 directed against short hairpin ChREBP to normalize hepatic ChREBP activity to levels observed in wild-type mice receiving AAV8-scrambled short hairpin RNA (shSCR). Hepatic ChREBP knockdown markedly increased liver weight and hepatocyte size in L-G6pc mice. This was associated with hepatic accumulation of G6P, glycogen, and lipids, whereas the expression of glycolytic and lipogenic genes was reduced. Enzyme activities, flux measurements, hepatic metabolite analysis and very low density lipoprotein (VLDL)-TG secretion assays revealed that hepatic ChREBP knockdown reduced downstream glycolysis and de novo lipogenesis but also strongly suppressed hepatic VLDL lipidation, hence promoting the storage of "old fat." Interestingly, enhanced VLDL-TG secretion in shSCR-treated L-G6pc mice associated with a ChREBP-dependent induction of the VLDL lipidation proteins microsomal TG transfer protein and transmembrane 6 superfamily member 2 (TM6SF2), the latter being confirmed by ChIP-qPCR.

Conclusions: Attenuation of hepatic ChREBP induction in GSD 1a liver aggravates hepatomegaly because of further accumulation of glycogen and lipids as a result of reduced glycolysis and suppressed VLDL-TG secretion. TM6SF2, critical for VLDL formation, was identified as a ChREBP target in mouse liver. Altogether, our data show that enhanced ChREBP activity limits NAFLD development in GSD 1a by balancing hepatic TG production and secretion.
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http://dx.doi.org/10.1002/hep.31198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702155PMC
November 2020

HILPDA Uncouples Lipid Droplet Accumulation in Adipose Tissue Macrophages from Inflammation and Metabolic Dysregulation.

Cell Rep 2020 02;30(6):1811-1822.e6

Nutrition, Metabolism and Genomics Group, Division of Human Nutrition and Health, Wageningen University, Stippeneng 4, 6708 WE Wageningen, the Netherlands. Electronic address:

Obesity leads to a state of chronic, low-grade inflammation that features the accumulation of lipid-laden macrophages in adipose tissue. Here, we determined the role of macrophage lipid-droplet accumulation in the development of obesity-induced adipose-tissue inflammation, using mice with myeloid-specific deficiency of the lipid-inducible HILPDA protein. HILPDA deficiency markedly reduced intracellular lipid levels and accumulation of fluorescently labeled fatty acids. Decreased lipid storage in HILPDA-deficient macrophages can be rescued by inhibition of adipose triglyceride lipase (ATGL) and is associated with increased oxidative metabolism. In diet-induced obese mice, HILPDA deficiency does not alter inflammatory and metabolic parameters, despite markedly reducing lipid accumulation in macrophages. Overall, we find that HILPDA is a lipid-inducible, physiological inhibitor of ATGL-mediated lipolysis in macrophages and uncouples lipid storage in adipose tissue macrophages from inflammation and metabolic dysregulation. Our data question the contribution of lipid droplet accumulation in adipose tissue macrophages in obesity-induced inflammation and metabolic dysregulation.
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http://dx.doi.org/10.1016/j.celrep.2020.01.046DOI Listing
February 2020

Nutritional ketosis improves exercise metabolism in patients with very long-chain acyl-CoA dehydrogenase deficiency.

J Inherit Metab Dis 2020 07 5;43(4):787-799. Epub 2020 Feb 5.

Neuroimaging Center, Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, Groningen, The Netherlands.

A maladaptive shift from fat to carbohydrate (CHO) oxidation during exercise is thought to underlie myopathy and exercise-induced rhabdomyolysis in patients with fatty acid oxidation (FAO) disorders. We hypothesised that ingestion of a ketone ester (KE) drink prior to exercise could serve as an alternative oxidative substrate supply to boost muscular ATP homeostasis. To establish a rational basis for therapeutic use of KE supplementation in FAO, we tested this hypothesis in patients deficient in Very Long-Chain acyl-CoA Dehydrogenase (VLCAD). Five patients (range 17-45 y; 4 M/1F) patients were included in an investigator-initiated, randomised, blinded, placebo-controlled, 2-way cross-over study. Patients drank either a KE + CHO mix or an isocaloric CHO equivalent and performed 35 minutes upright cycling followed by 10 minutes supine cycling inside a Magnetic Resonance scanner at individual maximal FAO work rate (fatmax; approximately 40% VO max). The protocol was repeated after a 1-week interval with the alternate drink. Primary outcome measures were quadriceps phosphocreatine (PCr), Pi and pH dynamics during exercise and recovery assayed by in vivo P-MR spectroscopy. Secondary outcomes included plasma and muscle metabolites and respiratory gas exchange recordings. Ingestion of KE rapidly induced mild ketosis and increased muscle BHB content. During exercise at FATMAX, VLCADD-specific plasma acylcarnitine levels, quadriceps glycolytic intermediate levels and in vivo Pi/PCr ratio were all lower in KE + CHO than CHO. These results provide a rational basis for future clinical trials of synthetic ketone ester supplementation therapy in patients with FAO disorders. Trial registration: ClinicalTrials.gov. Protocol ID: NCT03531554; METC2014.492; ABR51222.042.14.
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http://dx.doi.org/10.1002/jimd.12217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384182PMC
July 2020

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

Mol Genet Metab 2020 03 9;129(3):171-176. Epub 2020 Jan 9.

Department of Pediatrics, Division of Metabolic Disorders, Emma Children's Hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Background: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes.

Methods: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [UC]Gal-1-P/ [C]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated.

Results: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001).

Conclusions: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.
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http://dx.doi.org/10.1016/j.ymgme.2020.01.002DOI Listing
March 2020

Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism.

Nucleic Acids Res 2020 01;48(2):770-787

Amsterdam UMC, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology and Metabolism, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.
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http://dx.doi.org/10.1093/nar/gkz1042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954397PMC
January 2020

Mutations in PCYT2 disrupt etherlipid biosynthesis and cause a complex hereditary spastic paraplegia.

Brain 2019 11;142(11):3382-3397

Manchester Centre for Genomics Medicine, St Mary's Hospital, Manchester University Hospital Foundation Trust, Health Innovation Manchester, Oxford Road, Manchester, UK.

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.
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http://dx.doi.org/10.1093/brain/awz291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821184PMC
November 2019

Industrial Trans Fatty Acids Stimulate SREBP2-Mediated Cholesterogenesis and Promote Non-Alcoholic Fatty Liver Disease.

Mol Nutr Food Res 2019 10 7;63(19):e1900385. Epub 2019 Aug 7.

Nutrition, Metabolism and Genomics Group, Division of Human Nutrition and Health, Wageningen University, 6708 WE, Wageningen, The Netherlands.

Scope: The mechanisms underlying the deleterious effects of trans fatty acids on plasma cholesterol and non-alcoholic fatty liver disease (NAFLD) are unclear. Here, the aim is to investigate the molecular mechanisms of action of industrial trans fatty acids.

Methods And Results: Hepa1-6 hepatoma cells were incubated with elaidate, oleate, or palmitate. C57Bl/6 mice were fed diets rich in trans-unsaturated, cis-unsaturated, or saturated fatty acids. Transcriptomics analysis of Hepa1-6 cells shows that elaidate but not oleate or palmitate induces expression of genes involved in cholesterol biosynthesis. Induction of cholesterogenesis by elaidate is mediated by increased sterol regulatory element-binding protein 2 (SREBP2) activity and is dependent on SREBP cleavage-activating protein (SCAP), yet independent of liver-X receptor and ubiquitin regulatory X domain-containing protein 8. Elaidate decreases intracellular free cholesterol levels and represses the anticholesterogenic effect of exogenous cholesterol. In mice, the trans-unsaturated diet increases the ratio of liver to gonadal fat mass, steatosis, hepatic cholesterol levels, alanine aminotransferase activity, and fibrosis markers, suggesting enhanced NAFLD, compared to the cis-unsaturated and saturated diets.

Conclusion: Elaidate induces cholesterogenesis in vitro by activating the SCAP-SREBP2 axis, likely by lowering intracellular free cholesterol and attenuating cholesterol-dependent repression of SCAP. This pathway potentially underlies the increase in liver cholesterol and NAFLD by industrial trans fatty acids.
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http://dx.doi.org/10.1002/mnfr.201900385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790681PMC
October 2019